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WO2013053008A2 - Molecules which bind clec9a - Google Patents

Molecules which bind clec9a Download PDF

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Publication number
WO2013053008A2
WO2013053008A2 PCT/AU2012/001233 AU2012001233W WO2013053008A2 WO 2013053008 A2 WO2013053008 A2 WO 2013053008A2 AU 2012001233 W AU2012001233 W AU 2012001233W WO 2013053008 A2 WO2013053008 A2 WO 2013053008A2
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Prior art keywords
actin
cells
fragment
polypeptide
compound
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/AU2012/001233
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French (fr)
Other versions
WO2013053008A3 (en
Inventor
Peter Malcolm Colman
Ross Leon Coppel
Peter Edward Czabotar
Mireille Hanna Lahoud
Nicos Anthony Nicola
Antonia Natalie POLICHENI
Kenneth Douglas Shortman
Kirsteen McInnes TULLETT
Jian-Guo Zhang
Jacob Baum
Wilson Wong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Walter and Eliza Hall Institute of Medical Research
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Walter and Eliza Hall Institute of Medical Research
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Publication of WO2013053008A2 publication Critical patent/WO2013053008A2/en
Publication of WO2013053008A3 publication Critical patent/WO2013053008A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention relates to the identification of molecules which bind the dendritic cell marker known as Clec9A.
  • the present invention provides new compounds for targeting therapeutic agents such as antigens to dendritic cells.
  • methods of modulating a humoral and/or T-cell mediated immune response to the antigen methods of delivery of a cytotoxic agent to dendritic cells thereof involved in diseased states, methods of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, and methods of modulating antigen recognition, processing and/or presentation, as well as immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • DCs Dendritic cells
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damaged cell-associated molecular patterns
  • the CD8 + DC (Shortman and Heath, 2010) are especially efficient at the uptake and processing of material from dead cells, and in "cross-presenting" such exogenous antigenic material on class I HC (Shortman and Heath, 2010).
  • Human CD141 + (BDCA-3 + ) DCs have been identified as the lineage and functional equivalents of the mouse CD8 + DCs (Jongbloed et al., 2010; Bachem et al., 2010; Crozat et al., 2010; Poulin et al., 2010), thus they would be expected to have a particular pattern of PAMP and DAMP receptors related to these functions.
  • Clec9A also called DNGR-1 (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008; Sancho et al., 2009; WO 2009/026660; WO 2009/137871; WO 2010/108215) which binds to dead cells.
  • Clec9A also regulates the cross-presentation of dead cell-associated antigens in a Syk-dependent manner (Sancho et al., 2009).
  • Clec9A has been found to be an especially effective target for delivery of antigens to DCs, so promoting immune responses (Caminschi et al., 2008; Sancho et al., 2008; Idoyaga et al., 2011; Lahoud et al., 2011). This has great promise as a strategy for enhancing the effectiveness of vaccines.
  • the present inventors have identified further ligands of Clec9A. These ligands can be used to target therapeutic agents or detectable labels to Clec9A expressing. cells such as dendritic cells.
  • the present invention provides a polypeptide complex conjugated to a therapeutic agent or a detectable label, wherein the polypeptide complex comprises
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A.
  • the first polypeptide can be selected from a large variety of proteins which bind actin. Examples include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titin, myosin, tubulin, catenin, keratin, cytokeratin, nestin, larnin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
  • the first polypeptide comprises
  • the second polypeptide consists of actin, or a fragment thereof which binds the actin binding domain.
  • the actin is cytoskeletal actin or muscle actin.
  • the actin is filamentous actin or a filamentous fragment thereof.
  • the second polypeptide comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 26 to 40; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 26 to 40; and/or
  • the actin is cytoskeletal actin.
  • the second polypeptide comprises
  • the Clec9A comprises
  • the polypeptide complex further comprises Clec9A.
  • the complex when bound to Clec9A, further comprises RNF41 bound to the Clec9A.
  • the RNF41 comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 21 to 25; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 21 to 25; and/or
  • Suitable therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
  • the antigen can be any molecule that induces an immune response in an animal. Examples include, but are not limited to, a cancer antigen, a self-antigen, an allergen, and or an antigen from a pathogenic and/or infectious organism.
  • the antigen from a pathogenic and/or infectious organism can be from, but not limited to, Plasmodium falciparum or Plasmodium vivax.
  • the first and second polypeptides are separate polypeptide chains which form the complex at least by the actin binding domain binding the actin.
  • first and second polypeptides form part of a single polypeptide chain.
  • the first and second polypeptides are separated by a peptide linker.
  • the present invention provides a compound that binds a polypeptide complex which comprises:
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A.
  • the compound is not an antibody which binds Clec9A alone or a fragment of Clec9A which binds Clec9A alone such as a soluble fragment.
  • the compound is a polypeptide.
  • the compound is an antibody or antigen-binding fragment thereof.
  • antibodies or antigen-binding fragment thereof include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the actin is filamentous actin or a filamentous fragment thereof.
  • the compound of the above aspect is detectably labelled.
  • the present invention provides a composition comprising a polypeptide complex of the invention, or a compound of the invention, and a pharmaceutically acceptable carrier.
  • composition further comprises an adjuvant.
  • the present invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject at least one of
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • the immune response to an antigen is induced and/or enhanced.
  • the immune response is modulated by enhancing a helper T-cell response.
  • the immune response is modulated by the activation of CD4 + and/or CD8 + T-cells.
  • the immune response is modulated by enhancing B-cell antibody production.
  • antibodies produced include, but are not necessarily limited to, IgGl, IgG2b, IgG2c, IgG3, IgG4 ⁇ IgM, IgAl, IgA2, IgE and/or IgD antibody isotypes.
  • the immune response is modulated by generating a memory response.
  • the subject is administered with a compound comprising the antigen.
  • an immune response to a self-antigen or allergen is reduced.
  • the immune response is modulated by suppressing a T-cell response and/or a B-cell antibody response.
  • the present invention provides a method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to at least one of
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • composition of the invention iv) a composition of the invention, and administering said cells to the subject.
  • the cells have been isolated from the subject.
  • a humoral and/or T-cell mediated response is modulated.
  • naive CD8 + T-cell activation, and/or naive CD4 + T- cell activation is modulated.
  • the humoral response comprises the production of IgGl, IgG2b, IgG2c, IgG3, IgG4, IgM, IgAl, IgA2, IgE, and/or IgD antibody isotypes.
  • the humoral response at least comprises the production of IgGl antibody isptype.
  • the dendritic cell is an animal dendritic cell or precursor of an animal dendritic cell. More preferably, the dendritic cell is a human dendritic cell. Even more preferably, the human dendritic cell is Necl-2+, HLA DR+ and/or BDCA-3+.
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • the infection is a Plasmodium sp., such as Plasmodium falciparum or Plasmodium vivax, infection.
  • the present invention provides a method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding.
  • the present invention provides a method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of i) a polypeptide complex comprising
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is increased.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased.
  • the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is increased.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased.
  • the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • diseases include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • the present invention provides a method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
  • actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the compound is an antibody or antigen-binding fragment thereof.
  • examples include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the compound is detectably labelled.
  • the method is performed in vivo on a subject. In an alternate embodiment, the method is performed in vitro on a sample obtained from a subject.
  • diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury .
  • GVHD graft versus host disease
  • SIRS systemic inflammatory reaction syndrome
  • the present invention provides a method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
  • the cell in step i) is in vivo. In an alternate embodiment, the cell in step i) is in vitro.
  • the therapy is administered to a subject.
  • the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
  • the subject has cancer or an infection.
  • step ii) is performed on a sample obtained from a subject.
  • the therapy can be any type of procedure. Examples include, but are not limited to, drug therapy or radiotherapy.
  • the present invention provides a method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
  • actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
  • the compound binding to the polypeptide indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the present invention provides a method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising
  • the cells obtained from step ii) are administered to a subject.
  • the cells are administered to treat and or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
  • the present invention provides a method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
  • the present invention provides a method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
  • the polypeptide complex or compound is detectably labelled.
  • a detectably labelled secondary antibody that binds the polypeptide complex or compound.
  • the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, Clec9A, XCRI and BDCA3.
  • the dendritic cells are human dendritic cells that express one or more of the following markers, Necl-2, HLADR and BDC A3.
  • the dendritic cells are murine dendritic cells that express one or more of the following markers, CD24, Necl-2, CD 11c, Clec9A and CD8.
  • the precursor dendritic cells are intermediate or late precursor
  • dendritic cells which are capable of differentiating into dendritic cells in culture and/or on transfer into irradiated recipients.
  • actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
  • the compound binding to the polypeptide complex indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying or is dead.
  • the compound is not an antibody which binds Clec9A, Clec9A per se or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the present inventors have also identified two tryptophan residues which are important for the biological function of Clec9A.
  • the present invention provides a compound which
  • ii) binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
  • actin or fragment thereof is bound to the actin binding domain, or
  • the compound is a polypeptide.
  • the compound is an antibody or antigen-binding fragment thereof.
  • antibodies or antigen-binding fragment thereof include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the compound does not affect the binding of RNF41 to Clec9A.
  • composition comprising a compound of the above aspect, and a pharmaceutically acceptable carrier.
  • present invention provides a method of reducing an immune response in a subject, the method comprising administering to the subject a compound of the above aspect and/or a composition of the above aspect.
  • the present invention provides a method of reducing an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to a compound of the above aspect and or a composition of the above aspect, and administering said cells to the subject.
  • the present invention provides a method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject a compound of the above aspect and/or a composition of the above aspect.
  • the disease is an autoimmune disease or inflammation.
  • the present invention provides a method of reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
  • the present invention provides a method of reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
  • the present invention provides a method of reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
  • a compound of the above aspect and/or a composition of the above aspect for use in at least one of 3 i) reducing an immune response in a subject,
  • the present invention provides an isolated and/or exogenous polynucleotide encoding
  • polypeptide complex of the invention wherein the first polypeptide and the second polypeptide form part of a single polypeptide chain
  • a vector comprising a polynucleotide of the invention.
  • the vector is an expression vector.
  • the present invention provides a host cell comprising a polynucleotide of the invention, and/or a vector of the invention.
  • the cell can be any cell type such as, but not limited to, a bacterial, yeast, animal, insect or plant cell.
  • the present invention provides an enriched population of dendritic cells and/or precursors thereof, obtained by a method of the invention.
  • the present invention provides an expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof of the invention.
  • composition comprising a polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or a cell population of the invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a crystal of the C-type lectin- like domain of human Clec9A.
  • the present invention provides a set of atomic coordinates, or subset thereof, provided in Appendix I.
  • the present invention provides a computer-readable medium having recorded thereon data representing the atomic coordinates, or subset thereof, provided in Appendix I and/or a model produced using the atomic coordinates.
  • the present invention provides a computer-assisted method of identifying a compound that binds Clec9A, the method comprising
  • the method further comprises synthesising or obtaining an identified candidate compound and determining if the compound binds Clec9A.
  • the present invention provides a computer-assisted method for identifying a compound which binds a polypeptide complex which comprises
  • the method comprises the steps of:
  • the method further comprises synthesising or obtaining a selected candidate compound and detennining if the compound binds the polypeptide complex.
  • the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
  • the present invention provides a method of identifying a compound which binds a polypeptide complex comprising
  • the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, the method comprising a) exposing the polypeptide complex to a binding partner which binds the polypeptide complex, and a candidate compound,
  • the present invention provides a method of identifying a compound which binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO: 1 , the method comprising
  • the present inventors have also identified that Clec9A can bind actin in the absence of a polypeptide which comprises an actin binding domain.
  • the present invention provides an isolated actin or a fragment thereof conjugated to a therapeutic agent or a detectable label, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain.
  • the actin or fragment thereof is filamentous actin (F- actin) or a filamentous fragment thereof.
  • the actin is cytoskeletal actin or muscle actin.
  • the actin is cytoskeletal actin.
  • the second polypeptide comprises
  • the Clec9A comprises
  • the antigen can be any molecule that induces an immune response in an animal. Examples include, but are not limited to, a cancer antigen, a self-antigen, an allergen, and/or an antigen from a pathogenic and/or infectious organism.
  • the conjugated actin or fragment thereof is encapsulated in, or exposed on the surface of, a liposome.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the immune response to an antigen is induced and/or enhanced.
  • the immune response is modulated by enhancing a helper T-cell response.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9 A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the cells have been isolated from the subject.
  • naive CD8 + T-cell activation, and/or naive CD4 + T- cell activation is modulated.
  • the method comprises administering a conjugated aetin or fragment thereof of the invention comprising the cytotoxic agent, drug and/or pharmacological agent.
  • diseases involving dendritic cells or precursors thereof include, but are not limited to, cancer, an infection, an autoimmune disease or an allergy.
  • the autoimmune disease is lupus erythematosus.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the present invention provides a method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the present invention provides a method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is increased. In an alternate embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is increased.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased.
  • the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is increased.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the present invention provides a method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the compound is an antibody or antigen-binding fragment thereof.
  • examples include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the compound is detectably labelled.
  • the method is performed in vivo on a subject. In an alternate embodiment, the method is performed in vitro on a sample obtained from a subject.
  • diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
  • GVHD graft versus host disease
  • SIRS systemic inflammatory reaction syndrome
  • the present invention provides a method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
  • the cell in step i) is in vivo. In an alternate embodiment, the cell in step i) is in vitro.
  • the therapy is administered to a subject.
  • the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
  • the subject has cancer or an infection.
  • step ii) is performed on a sample obtained from a subject.
  • the compound binding to the actin or a fragment thereof indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the cells obtained from step ii) are administered to a subject.
  • the cells are administered to treat and/or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
  • the present invention provides a method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
  • the present invention provides a method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
  • the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, Clec9A, XCRI and BDCA3.
  • the dendritic cells are human dendritic cells that express one or more of the following markers, Necl-2, HLADR and BDCA3.
  • the dendritic cells are murine dendritic cells that express one or more of the following markers, CD24, Necl-2, CDl lc, Clec9A and CD8.
  • the precursor dendritic cells are intermediate or late precursor dendritic cells which are capable of differentiating into dendritic cells in culture and/or on transfer into irradiated recipients.
  • the present invention provides a method of detecting a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
  • the compound binding to the actin or fragment thereof indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying, or is dead.
  • the compound is not an antibody which binds Clec9A, Clec9A per se or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the present invention provides an enriched population of dendritic cells and/or precursors thereof, obtained by a method of the invention.
  • the present invention provides an expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof of the invention.
  • composition comprising a cell population of the invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a method of identifying a compound which binds actin or a fragment thereof, the method comprising
  • the present invention provides a kit comprising one or more of a conjugated actin or fragment thereof of the invention, a cell population of the invention, and a composition of the invention.
  • FIG. 1 A schematic representation of the endogenous and recombinant soluble mClec9A proteins.
  • the endogenous protein includes the Clec9A extracellular domains, the transmembrane (TM) and the cytoplasmic (Cyto) domains.
  • mClec9A-ecto which consists of the full Clec9A ectodomain, a FLAG tag and a biotinylation consensus sequence (predicted mol wt of 27 kDa);
  • mClec9A-CTLD which consists of the Clec9A-CTLD, FLAG tag and biotinylation consensus sequence (predicted mol wt of 19.7 kDa);
  • mClec9A-stalk which consists of the Clec9A-stalk region, FLAG tag and biotinylation consensus sequence (predicted mol wt of 12 kDa).
  • (C) Viable or freeze-thawed mouse fibroblasts (3T3 cell line) were incubated with biotinylated mClec9A or hCLEC9A ectodomains (solid line), or with biotinylated control (Cire-ecto, dashed line). Binding in (C)-(F) was detected using SA-PE and flow cytometry.
  • Control untreated MEFs (viable) and ABT-737 treated MEFs (2.5 ⁇ ABT- 737 for 16 h; late apoptotic) were incubated in PBS alone or in the presence of DNasel, RNaseA, protease K or trypsin. Cells were washed extensively to remove nucleases and proteases, then incubated with biotinylated mClec9A-ecto (solid line), or biotinylated Cire-ecto as a control (dashed line). 80% of the untreated MEFs were viable based on normal FSC and PI exclusion, whereas 97% of the ABT-737 treated MEFs were dead based on reduced FSC and high PI staining.
  • Clec9A-ecto binding to the ⁇ N-4 + muscle actin was significantly greater than binding to ⁇ N-4 alone (*** p ⁇ 0.0001) or to muscle actin alone (*** p ⁇ 0.0005).
  • Clec9A-ecto binding to ⁇ N + muscle actin was significantly greater than binding to ⁇ N alone or to muscle actin alone (*** pO.OOOl);
  • Clec9A-ecto binding to ⁇ N-4 + platelet actin and to ⁇ N + platelet actin was significantly greater than binding to ⁇ N or ⁇ N-4 alone, respectively, or to platelet actin alone (*** p ⁇ 0.0001).
  • C Clec9A binding to dead cells is inhibited by pre-incubation of Clec9A with GST-tagged erythrocytic spectrin ⁇ N plus actin. Pre-associated spectrin ⁇ N plus platelet actin, was incubated with mClec9A-ecto (0.5 h, 21°C), before incubation with freeze-thawed 293F cells.
  • Clec9A-ecto binding to the spectrin ⁇ N-4 + actin was significantly greater than binding to ⁇ N-4 alone (*** pO.0001) or to actin alone (*** p ⁇ 0.0005).
  • Clec9A-ecto binding to the spectrin ⁇ N + actin was significantly greater than binding to ⁇ N or to actin alone (*** p ⁇ 0.0001);
  • Clec9A- ecto binding to a-actinin- 1 N + actin was significantly greater than binding to a- actinin- 1 N or to actin alone (* * * p ⁇ 0.0001 ).
  • human 293F cells were freeze-thawed to induce membrane disruption then incubated with the original hCLEC9A-CTLD, or hCLEC9A-CTLD (S225D) at 5 ⁇ g/ml (solid lines). Binding was detected using FITC-conjugated anti- FLAG mAb, and flow cytometry. Staining with secondary reagents alone is demonstrated (dashed line). This confirmed that the hCLEC9A-CTLD (S225D) maintained binding activity to dead cells.
  • FIG. 1 Schematic representation of RNF41 protein.
  • A, B Mouse Clec9A-ecto binding to GST-RNF41.
  • GST-tagged RNF41-C- terminal domain (CTD), RNF41-N -terminal domains (RBCC) and GST control were coated onto ELISA plates at 10 ⁇ g/ml.
  • FLAG-tagged mClec9A-ecto and mClecl2A- ecto were incubated on the ELISA plates at concentrations from ⁇ to 0.04 g/ml.
  • Clec binding to RNF41 was detected using anti-FLAG-HRP. Cumulative data of 3 experiments is shown, demonstrating the means of absorbance values.
  • C, D Human CLEC9A-ecto binding to GST-RNF41.
  • GST-tagged RNF41-C- terminal domain (CTD), RNF41-N-terminal domains (RBCC) and GST control were coated onto ELISA plates at ⁇ g/ml.
  • FLAG-tagged wildtype CLEC9A (hCLEC9A- ecto) and mutant CLEC9A (W131A, W227A, represented as hCLEC9A-ecto (W->A)) were incubated on the ELISA plates at concentrations from 10 ⁇ g/ml to 0.12 ⁇ / ⁇ 1. CLEC9A binding to RNF41 was detected using anti-FLAG-HRP. Data is shown for 1 representative experiment.
  • mClec9A-ecto 5 ⁇ g ml
  • control mClecl2A-ecto
  • platelet actin 25 ⁇ g/ml
  • GST-tagged ABD 25ug/ml
  • eythrocytic spectrin spectrin ⁇ N
  • non erthyroctic spectrin spectrin ⁇ N
  • a-actinin-1 a-actinin-1 N
  • Wildtype hCLEC9A-ecto, mutant hCLEC9A-ecto (W131A, W227A) and control mClecl2A-ecto ⁇ g/ml) were pre-incubated with platelet actin (25 ⁇ g ml) complexed with GST-tagged ABD (25 ⁇ g ml) of eythrocytic spectrin (spectrin ⁇ N), non erthyroctic spectrin (spectrin ⁇ N) and a-actinin-1 (a-actinin-1 N).
  • CLEC-ABD-Actin complexes were incubated onto ELISA plates and binding to RNF41 was detected using anti-FLAG- HRP. Cumulative data of 2 experiments is shown, demonstrating the means of absorbance values.
  • Ectodomains of mClec9A (5 ⁇ ) were incubated with various concentrations of actin in the form of preformed muscle actin filaments, then centrifuged.
  • SEQ ID NO: 9 Open reading frame encoding human Clec9A.
  • SEQ ID NO: 10 Open reading frame encoding murine Clec9A.
  • SEQ ID NO: 12 Open reading frame encoding rhesus monkey Clec9A.
  • SEQ ID NO: 16 Open reading frame encoding rat Clec9A.
  • SEQ ID NO: 17 Soluble mouse Clec9A including stalk.
  • SEQ ID NO:20 Soluble human Clec9A without stalk.
  • SEQ ID NO:21 Human RNF41 RING(Really Interesting New Gene) finger protein 41 (isoform 1).
  • SEQ ID NO:26 Human muscle alpha actin.
  • SEQ ID NO:27 Human cytoplasmic beta actin.
  • SEQ ID NO:28 Human cytoplasmic gamma actin.
  • SEQ ID NO:31 Mouse cytoplasmic gamma actin.
  • SEQ ID NO:34 Chimpanzee cytoplasmic gamma actin.
  • SEQ ID NO:36 Rat cytoplasmic beta actin.
  • SEQ ID NO:37 Rat cytoplasmic gamma actin.
  • SEQ ID NO:38 Horse muscle alpha actin.
  • SEQ ID NO:39 Horse cytoplasmic beta actin.
  • SEQ ID NO:41 Human erythrocytic spectrin, alpha 1 (elliptocytosis 2 or SPTA1).
  • SEQ ID NO:42 Human non-erythrocytic spectrin, alpha 1 (alpha-fodrin or SPTANl)
  • SEQ ID NO:43 Human non-erythrocytic spectrin, alpha 1 (alpha-fodrin or SPTANl) 30 (isoform 2)
  • SEQ ID NO:46 Human non-erythrocytic beta spectrin 1, or SPTBN1 (isoform 1)
  • SEQ ID NO:47 Human non-erythrocytic beta spectrin 1
  • SPTBN1 isoform 2
  • SEQ ID NO-.48 Human non-erythrocytic beta spectrin 2
  • SEQ ID NO:49 Mouse spectrin alpha 1 , or SPNA 1.
  • SEQ ID NO:50 Mouse spectrin alpha 2, or SPNA2.
  • SEQ ID NO:51 Mouse spectrin beta 1 , or SPNB 1.
  • SEQ ID NO:54 Mouse spectrin beta 3, or SPNB3.
  • SEQ ID NO:56 Mouse spectrin beta 5, or SPNB5.
  • SEQ ID NO:57 Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 1).
  • SEQ ID NO:58 Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 2).
  • SEQ ID NO:59 Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 3).
  • SEQ ID NO:60 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 1).
  • SEQ ID NO:61 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 2).
  • SEQ ID NO:62 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 3).
  • SEQ ID NO:63 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 4).
  • SEQ ID NO:64 Chimpanzee non-erythrocytic beta spectrin 1, or SPTBNl (isoform 1).
  • SEQ ID NO:65 Chimpanzee non-erythrocytic beta spectrin 2, or SPTBN2 (isoform 1).
  • SEQ ID NO:66 Horse erythrocytic alpha spectrin 1 (elliptocytosis 2 or SPTA1).
  • SEQ ID NO:67 Horse erythrocytic beta spectrin, or SPTB.
  • SEQ ID NO:68 Horse non-erythrocytic beta spectrin 1 , or SPTBN 1.
  • SEQ ID NO:73 Human filamin isoform 1.
  • SEQ ID NO:74 Human dystrophin isoform 1.
  • SEQ ID NO:75 Human actinin.
  • SEQ ID NO:76 Human alpha actinin 2.
  • SEQ ID NO:78 Human calponin-1 actin binding domain.
  • SEQ ID NO:79 Human calponin-2 actin binding domain.
  • SEQ ID NO:80 Human calponin-3 actin binding domain.
  • SEQ ID NO:81 Human plectin isoform 1 actin binding domain.
  • SEQ ID NO:83 Human dystrophin isoform 1 actin binding domain.
  • SEQ ID NO:86 Human alpha actinin 1 (isoform a) actin binding domain.
  • SEQ ID NO:88 Human spectrin beta chain brain 1 isoform 2 actin binding domain.
  • SEQ ID NO:89 Human spectrin beta chain erythrocyte isoform b actin binding domain.
  • the term about refers to +/- 20%, more preferably +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.
  • cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells includes situations where the cell can have, where possible, one or more of these features.
  • the cell could have a disrupted membrane, be infected with a pathogen and be dying.
  • eyely stage apoptotic cell or “early stage apoptotic cells” includes cells that are AnnexinV + and ⁇ .
  • the term "dead cell” or “dead cells” refers to cell(s) that has passed a point of no return in the death prbcess and which changes cannot be reversed. The cell(s) may have died through apoptosis or necrosis.
  • pathogen includes any organism which can infect a cell. Examples include, but are not limited to, viruses, protozoa and bacteria.
  • the term "uptake and/or clearance" of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof refers to the removal of cellular material, such a proteins or fragments thereof, of the cells.
  • dendritic cells are responsible, at least in part, for the uptake and/or clearance of the cells.
  • the dendritic cells are Clec9A + .
  • the term “surrounding cells” refers to cells in close proximity to one or more of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
  • treating include administering a therapeutically effective amount of a molecule of the invention (such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein) useful for the invention sufficient to reduce or eliminate at least one symptom of the specified condition.
  • a molecule of the invention such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein
  • preventing include administering a therapeutically effective amount of a molecule of the invention (such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein) useful for the invention sufficient to stop or hinder the development of at least one symptom of the specified condition.
  • a molecule of the invention such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein
  • diagnosis refers to the detection of a disease.
  • the term “prognosing” or variations thereof refers to an assessment of the future outcome of a disease.
  • monitoring the status refers to determining the stage of a disease.
  • the status can be determined before, during and/or after a subject has been administered with a treatment for the disease.
  • Clec9A refers to a polypeptide which comprises; i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8;
  • the polypeptide is at least expressed on a subset of dendritic cells.
  • the dendritic cells also express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, XCR1, Clec9A and BDCA3.
  • markers CD8, CD24, Necl-2, CD1 lc, HLADR, XCR1, Clec9A and BDCA3.
  • soluble fragments include those with the CTLD domain (for instances fragments comprising sequences as provided in SEQ ID NOs 17 to 20).
  • Clec9A has also been referred to in the art as 5B6 and HEEE9341.
  • tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l refer to the relative position of the amino acid compared to surrounding amino acids.
  • a polypeptide of the invention may have deletional or substitutional mutations which alters the relative positioning, of the amino acid when aligned against, for example, SEQ ID NO:l.
  • tryptophan residues 131 and 227 of full length human Clec9A correspond to residues 65 and 161 respectively of the fragment provided as SEQ ID NO: 18.
  • the position of the tryptophan residues may vary between orthologs of Clec9A.
  • the phrase "tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l" encompasses tryptophan residues 155 and 250 respectively of SEQ ID NO:2.
  • the polypeptide comprises the defined amino acid at the nominated residue number.
  • the "sample” can be any biological material suspected of having cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • the “sample” can be any biological material suspected of having Clec9A + dendritic cells Examples include, but are not limited to, blood, for example, whole peripheral blood, cord blood, foetus blood, bone marrow, plasma, serum, urine, cultured cells, saliva or urethral swab, lymphoid tissues, for example tonsils, peyers patches, appendix, thymus, spleen and lymph nodes, and any biopsy samples taken for routine screening, diagnostic or surgical reason such as tumour biopsy or bioposy of inflamed organs/ tissues.
  • the sample may be tested directly or may require some form of treatment prior to testing.
  • a biopsy sample may require homogenization to produce a cell suspension prior to testing.
  • a reagent such as a buffer
  • the mobilizing reagent may be mixed with the sample prior to placing the sample in contact with a compound as defined herein.
  • conjugate As used herein, the terms “conjugate”, “conjugated” or variations thereof are used broadly to refer to any form to covalent or non-covalent association between a compound useful for the invention and a therapeutic agent or a detectable label, or to placing a compound useful for the invention and a therapeutic agent or detectable label in close proximity to each other such as in a liposome.
  • the term "immune response” refers to an alteration in the reactivity of the immune system of a subject in response to an antigen and may involve antibody production, induction of cell-mediated immunity, complement activation and/or development of immunological tolerance.
  • the term "subject” preferably relates to an animal. More preferably, the subject is a mammal such as a human, dog, cat, horse, cow, or sheep. Most preferably, the subject is a human.
  • the present inventors have identified protein complexes which bind Clec9A.
  • the protein complexes comprise
  • actin or fragment thereof is bound to the actin binding domain.
  • present inventors have also identified that when bound to the protein complex, Clec9A is still capable of binding RNF41.
  • Clec9A binds actin without the need for an actin binding protein.
  • ABS actin binding domain
  • proteins include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titiii, myosin, tubulin, catenin, keratin, cytokeratin, nestin, lamin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
  • Actin binding domains relevant to the invention comprise a calponin homology domain (CHD).
  • CHD calponin homology domain
  • the ABD is typically about 100 residues.
  • Three major groups of ABD proteins have been recognized on the basis of sequence analysis, namely i) proteins containing a single N-terminal ABD including calponin, Vav, IQGAP and Cdc24, ii) proteins with an F-actin binding domain composed of two ABDs in tandem including spectrins, dystrophin, filamins and plakins, and iii) proteins of the fimbrin plastin family which also contain two ABDs in tandem (Gimona et al., 2002; Korenbaum and Rivero, 2002; Banuelos et al., 1998; Stradal et al., 1998).
  • a first polypeptide as defined herein can comprise one, two or possibily more calponin homology domains.
  • an actin binding domain relevant to the invention comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 78 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 78 to 89.
  • the first polypeptide comprises
  • the first polypeptide comprises
  • variant thereof which binds actin generally refers to naturally occurring proteins, or fragments thereof comprising an actin binding domain, which bind actin and which have been altered at one or more amino acids but still maintain the ability to bind actin.
  • variants are at least 75%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to a naturally occurring actin binding protein, preferably a mammalian actin binding protein such as those described herein.
  • the first polypeptide is itself a protein complex of two or more subunits (for instance spectrin may be present as a complex), in other embodiments the first polypeptide is a single polypeptide chain.
  • spectrin refers to membrane-associated cytoskeletal proteins involved in the crosslinking of filamentous actin which act as molecular scaffold proteins to link the actin cytoskeleton to the plasma membrane, and function in the determination of cell shape, arrangement of transmembrane proteins, and organization of organelles (Broderick and Winder, 2005).
  • Spectrins are traditionally divided into erythrocytic and non-erythrocytic forms, the former being exclusive to red blood cells and being responsible for the elasticity of BCs.
  • Spectrins are ubiquitous in cells and different isoforms may be expressed in different tissues in different organisms.
  • Spectrins are highly modular proteins, containing many repeating alpha-helical 106-amino acid units (or 'spectrin repeats').
  • Alpha forms generally contain 20 spectrin repeats and, in contrast to the beta forms, generally lack an actin binding domain (ABD).
  • Most alpha forms contain an SH3 (Src homology 3 domains) for binding polyproline-containing proteins.
  • Non- erythrocytic alpha isoforms generally contain an EF-hand motif for binding calcium.
  • Examples of erythrocytic alpha forms of spectrin are given as SEQ ID NOs: 41, 57-59 and 66.
  • Examples of non-erythrocytic alpha forms of spectrin are given as SEQ ID NOs: 42-43.
  • Mutations in the human SPTA1 gene (encoding erythrocytic spectrin alpha 1) are the cause of elliptocytosis type 2 (EL2), an autosomal dominant hematological disorder characterised by hemolytic anemia and elliptical or oval RBC shape.
  • SPTA1 mutations also cause hereditary pyropoikilocytosis (HPP) and spherocytosis type III (SPH3), both being hemolytic disorders.
  • HPP hereditary pyropoikilocytosis
  • SPH3 spherocytosis type III
  • SPTANl non- erythrocytic alpha 1 gene
  • Sjogrens syndrome Sjogrens syndrome
  • autoimmune diseaeses rheumatoid arthritis
  • multiple sclerosis multiple sclerosis
  • neurodegenerative diseases and xerostomia Non-erythrocytic forms of alpha 1 spectrin (encoded by the SPTANl gene) are also known as alpha-fodrin.
  • Beta forms generally contain 17 spectrin repeats and an actin binding domain (ABD).
  • ABDs generally contain two CH (calponin homology) domains, which enable beta forms of spectrin to interact with F-actin.
  • Non-erythrocytic forms of beta spectrin contain a PH (pleckstrin homology) domain for interaction with membrane phospholipids.
  • Beta forms of spectrin generally lack EF-hand motifs. Examples of erythrocytic beta forms of spectrin are given as SEQ ID NOs: 44-45, 60-63 and 67. , Examples of non-erythrocytic beta forms of spectrin are given as SEQ ID NOs: 46-48, 64-65 and 68.
  • erythrocytic spectrin beta Mutations in the human SPTB gene (encoding erythrocytic spectrin beta) are the cause of RBC disorders including elliptocytosis type 3 (EL3), spherocytosis type I (SPH1), muscular dystrophy, various anemic disease and pyropoikilocytosis. Mutations in the non-erythrocytic beta 1 gene (SPTBN1) cause neurofibromatosis type 2 and leukemia. Non-erythrocytic forms of beta 1 spectrin (encoded by the SPTBN1 gene) are also known as beta-fodrin.
  • Spectrin functions as a tetramer of alpha and beta dimers linked in a head-to- head arrangement.
  • Alpha and beta spectrin interact to form a dimer and two heterodimers form the functional tetramer.
  • Tetramers bind via their tail ends to a junctional complex consisting of filamentous actin and band 4.1 protein.
  • Spectrin also binds to integral membrane proteins via ankyrin and band 3 protein (especially in RBCs) and also via protein 4.1 and glycophorin C. Interactions also occur with phospholipids via the PH domains of beta spectrin.
  • the first polypeptide is not spectrin.
  • the polypeptide does not comprise
  • Actin is a well known globular protein about 42 kDa in size found in close to all eukaryotic cells (Rohn and Baum et al., 2010). Actin is one of the most highly- conserved proteins, differing by as little as 20% in primary amino acid sequence in species as diverse as algae and humans. Actin is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Actin participates in many important cellular processes including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape.
  • actin isoforms
  • alpha actins
  • beta actins
  • gamma actins
  • the alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus (examples provided as SEQ ID NOs 26, 29, 32, 35 and 38).
  • the beta and gamma actins co-exist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility (examples provided as SEQ ID NOs 27, 28, 30, 31 , 33, 34, 36, 37, 39 and 40).
  • G-actin is the monomeric form of the globular protein which assembles into actin filaments.
  • F-actin is the polymeric form of the globular, protein assembled into actin filaments.
  • the actin is filamentous actin or a filamentous fragment thereof.
  • the actin is a polymer comprising at least two actin monomer units or fragments thereof.
  • Determining whether a compound will bind acti can be determined using standard techniques such as using a simple actin binding assay (Corrado et al., 1994). Briefly, the candidate protein is expressed as fusion proteins with a tag, F-actin is absorbed on to microtiter plates, the candidate protein is incubated on the F-actin coated microtiter plates, the plates are washed, anti-tag rabbit antibody is added, anti- rabbit antibody conjugated to a detectable marker is added, and the plates screened for the detectable marker. As the skilled person would readily appreciate, a similar method can be used to identify fragments of actin which bind Clec9A.
  • RNF-41 protein is also known as RING (Really Interesting New Gene) finger protein, neuregulin receptor degradation protein- 1 (NRDP1), or fetal liver RING protein (FLRF), refers to a protein which acts as an E3-ubiquitin ligase and regulates the degradation of target proteins.
  • Target proteins for RNF-41 include members of the EGF (epidermal growth factor) receptor family, for example ErbB3 (or Her3).
  • Other targets of RNF-41 include ErbB4, ubiquitin-specific protease 8 (Usp8), Birc6 and reticulon 4 (Rtn4, also known as NogoA). Mutations in RNF-41 have been linked to tumour diseases. Overexpression of RNF-41 has been shown to decrease ErbB3 and inhibition of breast cancer growth. Decreased levels of RNF-41 are inversely correlated with ErbB3 levels in primary human breast cancer tissue.
  • Clec9A is expressed by a subset of dendritic cells, can be targetted to modulate an immune response, and binds a ligand on cells with a disrupted cell membrane, cells infected with a pathogen, dying cells and dead cells (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008; Sancho et al., 2009; WO 2009/026660; WO 2009/137871; WO 2010/108215).
  • the present inventors have now identified further molecules which bind Clec9A which can be used to, inter alia, target therapeutic molecules to dendritic cells.
  • the invention relates to a polypeptide complex comprising a first polypeptide comprising an actin binding domain and a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and which has been modified to deliver a therapeutic agent.
  • the invention in another embodiment, relates to a compound that binds a polypeptide complex which comprises a first polypeptide comprising an actin binding domain and a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A.
  • the present invention provides a compound which
  • ii) binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
  • actin or fragment thereof is bound to the actin binding domain, or
  • iii) competes with the one or both tryptophan residues of CIec9A for binding to the polypeptide complex.
  • the binding may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.
  • the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of hydrophilic/lipophilic interactions.
  • the compound is a purified and/or recombinant polypeptide (which can be a polypeptide complex).
  • the polypeptide complex, conjugate or compound may bind specifically a target molecule.
  • the phrase "specifically binds”, means that under particular conditions, the polypeptide complex, conjugate or compound binds the target and does not bind to a significant amount to other, for example, proteins or carbohydrates.
  • the polypeptide complex, conjugate or compound specifically binds the target and not other molecules in a sample obtained from a subject comprising, for example, cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • a polypeptide complex, conjugate or compound is considered to "specifically binds" if there is a greater than 2-fold difference, and preferably a 5, 25, 50 or 100 fold greater difference between the binding of the polypeptide complex, conjugate or compound when compared to another protein.
  • a compound of, and/or useful for, the invention is an antibody or antigen-binding fragment thereof.
  • antibodies and “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds.
  • L light
  • H heavy
  • the structure of immunoglobulins has been well characterized, see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
  • each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as C H ).
  • the heavy chain constant region typically is comprised of three domains, CH I , Cm, and CH3.
  • Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL).
  • the light chain constant region typically is comprised of one domain, CL-
  • the VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and V L is typically composed of three CDRs and four FRs, arranged from amirio-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, 1987).
  • the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (phrases such as variable domain residue numbering as in Kabat or according to Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable domains).
  • the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • humanized antibody refers to herein an antibody derived from a non-human antibody, typically murine, that retains or substantially retains the antigen-binding properties of the parent antibody but which is less immunogenic in humans.
  • complementarity determining region refers to amino acid sequences which together define the binding affinity and specificity of a variable fragment (Fv) region of a immunoglobulin binding site.
  • framework region refers to amino acid sequences interposed between CDRs. These portions of the antibody serve to hold the CDRs in appropriate orientation (allows for CDRs to bind antigen).
  • a variable region either light or heavy, comprises a framework and typically three CDRs.
  • constant region refers to the portion of the antibody molecule which confers effector functions.
  • the constant regions of the subject humanized antibodies are derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, antibodies with desired effector function can be produced.
  • Preferred heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3) and gamma 4 (IgG4), more preferably gamma 4 (IgG4).
  • the light chain constant region can be of the kappa or lambda type, preferably of the kappa type.
  • Antibodies may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CHI domain.
  • domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CHI domain.
  • a scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody”. Also encompassed are fragments of antibodies such as Fab, (Fab') 2 and FabFc 2 fragments which contain the variable regions and parts of the constant regions. CDR-grafted antibody fragments and oligomers of antibody fragments are also encompassed.
  • the heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region.
  • the antibody may be of animal (for example mouse, rabbit or rat) or human origin or may be chimeric (Morrison et al., 1984) or humanized (Jones et al., 1986; UK 8707252).
  • the term "antibody” includes these various forms. Using the guidelines provided herein and those methods well known to those skilled in the art which are described in the references cited above and in such publications as Harlow & Lane ⁇ supra) the antibodies for use in the methods of the present invention can be readily made.
  • an "antigen-binding fragment” refers to a portion of an antibody as defined herein that is capable of binding the same antigen as the full length molecule.
  • Antibodies or antigen-binding fragments of, and/or useful for, the invention which are not from a natural source, such as a humanized antibody, preferably retain a significant proportion of the binding properties of the parent antibody.
  • such antibodies or fragments of, and/or useful for, the invention retain the ability to specifically bind the antigen recognized by the parent antibody used to produce the antibody or fragment such as a humanized antibody.
  • the antibody or fragment exhibits the same or substantially the same antigen-binding affinity and avidity as the parent antibody.
  • the affinity of the antibody or fragment will not be less than 10% of the parent antibody affinity, more preferably not less than about 30%, and most preferably the affinity will not be less than 50% of the parent antibody.
  • Methods for assaying antigen-binding affinity are well known in the art and include half-maximal binding assays, competition assays, and Scatchard analysis.
  • immunoassay formats may be used to select antibodies or fragments that are specifically immunoreactive with the ligand.
  • surface labelling and flow cytometric analysis or solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See Harlow & Lane (supra) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • the antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain.
  • VL variable light
  • VH variable heavy chain
  • the light and heavy chains may be joined directly or through a linker.
  • a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed.
  • Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.
  • recombinantly produced single chain scFv antibody preferably a humanized scFv
  • scFv antibody preferably a humanized scFv
  • Monoclonal antibodies of, and/or useful for, the invention can be readily produced by one skilled in the art.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against target epitopes can be screened for various properties; i.e. for isotype and epitope affinity.
  • Animal-derived monoclonal antibodies can be used for both direct in vivo and extracorporeal immunotherapy. However, it has been observed that when, for example, mouse-derived monoclonal antibodies are used in humans as therapeutic agents, the patient produces human anti-mouse antibodies. Thus, animal-derived monoclonal antibodies are not preferred for therapy, especially for long term use. With established genetic engineering techniques it is possible, however, to create chimeric or humanized antibodies that have animal-derived and human-derived portions.
  • the animal can be, for example, a mouse or other rodent such as a rat.
  • variable region of the chimeric antibody is, for example, mouse-derived while the constant region is human-derived
  • the chimeric antibody will generally be less immunogenic than a "pure" mouse-derived monoclonal antibody. These chimeric antibodies would likely be more suited for therapeutic use, should it turn out that "pure" mouse-derived antibodies are unsuitable.
  • the light and heavy chains can be expressed separately, using, for example, immunoglobulin light chain and immunoglobulin heavy chains in separate plasmids. These can then be purified and assembled in vitro into complete antibodies; methodologies for accomplishing such assembly have been described (see, for example, Sun et al., 1986).
  • a DNA construct may comprise DNA encoding functionally rearranged genes for the variable region of a light or heavy chain of an antibody linked to DNA encoding a human constant region. Lymphoid cells such as myelomas or hybridomas transfected with the DNA constructs for light and heavy chain can express and assemble the antibody chains.
  • the antibody is humanized, that is, an antibody produced by molecular modeling techniques wherein the human content of the antibody is maximised while causing little or no loss of binding affinity attributable to the variable region of, for example, a parental rat, rabbit or murine antibody.
  • variable domain framework residues have little or no direct contribution.
  • the primary function of the framework regions is to hold the CDRs in their proper spatial orientation to recognize antigen.
  • substitution of animal, for example, rodent CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework is highly homologous to the animal variable domain from which they originated.
  • a human variable domain should preferably be chosen therefore that is highly homologous to the animal variable domain(s).
  • a suitable human antibody variable domain sequence can be selected as follow.
  • Step 1 Using a computer program, search all available protein (and DNA) databases for those human antibody variable domain sequences that are most homologous to the animal-derived antibody variable domains.
  • the output of a suitable program is a list of sequences most homologous to the animal-derived antibody, the percent homology to each sequence, and an alignment of each sequence to the animal- derived sequence. This is done independently for both the heavy and light chain variable domain sequences. The above analyses are more easily accomplished if only human immunoglobulin sequences are included.
  • Step 2. List the human antibody variable domain sequences and compare for J homology. Primarily the comparison is performed on length of CDRs, except CDR3 of the heavy chain which is quite variable.
  • Human heavy chains and Kappa and Lambda light chains are divided into subgroups; Heavy chain 3 subgroups, Kappa chain 4 subgroups, Lambda chain 6 subgroups.
  • the CDR sizes within each subgroup are similar but vary between subgroups. It is usually possible to match an animal-derived antibody CDR to one of the human subgroups as a first approximation of homology. Antibodies bearing CDRs of similar length are then compared for amino acid sequence homology, especially within the CDRs, but also in the surrounding framework regions.
  • the human variable domain which is most homologous is chosen as the framework for humanisation.
  • An antibody may be humanized by grafting the desired CDRs onto a human framework according to standard procedures such as those decribed in EP 0239400.
  • a DNA sequence encoding the desired reshaped antibody can therefore be made beginning with the human DNA whose CDRs it is wished to reshape.
  • the animal- derived variable domain amino acid sequence containing the desired CDRs is compared to that of the chosen human antibody variable domain sequence.
  • the residues in the human variable domain are marked that need to be changed to the corresponding residue in the animal to make the human variable region incorporate the animal-derived CDRs. There may also be residues that need substituting in, adding to or deleting from the human sequence.
  • Oligonucleotides are synthesized that can be used to mutagenize the human variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.
  • Synthetic gene sequences such as those encoding humanized antibodies or fragments thereof, can be commercially ordered through any of a number of service companies, including DNA 2.0 (Menlo Park, Calif.), Geneart (Regensburg, Germany), CODA Genomics (Irvine, Calif.), and GenScript, Corporation (Piscataway, N.J.).
  • humanisation may be achieved using the recombinant polymerase chain reaction (PCR) methodology of WO 92/07075.
  • PCR polymerase chain reaction
  • a CDR may be spliced between the framework regions of a human antibody.
  • the technique of WO 92/07075 can be performed using a template comprising two human framework regions, AB and CD, and between them, the CDR which is to be replaced by a donor CDR.
  • Primers A and B are used to amplify the framework region AB, and primers C and D used to amplify the framework region CD.
  • the primers B and C each also contain, at their 5' ends, an additional sequence corresponding to all or at least part of the donor CDR sequence.
  • Primers B and C overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a PCR to be performed.
  • the amplified regions AB and CD may undergo gene splicing by overlap extension to produce the humanized product in a single reaction.
  • the mutagenised DNAs can be linked to an appropriate DNA encoding a light or heavy chain constant region, cloned into an expression vector, and transfected into host cells, preferably mammalian cells. These steps can be carried out in routine fashion.
  • a reshaped antibody may therefore be prepared by a process comprising:
  • the DNA sequence in step (a) encodes both the variable domain and each constant domain of the human antibody chain.
  • the humanized antibody can be prepared using any suitable recombinant expression system.
  • the cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line.
  • the cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus.
  • the immortalised cell line is a myeloma cell line or a derivative thereof.
  • the CHO cells used for expression of the antibodies may be dihydrofolate reductase (DHFR) deficient and so dependent on thymidine and hypoxanthine for growth.
  • DHFR dihydrofolate reductase
  • the parental DHFR * CHO cell line is transfected with the DNA encoding the antibody and DHFR gene which enables selection of CHO cell transformants of DHFR positive phenotype. Selection is carried out by culturing the colonies on media devoid of thymidine and hypoxanthine, the absence of which prevents untransformed cells from growing and transformed cells from resalvaging the folate pathway and thus bypassing the selection system.
  • These transformants usually express low levels of the DNA of interest by virtue of co-integration of transfected DNA of interest and DNA encoding DHFR.
  • the expression levels of the DNA encoding the antibody may be increased by amplification using methotrexate (MTX).
  • MTX methotrexate
  • This drug is a direct inhibitor of the enzyme DHFR and allows isolation of resistant colonies which amplify their DHFR gene copy number sufficiently to survive under these conditions. Since the DNA sequences encoding DHFR and the antibody are closely linked in the original transformants, there is usually concomitant amplification, and therefore increased expression of the desired antibody.
  • GS glutamine synthetase
  • Msx methionine sulphoximine
  • the cell line used to produce the humanized antibody is preferably a mammalian cell line
  • any other suitable cell line such as a bacterial cell line or a yeast cell line
  • E. co/i ' -derived bacterial strains could be used.
  • the antibody obtained is checked for functionality. If functionality is lost, it is necessary to return to step (2) and alter the framework of the antibody.
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be recovered and purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (See, generally, Scopes, R., Protein Purification, Springer- Verlag, N.Y. (1982)).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • a humanized antibody may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (See, generally, Lefkovits and Pernis (editors), Immunological Methods, Vols. I and II, Academic Press, ( 1979 and 1981 )). Studies carried out by Greenwood et al. (1993) have demonstrated that recognition of the Fc region of an antibody by human effector cells can be optimised by engineering the constant region of the immunoglobulin molecule.
  • variable region genes of the antibody with the desired specificity, to human constant region genes encoding immunoglobulin isotypes that have demonstrated effective antigen dependent cellular cytotoxicity (ADCC) in human subjects, for example the IgGl and IgG3 isotypes (Greenwood and Clark, Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man. Mike Clark (editor), Academic Titles, Section II, p.85-l l3, (1993)).
  • the resulting chimeric or humanized antibodies should be particularly effective in modulating humoral immunity and/or T-cell mediated immunity.
  • Antibodies with fully human variable regions can also be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Various subsequent manipulations can be performed to obtain either antibodies per se or analogs thereof (see, for example, US 6,075,181).
  • Genes encoding antibodies, both light and heavy chain genes or portions thereof, e.g., single chain Fv regions, may be cloned from a hybridoma cell line. They may all be cloned using the same general strategy such as RACE using a commercially available kit, for example as produced by Clontech. Typically, for example, poly(A) + mRNA extracted from the hybridoma cells is reverse transcribed using random hexamers as primers. For Fv regions, the VH and VL domains are amplified separately by two polymerase chain reactions (PCR).
  • PCR polymerase chain reactions
  • Heavy chain sequences may be amplified using 5' end primers which are designed according to the amino-terminal protein sequences of the heavy chains respectively and 3' end primers according to consensus immunoglobulin constant region sequences (Kabat et al., Sequences of Proteins of Immunological Interest. 5th edition. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Light chain Fv regions are amplified using 5' end primers designed according to the amino- terminal protein sequences of light chains and in combination with the primer C-kappa.
  • One of skill in the art would recognize that many suitable primers may be employed to obtain Fv regions.
  • the PCR products are subcloned into a suitable cloning vector. Clones containing the correct size insert by DNA restriction are identified. The nucleotide sequence of the heavy or light chain coding regions may then be determined from double stranded plasmid DNA using sequencing primers adjacent to the cloning site. Commercially available kits (e.g., the SequenaseTM kit, United States Biochemical Corp., Cleveland, Ohio, USA) may be used to facilitate sequencing the DNA. DNA encoding the Fv regions may be prepared by any suitable method, including, for example, amplification techniques such as PCR and LCR.
  • amplification techniques such as PCR and LCR.
  • Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. While it is possible to chemically synthesize an entire single chain Fv region, it is preferable to synthesize a number of shorter sequences (about 100 to 150 bases) that are later ligated together.
  • sub-sequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.
  • the sequences may be ligated together, either directly or through a DNA sequence encoding a peptide linker, using techniques well known to those of skill in the art.
  • heavy and light chain regions are connected by a flexible peptide linker (e.g., (Gh/ 4 Ser)3) which starts at the carboxyl end of the heavy chain Fv domain and ends at the amino terminus of the light chain Fv domain.
  • the entire sequence encodes the Fv domain in the form of a single-chain antigen-binding protein.
  • Polypeptide complexes, conjugates and compounds defined herein can be used to deliver a therapeutic agent.
  • therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
  • the therapeutic agent may be a polypeptide fused to the polypeptide complex, actin or a fragment thereof, or compound as defined herein.
  • Fusion polypeptides comprising the polypeptide complex or compound may be prepared by methods known to one of skill in the art. For example, a gene encoding a polypeptide chain is fused to a gene encoding a therapeutic agent.
  • the gene is linked to a segment encoding a peptide connector.
  • the peptide connector may be present simply to provide space between the compound and the therapeutic agent or to facilitate mobility between these regions to enable them to each attain their optimum conformation.
  • the DNA sequence comprising the connector may also provide sequences (such as primer sites or restriction sites) to facilitate cloning or may preserve the reading frame between the sequence encoding the binding moiety and the sequence encoding the therapeutic agent.
  • sequences such as primer sites or restriction sites
  • the design of such connector peptides is well known to those of skill in the art.
  • Polypeptide complexes, actins or compounds useful for the invention may be fused to, or otherwise bound to the therapeutic agent by any method known and available to those in the art.
  • the two components may be chemically bonded together by any of a variety of well-known chemical procedures.
  • the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like.
  • Production of various immunotoxins, as well as chemical conjugation methods are well-known within the art (see, for example, “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, p. 168-190 (1982); Waldmann, 1991 ; Vitetta et al., 1987; Pastan et al., 1986; and Thorpe et al., 1987).
  • drugs and/or pharmacological agents include, but are not limited to, agents that promote DC activation (e.g. TLR ligands), agents that suppress DC activation or function (e.g. specific inhibitors or promotors of DC signalling molecules such as kinases and phosphatases), and agents that modulate DC death (e.g. promotors or suppressors of apoptosis).
  • agents that promote DC activation e.g. TLR ligands
  • agents that suppress DC activation or function e.g. specific inhibitors or promotors of DC signalling molecules such as kinases and phosphatases
  • agents that modulate DC death e.g. promotors or suppressors of apoptosis.
  • Such drugs and/or pharmacological agents are well known to those skilled in the art.
  • polypeptide toxins that are suitable for use as cytotoxic agents in the methods of the invention.
  • polypeptides include, but are not limited to, polypeptides such as native or modified Pseudomonas exotoxin (PE), diphtheria toxin (DT), ricin, abrin, gelonin, momordin II, bacterial RIPs such as shiga and shiga-Iike toxin a-chains, luffm, atrichosanthin, momordin I, Mirabilis anti- viral protein, pokeweed antiviral protein, byodin 2 (U.S. 5,597,569), gaporin, as well as genetically engineered variants thereof.
  • PE native or modified Pseudomonas exotoxin
  • DT diphtheria toxin
  • ricin abrin
  • gelonin gelonin
  • momordin II bacterial RIPs
  • shiga and shiga-Iike toxin a-chains such as shiga
  • Native PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • Pseudomonas exotoxin and DT are modified into a form that removes the native targeting component of the toxin, e.g., domain la of Pseudomonas exotoxin and the B chain of DT.
  • the invention is not limited to a particular cytotoxic agent.
  • cytotoxic agents include, but are not limited to, agents such as bacterial or plant toxins, drugs, e.g., cyclophosphamide (CTX; Cytoxan), chlorambucil (CHL; leukeran), cisplatin (CisP; CDDP; platinol), busulfan (myleran), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and other alkylating agents; methotrexate (MTX), etoposide (VP- 16; vepesid), 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5FU), dacarbazine (DTIC), 2-chlorodeoxyadenosine (2-CdA), and other antimetabolites; antibiotics including actinomycin D, doxorubicin (DXR; a
  • radioisotopes and chemocytotoxic agents that can be coupled to compounds of the invention by well known techniques, and delivered to specifically destroy dendritic cells (see, e.g., U.S. 4,542,225).
  • photo-activated toxins include dihydropyridine-and omega- conotoxin.
  • cytotoxic reagents that can be used include ,25 I, ,31 I, m In, m l, "mTc, and 32 P.
  • the antibody can be labeled with such reagents using techniques known in the art. For example, see Wenzel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y.
  • the linker-chelator tiuexutan is conjugated to the compound by a stable thiourea covalent bond to provide a high-affinity chelation site for Indium- 111 or Yttrium-90.
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
  • Polypeptide complexes, conjugates or compounds useful for the methods of the invention may also be conjugated to an "antigen”.
  • antigen is further intended to encompass peptide or. protein analogs of known or wild-type antigens such as those described above.
  • the analogs may be more soluble or more stable than wild type antigen, and may also contain mutations or modifications rendering the antigen more immunologically active.
  • Also useful in the present invention are peptides or proteins which have amino acid sequences homologous with a desired antigen's amino acid sequence, where the homologous antigen induces an immune response to the respective tumor or organism.
  • a “cancer antigen,” as used herein is a molecule or compound (e.g., a protein, peptide, polypeptide, lipid, glycolipid, carbohydrate and/or DNA) associated with a tumor or cancer cell and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule.
  • Cancer antigens include self-antigens, as well as other antigens that may not be specifically associated with a cancer, but nonetheless induce and/or enhance an immune response to and/or reduce the growth of a tumor or cancer cell when administered to an animal.
  • an "antigen from a pathogenic and/or infectious organism” as used herein, is an antigen of any organism and includes, but is not limited to, infectious virus, infectious bacteria, infectious parasites including protozoa (such as Plasmodium sp.) and worms and infectious fungi.
  • the antigen is a protein or antigenic fragment thereof from the organism, or a synthetic compound which is identical to or similar to naturally-occurring antigen which induces an immune response specific for the corresponding organism.
  • Compounds or antigens that are similar to a naturally-occurring organism antigens are well known to those of ordinary skill in the art.
  • a non-limiting example of a compound that is similar to a naturally- occurring organism antigen is a peptide mimic of a polysaccharide antigen.
  • cancer antigens include, e.g., mutated antigens such as the protein products of the Ras p21 protooncogenes, tumor suppressor p53 and HER- 2/neu and BCR-abl oncogenes, as well as CD 4, MUM1, Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, Aldolase A, FRAME, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionic gonadotropin (hCG); self-antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens such as Mart 1 Melan A, gplOO, gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryo
  • Cancer antigens and their respective tumor cell targets include, e.g., cytokeratins, particularly cytokeratin 8, 18 and 19, as antigens for carcinoma.
  • Epithelial membrane antigen (EM A) human embryonic antigen (HEA-125), human milk fat globules, MBrl, MBr8, Ber-EP4, 17-1 A, C26 and T16 are also known carcinoma antigens.
  • Desmin and muscle-specific actin are antigens of myogenic sarcomas.
  • Placental alkaline phosphatase, beta-human chorionic, gonadotropin, and alpha- fetoprotein are antigens of trophoblastic and germ cell tumors.
  • Prostate specific antigen is an antigen of prostatic carcinomas, carcinoembryonic antigen of colon adenocarcinomas.
  • HMB-45 is an antigen of melanomas.
  • useful antigens could be encoded by human papilloma virus.
  • Chromagranin-A and synaptophysin are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid tumor masses having necrotic areas. Antigens derived from pathogens known to predispose to certain cancers may also be advantageously used in the present invention.
  • Pathogens of particular interest for use in the cancer vaccines provided herein include the hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EBV) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), HTLVL (adult T-cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B-cell gastric lymphoma).
  • EBV Epstein Barr virus
  • HTLVL adult T-cell leukemia
  • HTLVL adult T-cell leukemia
  • oncogenic human papilloma viruses types 16, 18, 33, 45 adult cervical cancer
  • Helicobacter pylori B-cell gastric lymphoma
  • Other medically relevant microorganisms that may serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g.,
  • Exemplary viral pathogens include, but are not limited to, infectious virus that infect mammals, and more particularly humans.
  • infectious virus include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-IH, LAV or HTLV-III LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g.
  • Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronoviridae e.g. coronaviruses such as the SARS coronavirus
  • Rhabdoviradae e.g. vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g. ebola viruses
  • Paramyxoviridae e.g. parairifluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g.
  • African swine fever virus African swine fever virus
  • gram negative and gram-positive bacteria may be targeted by the subject compositions and methods in vertebrate animals.
  • Such gram-positive bacteria include, but are not limited to Pasteurella sp, Staphylococci sp., and Streptococcus sp.
  • Gram- negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas sp., and Salmonella sp.
  • infectious bacteria include but are not limited to: Helicobacter pyloris, Borella burgdorferi, Legionella pneumophilia, Mycobacteria sp. (e.g. M. tuberculosis, M. avium, M: intracellular , M. kansaii, M.
  • antigens can be isolated or prepared recombinantly or by any other means known in the art.
  • pathogens further include, but are not limited to, infectious fungi and parasites that infect mammals, and more particularly humans.
  • infectious fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
  • Examples of parasites include intracellular parasites and obligate intracellular parasites.
  • Examples of parasites include but are not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense, Wuchereria bancrofti, Brugia malayi, Brugia timori, Ascaris lumbricoides, Onchocerca volvulus and Schistosoma mansoni.
  • compositions and methods of the present invention are useful for treating infections of nonhuman mammals.
  • non-human pathogens include, but are not limited to, mouse mammary tumor virus (“MMTV”), Rous sarcoma virus (“RSV”), avian leukemia virus (“ALV”), avian myeloblastosis virus (“AMV”), murine leukemia virus (“MLV”), feline leukemia virus (“FeLV”), murine sarcoma virus (“MSV”), gibbon ape leukemia virus (“GALV”), spleen necrosis virus (“SNV”), reticuloendotheliosis virus (“RV”), simian sarcoma virus (“SSV”), Mason-Pfizer monkey virus (“MPMV”), simian retrovirus type 1 (“SRV-l”), lentiviruses such as HIV-1, HIV-2, SIV, Visna virus, feline immunodeficiency virus (“FIV”), and equine infectious anemia virus (“EIAV”), T-cell leukemia viruses such as HTLV-1, HT
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen.
  • Polypeptide complexes, conjugates or compounds useful for the invention may be employed in a range of detection systems.
  • the polypeptide complex, conjugate or compound may be used in methods for imaging an internal region of a subject and/or diagnosing the presence or absence of a disease in a subject.
  • diagnostic, prognostic and/or monitoring methods of the present invention involve a degree of quantification to determine levels of Clec9A, Clec9A expressing cells, ligand and/or ligand expressing cells present in patient samples. Such quantification is readily provided by the inclusion of appropriate control samples.
  • internal controls are included in the methods of the present invention.
  • a preferred internal control is one or more samples taken from one or more healthy individuals.
  • Polypeptide complexes, conjugates or compounds useful for the present invention when used diagnosticall may be linked to a diagnostic reagent such as a detectable label to allow easy detection of binding events in vitro or in vivo.
  • a diagnostic reagent such as a detectable label to allow easy detection of binding events in vitro or in vivo.
  • Suitable labels include radioisotopes, or non-radioactive labels such as biotin, enzymes, chemiluminescent molecules, fluorophores, dye markers or other imaging reagents for detection and or localisation of target molecules.
  • a second labelled molecule such as an antibody which binds the polypeptide complex, actin or fragment thereof, conjugate or compound can be used for detection.
  • an enzyme can be conjugated to the second molecule, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above.
  • fluorescent compounds such as but not limited to fluorecein and rhodamine amongst others, may be chemically coupled to, for examples, antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the compounds coupled to imaging agents can be used in the detection of Clec9A in histochemical tissue sections.
  • the polypeptie complexes, actin, or compound (for example) may be covalently or non- covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, echogenic, radioactive, or non-radioactive labels such as biotin or avidin.
  • Cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells can be detected in a sample by a variety of techniques well known in the art, including cell sorting, especially fluorescence-activated cell sorting (FACS), by using an affinity reagent bound to a substrate (e.g., a plastic surface, as in panning), or by using an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles).
  • FACS fluorescence-activated cell sorting
  • any detectable substance which has the appropriate characteristics for the cell sorter may be used (e.g., in the case of a fluorescent dye, a dye which can be excited by the sorter's light source, and an emission spectra which can be detected by the cell sorter's detectors).
  • a beam of laser light is projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties.
  • Cells labelled with a suitable dye are excited by the laser beam, and emit light at characteristic wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital signals, allowing for their storage, analysis and display.
  • FACS fluorescence- activated cell sorters
  • the instruments electronics interprets the signals collected for each cell as it is interrogated by the laser beam and compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination.
  • the cells can automatically be deposited in collection vessels as single cells or as a plurality of cells, e.g. using a laser, e.g. an argon laser (488 run) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA).
  • a laser e.g. an argon laser (488 run)
  • a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA).
  • FACS machines include, but are not limited to, MoFloTM High-speed cell sorter (Dako-Cytomation ltd), FACS AriaTM (Becton Dickinson), FACS Diva (Becton Dickinson), ALTRA Hyper sort (Beckman Coulter) and CyFlow sorting system (Partec GmbH).
  • any particle with the desired properties may be utilized.
  • large particles e.g., greater than about 90-100 ⁇ in diameter
  • the particles are "magnetic particles" (i.e., particles which can be collected using a magnetic field). Labelled cells are retained in the column (held by the magnetic field), whilst unlabelled cells pass straight through and are eluted at the other end.
  • Magnetic particles are now commonly available from a variety of manufacturers including Dynal Biotech (Oslo, Norway) and Milteni Biotech GmbH (Germany).
  • An example of magnetic cell sorting (MACS) is provided by Al-Mufti et al. (1999).
  • Laser-capture microdissection can also be used to selectively detect labelled cells on a slide using methods of the invention.
  • Methods of using laser-capture microdissection are known in the art (see, for example, U.S. 20030227611 and Bauer et al., 2002).
  • the terms “enriching” and “enriched” are used in their broadest sense to encompass the isolation of dendritic cells or precursors thereof such that the relative concentration of dendritic cells or precursors thereof to non-dendritic cells or precursors thereof in the treated sample is greater than a comparable untreated sample.
  • the enriched dendritic cells and/or precursors thereof are separated from at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% of the non-dendritic cells or precursors thereof in the sample obtained from the original sample.
  • the enriched cell population contains no non-dendritic cells or precursors thereof (namely, pure).
  • the terms "enrich” and variations thereof are used interchangeably herein with the term "isolate” and variations thereof.
  • a population of cells enriched using a method of the invention may only comprise a single dendritic cell or precursor thereof.
  • the enrichment methods of the invention may be used to isolate a single dendritic cell or precursor thereof.
  • Dendritic cells or precursors thereof can be enriched from the sample by a variety of techniques well known in the art, including cell sorting, especially fluorescence-activated cell sorting (FACS), by using an affinity reagent bound to a substrate (e.g., a plastic surface, as in panning), or by using an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles).
  • FACS fluorescence-activated cell sorting
  • a substrate e.g., a plastic surface, as in panning
  • an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles).
  • the procedure used to enrich the dendritic cells ' and/or precursors thereof will depend upon how the cells have been labelled.
  • any detectable substance which has the appropriate characteristics for the cell sorter may be used (e.g., in the case of a fluorescent dye, a dye which can be excited by the sorter's light source, and an emission spectra which can be detected by the cell sorter's detectors).
  • a beam of laser light is projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties.
  • Cells labelled with a suitable dye are excited by the laser beam, and emit light at characteristic wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital signals, allowing for their storage, analysis and display.
  • FACS fluorescence- activated cell sorters
  • the instruments electronics interprets the signals collected for each cell as it is interrogated by the laser beam and compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to "the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination.
  • the cells can automatically be deposited in collection vessels as single cells or as a plurality of cells, e.g. using a laser, e.g. an argon laser (488 nm) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA).
  • a laser e.g. an argon laser (488 nm) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA.
  • FACS machines include, but are not limited to, MoFloTM High-speed cell sorter (Dako-Cytomation ltd), FACS AriaTM (Becton Dickinson), FACS Diva (Becton Dickinson), ALTRATM Hyper sort (Beckman Coulter) and CyFlowTM sorting system (Partec GmbH).
  • any particle with the desired properties may be utilized.
  • large particles e.g., greater than about 90-100 ⁇ in diameter
  • the particles are "magnetic particles" (i.e., particles which can be collected using a magnetic field). Labelled cells are retained in the column (held by the magnetic field), whilst unlabelled cells pass straight through and are eluted at the other end.
  • Magnetic particles are now commonly available from a variety of manufacturers including Dynal Biotech (Oslo, Norway) and Milteni Biotech GmbH (Germany).
  • An example of magnetic cell sorting (MACS) is provided by Al- Mufti et al. (1999).
  • Laser-capture microdissection can also be used to selectively enrich labelled dendritic cells or precursors thereof on a slide using methods of the invention. Methods of using laser-capture microdissection are known in the art (see, for example, U.S. 20030227611 and Bauer et al., 2002).
  • the cells can be used immediately or cultured in vitro to expand dendritic cells and/or precursors thereof numbers using techniques known in the art. Furthermore, dendritic cell precursors can be cultured to produce mature dendritic cells. Identification of Compounds
  • Methods of screening test compounds are described which can identify a compound, for example, that binds to a polypeptide complex defined herein, that binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l, that binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex defined herein, or which competes with the one or both tryptophan residues of Clec9A for binding to the polypeptide complex, and are thus useful in a method of the invention.
  • Compounds may be screened by resort to assays and techniques useful in identifying molecules capable of binding to the ligand (such as a polypeptide complex of the invention) and thereby inhibiting its biological activity by blocking Clec9A binding.
  • assays include the use of mammalian cell lines (for example, CHO cells or 293T-cells) for phage display system for expressing the polypeptide complex and using a culture of transfected mammalian or E. coli or other microorganism to produce proteins for binding studies of potential binding compounds.
  • a method for identifying compounds which specifically bind to the polypeptide complex can include simply the steps of contacting a selected cell expressing the complex with a test compound to permit binding of the test compound to the complex, and determining the amount of test compound, if any, which is bound to the complex.
  • Such a method involves the incubation of the test compound and the complex immobilized on a solid support.
  • the surface containing the immobilized compound is permitted to come into contact with a solution containing the complex and binding is measured using an appropriate detection system. Suitable detection systems are known in the art, some of which are described herein.
  • crystal means a structure (such as a three dimensional
  • crystal refers in particular to a solid physical crystal form such as an experimentally prepared crystal.
  • any reference herein to the atomic coordinates or subset of the atomic coordinates shown in Appendix I shall include, unless specified otherwise, atomic coordinates having a root mean square deviation of backbone atoms of not more than 1.5 A, preferably not more than 1 A, when superimposed on the corresponding backbone atoms described.by the atomic coordinates shown in Appendix I.
  • the following defines what is intended by the term "root mean square deviation (RMSD)" between two data sets. For each element in the first data set, its deviation from the corresponding item in the second data set is computed. The squared deviation is the square of that deviation, and the mean squared deviation is the mean of all these squared deviations.
  • the root mean square deviation is the square root of the mean squared deviation.
  • Preferred variants are those in which the RMSD of the x, y and z coordinates for all backbone atoms other than hydrogen is less than 1.5 A (preferably less than 1 A, 0.7 A or less than 0.3 A) compared with the coordinates given in Appendix I. It will be readily appreciated by those skilled in the art that a 3D rigid body rotation and/or translation of the atomic coordinates does not alter the structure of the molecule concerned.
  • a "subset" of the atomic coordinates provided in Appendix I refers to a group of the co-ordinates which can be used in a method of the invention such as a computer-assisted method of identifying a compound that binds Clec9A, or a computer-assisted method for identifying a compound which binds a polypeptide complex as defined herein.
  • the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
  • Clec9A defined, herein can be used to identify antagonists or agonists through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., 1997).
  • Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of a candidate compound to the polypeptide.
  • the tighter the fit e.g., the lower the steric hindrance, and/or the greater the attractive force
  • the more potent the potential agonist or antagonist will be since these properties are consistent with a tighter binding constant.
  • the more specificity in the design of a potential agonist or antagonist the more likely that it will not interfere with other proteins.
  • a potential compound could be obtained, for example, using methods of the invention such as by screening a random peptide library produced by a recombinant bacteriophage or a chemical library. A compound selected in this manner could then be systematically modified by computer modeling programs until one or more promising potential compounds are identified.
  • Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful agonist or antagonist.
  • Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized.
  • a large number of these compounds can be rapidly screened on the computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • Exemplary forcefields that are known in the art and can be used in such methods include, but are not limited to, the Constant Valence Force Field (CVFF), the AMBER force field and the CHARM force field.
  • CVFF Constant Valence Force Field
  • AMBER AMBER force field
  • CHARM CHARM force field
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behaviour of molecules with each other. c Diseases Associated with Cells with a Disrupted Cell Membrane. Cells Infected with a Pathogen. Dying Cells or Dead Cells
  • diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not necessarily limited to, the following:
  • GVHD graft versus host disease
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • Sjogren's syndrome multiple sclerosis
  • insulin dependent diabetes mellitus ulcerative colitis
  • VAHS virus associated hemophagocytic syndrome
  • HCV virus associated hemophagocytic syndrome
  • Leukemia for example, acute lymphatic leukemia.
  • SIRS Systemic inflammatory reaction syndrome
  • the invention is useful for GVHD, human immunodeficiency virus (HIV), hemophagocytic syndrome (HPS), especially virus associated hemophagocytic syndrome (VAHS), acute lymphatic leukemia, influenzal encephalitis, encephalopathy, and malaria.
  • HIV human immunodeficiency virus
  • HPS hemophagocytic syndrome
  • VAHS virus associated hemophagocytic syndrome
  • polypeptide and protein are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
  • proteins and polypeptides as used herein may also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein.
  • the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 200 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 200 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • biologically active fragment is a portion of a polypeptide as described herein which maintains a defined activity of the full-length polypeptide.
  • Biologically active fragments can be any size as long as they maintain the defined activity.
  • biologically active fragments are at least 100 amino acids in length.
  • soluble fragment refers to a portion of Clec9A which lacks the membrane spanning region.
  • the soluble fragment does not comprise at least about 40, at least about 50, at least about 55, or at least about 100, N-terminal residues of any one of SEQ ID NOs 1 to 8.
  • the soluble fragment comprises the C-type lectin-like domain of a polypeptide which comprises:
  • the soluble fragment comprises:
  • the soluble fragment does not comprise at least the about 40 N-terminal residues of any one of SEQ ID NOs 1 to 8.
  • the polypeptide comprises an amino acid sequence which is at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nomin
  • Amino acid sequence mutants of a polypeptide described herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide.
  • Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
  • a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
  • Mutant (altered) polypeptides can be prepared using any technique known in the art.
  • a polynucleotide described herein can be subjected to in vitro mutagenesis.
  • in vitro mutagenesis techniques may include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations.
  • the polynucleotides defined herein are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they are able to confer the desired phenotype.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical, and/or those in which particular residues obtained from related proteins are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1.
  • polypeptides which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide.
  • Polypeptides described herein can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides.
  • an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
  • a preferred cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production.
  • An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, tissue culture flasks, shake flasks, test tubes, microtiter dishes, and Petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • polynucleotide is used interchangeably herein with the term “nucleic acid”.
  • monomers of a polynucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units.
  • Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate and phosphoramidate.
  • Recombinant vectors useful for the invention can include at least one polynucleotide molecule described herein, and/or a polynucleotide encoding a polypeptide as described herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell.
  • a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.
  • One type of recombinant vector comprises the polynucleotide(s) operably linked to an expression vector.
  • the phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Vectors can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence.
  • a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system.
  • promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cw-acting.
  • some transcriptional regulatory elements, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance .
  • expression vectors contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or animal cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma
  • a recombinant cell comprising a host cell transformed with one or more recombinant molecules described herein or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention.
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides described herein or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as described herein.
  • Host cells of the present invention can be any cell capable of producing at least one protein defined herein, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, animal and plant cells.
  • host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, CHO cells, 293 cells, EL4 cells, MDCK cells, CRFK cells,. CV-1 cells, COS (e.g., COS-7) cells, and Vero cells.
  • E. coli including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are. translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • compositions comprising the polypeptide complex, actin or fragment thereof, conjugate or compound together with an acceptable carrier or diluent are useful in the methods of the present invention.
  • Therapeutic compositions can be prepared by mixing the desired component having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, A.ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions.
  • Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine,
  • Such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes
  • protamine sulfate disodium hydrogen phosphate
  • potassium hydrogen phosphate sodium chloride
  • colloidal silica magnesium trisilicate
  • compositions to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • the composition may be stored in lyophilized form or in solution if administered systemically. If in lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use.
  • An example of a liquid formulation is a sterile, clear, colorless unpreserved solution filled in a single-dose vial for subcutaneous injection.
  • compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the compositions are preferably administered subcutaneously, intramuscularly or parenterally, for example, as intravenous injections or infusions or administered into a body cavity.
  • the polypeptide complex, actin or fragment thereof, conjugate or compound may be administered in an amount of about 0.001 to 2000 mg kg body weight per dose, and more preferably about O.pi to 500 mg/kg body weight per dose. Repeated doses may be administered as prescribed by the treating physician.
  • compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease or immune response required, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference, 56 th ed. (2002). Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.,
  • a polypeptide complex, actin or fragment thereof, conjugate or compound useful for the methods of the invention comprises an antigen, such as a cancer antigen or an antigen of a pathogen or infectious organism, and can be delivered by intramuscular, subcutaneous or intravenous injection, or orally, as a vaccine to enhance humoral and/or T-cell mediated immune responses.
  • the antigen is a self-antigen or allergenic antigen which can used to diminish immune responses similar to that described for 33D1 and DEC-205 (Bonifaz et al., 2002; Finkelman et al., 1996).
  • a radiolabeled form of the polypeptide complex, actin or fragment thereof, conjugate or compound is delivered by intravenous injection as a therapeutic agent to target cells that express Clec9A.
  • radiolabeled antibodies and the methods for their administration to patients as therapeutics are known to those skilled in the art. Examples include Iodine 131 labeled Lym-1, against the ⁇ subunit of HLA-DR and the anti-CD20 Indium 111 and Yttrium 90 labeled Ibritumomab Tiuxetan (IDEC-Y2B8, ZEVALIN ® ) and Iodine 1 131 Tositumomab (BEXXAR ® ).
  • the composition does not comprise an adjuvant.
  • the composition does comprise an adjuvant.
  • adjuvants include, but are not limited to, aluminium hydroxide, aluminium phosphate, aluminium potassium sulphate (alum), muramyl dipeptide, bacterial endotoxin, lipid X, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • the composition comprises liposomes or membrane vesicles.
  • liposomes are described in US 2007/0026057, Leserman (2004) and van Broekhoven et al. (2004).
  • the polypeptide complex, conjugate or compound can be used to target the liposome to enhance the delivery of an agent of interest.
  • processes for the preparation of membrane vesicles for use in the invention are described in WO 00/64471.
  • compositions for detection of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, modulating an immune response, and/or antigen recognition, processing and/or presentation are conventionally administered parenterally, by injection, for example, subcutaneously, intramuscularly or intravenously.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
  • Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
  • the therapeutic composition may be administered to a patient either singly or in a cocktail containing other therapeutic agents, compositions, or the like.
  • the immune response is modulated by using a DNA vaccine encoding a polypeptide complex, actin or fragment thereof, conjugate or compound of the invention conjugated to an antigen.
  • DNA vaccination involves the direct in vivo introduction of DNA encoding the antigen into tissues of a subject for expression of the antigen by the cells of the subject's tissue. Such vaccines are termed herein "DNA vaccines” or "nucleic acid-based vaccines”. DNA vaccines are described in US 5,939,400, US 6,110,898, WO 95/20660, WO 93/19183, Deband et al. (2005) and Nchinda et al. (2008).
  • CMV cytomegalovirus
  • Vectors containing the nucleic acid-based vaccine of the invention may also be introduced into the desired host by other methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter.
  • Clec9A-ecto; stalk and CTLD Clec9A-ecto; stalk and CTLD
  • Clec9A-CTLD Clec9A CTLD only
  • Clec9A-stalk Clec9A stalk only
  • cDNA containing the required ectodomain region was amplified from the original Clec9A cDNA using Advantage high fidelity 2 polymerase (Clontech, MountainView, CA) or HotStar HiFidelity polymerase (Qiagen, Victoria, Australia) and the listed primers (Table 2).
  • Recombinant proteins were expressed in mammalian 293T or FreeStyle 293F cells by transient transfection, followed by culture in protein-free/serum-free media: X- Vivo- 10 (BioWhittaker, Walkersville, MD) or FreeStyle Expression Media (Invitrogen, Victoria, Australia) respectively.
  • Media containing the secreted recombinant protein was assayed for the presence of soluble mClec9A by reactivity with anti-mClec9A mAb (24/04- 10B4), concentrated 100-fold using a 10,000 molecular weight cutoff centrifugal device (Millipore, Billerica, MA) and either used directly or en2ymatically biotinylated using BirA enzyme (Avidity).
  • Clec9A soluble proteins were purified by affinity chromomatography using an anti-FLAG M2 agarose resin (Sigma, Castle Hill, Australia) and elution with 100 g/ml FLAG peptide (Auspep, Victoria, Australia), and further purified by size-exclusion chromatography using a pre-packed Superdex 200 column (GE Healthcare, Rydalmere, Australia). Table 2 - Construct s nthesis.
  • the resulting fusion constructs for hCLEC9A-CTLD (S225D) thus included (in order of N-terminus): the IL-3 signal sequence (to ensure secretion), a FLAG-tag, and hCLEC9A-CTLD (S225D).
  • the resulting fusion constructs for hCLEC9A-ecto included the IL-3 signal sequence, the biotinylation consensus peptide sequence, a FLAG-tag, and hCLEC9A-ecto (WT31A; W227A).
  • Recombinant proteins were expressed in FreeStyle 293F cells and hCLEC9A soluble proteins purified from the secreted media by affinity chromomatography using an anti-FLAG M2 agarose resin, and further purified by size- exclusion chromatography using a pre-packed Superdex 200 column.
  • Circular Dichroism was performed to compare the structural profiles of the
  • Platelets and red blood cells were purified from mouse blood. Binding studies were performed by incubating cells with purified or biotinylated Clec ectodomains and binding detected using fluorescently conjugated secondary reagents and flow cytometric analysis.
  • Isolation of cellular spectrin complexes was performed essentially as in Ungewickell and Gratzer (1978) and Gratzer (1982), and further purified by SEC.
  • erythrocytes were lysed for 30 min at 4°C in a hypotonic lysis buffer (5mM sodium phosphate, pH7.6, lmM PMSF, 1 x EDTA-free protease cocktail (Roche)) and erythrocyte membranes washed repeatedly using the lysis buffer.
  • a hypotonic lysis buffer 5mM sodium phosphate, pH7.6, lmM PMSF, 1 x EDTA-free protease cocktail (Roche)
  • Spectrin was extracted by resuspension of erythrocyte ghosts in a spectrin extraction buffer (0.3mM sodium phosphate, 0.1 mM EDTA, 0.1 mM PMSF) and overnight dialysis at 4°C against spectrin extraction buffer. Membrane fragments were removed by ultracentrifugation (90000g, 30 min, 4°C) and the supernatant containing spectrin harvested for further analysis. Isolation of non-erythroid spectrin from frozen pellets of human 293F cells (1 x 10 8 cells) was performed as for erythrocytic spectrin, with the addition of 50U/ml benzonase (Novagen) in the lysis buffer.
  • a spectrin extraction buffer 0.1% sodium phosphate, 0.1 mM EDTA, 0.1 mM PMSF
  • Spectrin samples were further purified by size exclusion chromatography using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min in PBS containing 10% glycerol, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF. Column fractions of 0.4 mL were collected for analysis.
  • Reassociation of cellular spectrins with actin was performed by collecting fractions of dissociated spectrin following SEC, and concentrating them in the presence of ⁇ of either bovine muscle actin or a control protein BSA, in PBS containing 10% glycerol, 0.1 mM PMSF, 1 mM EDTA, 1 mM DTT.
  • the spectrin-actin BSA samples were then incubated at 30°C for 2.5 h, then further purified by SEC using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min as above, and 0.4 ml fractions collected for analysis.
  • spectrin ⁇ Recombinant GST-tagged fragments of erythrocytic spectrin (spectrin ⁇ ) were expressed as previously described (Pei et al., 2005).
  • the Actin binding domains (ABD) of human Spectrin beta II (spectrin beta chain, brain 1 isoform 1; beta-fodrin, NM_003128.2) and of human alpha-actinin I (alpha-actinin-1 isoform a; alpha-actinin cytoskeletal isoform, NM_001130004.1) were expressed as glutathione S-transferase (GST) fusion proteins using E. coli BL21 (DE3).
  • cDNA containing the ABD regions were amplified from 293F cell cDNA using HotStar HiFidelity polymerase (Qiagen) using the primers listed below (Table 3), and subcloned into a modified pGex2T vector (GE Healthcare).
  • IPTG isopropyl- ⁇ - thiogalactopyranoside
  • GST-tagged proteins were purified by affinity chromatography using Glutathione-Sepharose 4B resin (GE Healthcare) and elution with 50mM Tris-HCl, pH8.0, 150mM NaCl containing 50mM reduced glutathione (Sigma), and further purified by size-exclusion chromatography using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min in 50mM Tris-HCl, pH 8.0 containing 150 mM NaCl.
  • Reassociation studies were performed by incubating GST-tagged spectrin and actinin proteins in the presence or absence of bovine muscle actin (Sigma) or platelet actin (>99% purity, Cytoskeleton) in PBS, for 2.5 h (30°C) at a concentration of 0.2mg/ml per protein. Proteins were diluted to ⁇ ⁇ in PBS for coating onto ELISA plates.
  • ELISA plates (Costar) were coated overnight at 4°C with either commercially available erythrocytic spectrin (Sigma), bovine muscle actin (Sigma) or platelet actin (>99% pure, Cytoskeleton) at ⁇ , or with cellular spectrins (K ⁇ g/ml) or complexes containing GST-tagged spectrin fragments, (l( ⁇ g spectrin fragment/ml) or with SEC fractions of spectrin (50 ⁇ 1). Bovine muscle actin and platelet actin appeared to have comparable purity on SDS-PAGE analysis. Unbound proteins were then washed away (PBS-0.05%Tween 20).
  • ELISA plates were blocked (1% BSA in PBS), then incubated with FLAG-tagged Clec9A ectodomain fragments or controls (mClecl2A-ecto , mCire DC-Sign-ecto). Binding was detected by incubation with an anti-FLAG antibody M2-conjugated to HRP (Sigma-Aldrich), and visualized by the addition of the HRP substrate ABTS, followed by measurement of absorbance at 405- 490 nm (Vmax Kinetic Microplate Reader, Molecular Devices).
  • Thermofluor assays were performed to determine protein stability (Phillips and de la Pena, 2011).
  • hCLEC9A-CTLD (S225D) was incubated in Tris buffered saline in the presence or absence of 6mM EDTA, 1.24mM MgS0 4 or 2.47mM CaCl 2 for lh at 4°C.
  • Thermofluor assays were performed using 18.4 ⁇ hCLEC9A-CTLD and Sypro Orange (1 :1250 dilution, Invitrogen) in a total volume of 25 ⁇ .
  • Thermal denaturation curves were measured on a CI 000 thermocycler equipped with a CFX384 Real-Time System fluorescence reader (BIORAD).
  • Tm melting temperature
  • Binding assays were performed in binding buffer (PBS containing 0.2%BSA and 0.02% sodium azide), on ice. Cells were washed 3 times with PBS to remove serum proteins, then resuspended in binding buffer. Cells were incubated with either (1) biotinylated soluble Clec9A and controls, and detected with Streptavidin (SA)-PE, or (2) soluble FLAG-tagged Clec9A and detected either with FITC conjugated rat anti- FLAG mAb 9H1, or with biotinylated rat anti-FLAG mAb 9H1 and SA-PE.
  • SA Streptavidin
  • Live cells were gated on forward and side scatter, or by PI exclusion, whereas dead cells were gated on forward and side scatter, or by PI inclusion.
  • Analysis of soluble Clec9 A binding was performed by flow cytometry using a FACScan (Becton Dickinson). The specificity of the binding was demonstrated by comparison to binding to other soluble FLAG-tagged C-type lectins, mouse Cire/mDCSign (Caminschi et al., 2001) and Clecl2A (Pyz et al., 2008).
  • Venous blood was obtained from C57B16 mice by cardiac puncture into 0.1 volume of Aster Jandl citrate-based anticoagulant (Aster and Jandl, 1964) (85 mM sodium citrate, 69 mM citric acid, 20 mg/ml glucose, pH 4.6). Platelet rich plasma was obtained by centrifugation of the murine blood at 125 x g for 8 min, followed by centrifugation of the supernatant buffy coat at 125 x g for 8 min.
  • Aster Jandl citrate-based anticoagulant Aster and Jandl, 1964
  • Platelet rich plasma was obtained by centrifugation of the murine blood at 125 x g for 8 min, followed by centrifugation of the supernatant buffy coat at 125 x g for 8 min.
  • Platelets were washed by two sequential centrifugations at 860 x g for 5 min in 140 mM NaCl, 5 mM C1, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM sucrose, pH 6.0 the platelet pellet resuspended in 10 mM HEPES, 140 Mm NaCl, 3 mM KC1, 0.5 mM MgCl 2 , 10 mM glucose and 0.5 mM NaHCC>3, pH 7.4. Washed platelets were treated with 0.5 ⁇ ABT-737 for 90 min at 37°C.
  • Control or ABT-737 treated platelets were incubated with 5 ⁇ g/ml of Flag-tagged recombinant proteins mClec9A or hClec9A for 30 min at room temperature.
  • Anti-Flag-FITC secondary antibodies were added to the platelets and incubated for 30 min at room temperature. Samples were diluted out in PBS containing 0.2% BSA and analyzed on a FACSCalibur flow cytometer.
  • ABT-737 treated platelets were confirmed to be AnnexinV-APC positive, and resting control platelets AnnexinV-APC negative.
  • Mouse fibroblasts (NIH 3T3) cells were cultured on glass coverslips, fixed, permeabilised using PBS containing 0.3% Triton X-100 and incubated either with biotinylated mClec9A-ecto, or biotinylated Cire-ecto as a background control and binding detected using SA-Alexa594. Fibroblasts were counterstained with DAPI, and analysed by confocal microscopy using a Leica SP2 (Wilson et al., 2003). Mass Spectrometry Analysis
  • Protein bands were excised from Simply blue (Invitrogen) stained gels and subjected to manual in-gel digestion.
  • the gel bands were reduced with lOmM DTT (Calbiochem) for 30 min, alkykated for 30 min with 50mM iodoacetic acid (Fluka) and digested with 375ng trypsin (Worthington) for 16hrs at 37°C.
  • the extracted peptide solutions (0.1% formic acid) were then concentrated to approximately ⁇ by centrifugal lyophilisation using a SpeedVac AES 1010 (Savant). Digests were then subjected to MS/MS analysis on the LTQ-Orbitrap (Thermo Fischer Scientific) mass spectrometer.
  • Extracted peptides were injected and fractionated by nanoflow reversed-phase liquid chromatography on a nano LC system (1200 series, Agilent, USA) using a nano flow reversed-phase-HPLC (Model 1200, Agilent). Fractionation was performed using a nano-Acquity (CI 8) 150 mm x 0.15 mm I.D. RP-UPLC column (Waters) developed with a linear 60 min gradient from 0-100% Buffer B (0.1% (v/v) aqueous formic acid / 60% (v/v) acetonitrile) with a flow rate of 0.8 ⁇ 7 ⁇ at 45°C, where Buffer A was 0.1% (v/v) aqueous formic acid.
  • the capillary HPLC was coupled on-line to the LTQ- Orbitrap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific). Positive ion mode was used for data-dependent acquisition.
  • Survey MS scans were acquired with the resolution set to 30,000. Each scan was recalibrated in real time by co-injecting an internal standard from ambient air into the C-trap (Olsen et al., 2005). The five most intense ions per cycle were fragmented and analysed in the linear trap. Target ions already selected for MS/MS were dynamically excluded for 180s.
  • Peak lists were searched against the LudwigNR protein sequence database (10753 sequence entries) (version Q409ml - www.ludwig.edu.au/archiveAudwigNR/ludwigNR.pdf) using the Mascot search algorithm (v2.2.04, Matrix Science, U.K.) (Perkins et al., 1 99).
  • the search parameters were as follows: carboxymethylation of cysteine as a . fixed modification (+58 Da) as well as variable modifications consisting of N3 ⁇ 4- terminal acetylation (+42 Da) and oxidation of methionine (+16 Da), and the allowance for up to three missed tryptic cleavage sites (trypsin P).
  • Precursor and fragment ion mass tolerance values were +/- 20ppm and 0.8Da respectively.
  • bacterial BL21 E. coli were electroporated with plasmid DNA and incubated at 37°C overnight on LB Agar plates containing Ampicillin (50 ⁇ g/ml). Bacterial culture broth containing ampicillin (100 ⁇ g ml) was inoculated with a single colony and incubated with shaking overnight at 37°C. Overnight bacterial cultures were diluted into Superbroth containing ampicillin (100 ⁇ g/ml), at 1:20 dilution and incubated in a shaking incubator at 30°C degrees until the cell density reached an ODioo of 0.8. Isopropyl-P-D- thiogalactopyranoside ( ⁇ ) was added to cultures to induce expression of proteins and cultures incubated at 19°C for 5 hours. Cells were harvested by centrifugation and stored at -80°C.
  • lysozyme lysis buffer phosphate buffered saline (PBS) containing 0.2mg/ml lysozyme, 30 ⁇ g/ml DNase I, 1 mM PMSF
  • Total lysates were subjected to sonication using the Bandelin sonoplus (50% duty cycle, 10 second pulse, 22 second cycle x5) and subsequently clarified by centrifugation.
  • the supernatant was incubated with 50% Giutathione-Sepharose resin slurry for 1 hour at 4°C on a rotating wheel and resin recovered in column (Bio-Rad Laboratories). Resin was washed extensively with PBS, and proteins eluted with 20mM reduced glutathione pH 7.0 in PBS.
  • ELISA plates (Costar) were coated with GST-RNF41 fusion proteins ( ⁇ g/ml) in tris buffered saline or phosphate buffered saline overnight at 4°C. Plates were washed with wash buffer (PBS/ 0.05% Tween20) to remove unbound proteins and then blocked for 1 hour in 5% skim milk in PBS at room temperature. Dilutions of Clec proteins in 5% skim milk in PBS were added and the plates incubated at RT for 2 hours. Plates were washed with wash buffer to remove unbound Clec proteins, and plates incubated with anti-FLAG 2 antibody conjugated to Horseradish Peroxidase (Sigma) for 2 hours at RT.
  • wash buffer PBS/ 0.05% Tween20
  • ELISA to measure enhancement of Clec9A binding to RNF41 by ABD-actin complexes were performed as follows. GST-tagged RNF41 C-terminal domain and control GST were coated on ELISA plates at ⁇ overnight at 4°C. Unbound proteins were washed away and plates blocked with 1% BSA in PBS. ABD-actin complexes of platelet actin with eythrocytic spectrin (spectrin ⁇ N), non-erthyroctic spectrin (spectrin ⁇ N) and a-actinin-1 (a-actinin-1 N) were pre-associated at 50 ⁇ g ml for 2.5 hours at 30°C, before incubation with Clec proteins for 1 hour at room temperature.
  • Clec9A mouse Clec9A
  • human CLEC9A human CLEC9A
  • mClec9A mouse Clec9A
  • hCLEC9A human CLEC9A
  • the inventors then investigated various stages of cell death, by following thymocytes undergoing apoptosis induced by ⁇ -irradiation or mouse embryonic fibroblasts (MEFs) undergoing apoptosis induced by BH3-only ligands (van Delft et al., 2006). Cells were stained with Annexin V, an early marker of apoptosis, and with PI, a late marker, which stains nuclei once the cell membrane is damaged.
  • MEFs mouse embryonic fibroblasts
  • Both mClec9A and hCLEC9A ectodomains bound to all dead mouse or human nucleated cells tested, including cultured cell lines and primary cells ( Figure 2). They also bound to freeze/thawed Chinese hamster (CHO) and African green monkey (Vero) cells (data not shown), and to freeze/thawed insect (SF21) cells, but not to freeze/thawed bacteria or yeast ( Figure 2F). Thus, recognition of the dead cell ligand was conserved across a wide evolutionary range.
  • Clec9A intracellular ligand for Clec9A could be a cytoskeletal component.
  • spectrin spectrin
  • filamin a suspension-adapted subline of human embryonic kidney cells
  • Clec9A interacting proteins isolated from mouse thymocytes and human 293 F cells.
  • Clec9A interacting proteins were isolated by incubation of FLAG-tagged Clec9A-ecto with lysates of mouse thymocytes or a suspension adapted subline of human 293 cells (Freestyle human 293 F) and affinity purification using anti-FLAG resin. Protein complexes were analysed by SDS-PAGE under reducing conditions, and a protein band of 220-300 kDa was excised and subjected to mass spectrometry analysis. The five most abundant proteins are described below.
  • Mass spectroscopic analysis revealed a majority of peptides corresponding to the expected spectrin al and spectrin ⁇ , but also peptides corresponding to spectrin-associated proteins including actin, band 4.1, adducins and tropomodulins (Table 5).
  • Example 8 - Clec9A binds to a higher order complex of erythrocyte spectrin
  • Spectrin extractions from mouse erythrocyte ghosts prepared by osmotic lysis were performed at 4°C, which enables the extraction of spectrin in the form of tetramers and higher order complexes, or at 37°C, which produces mainly spectrin dimers (Ungewickell and Gratzer, 1978; Gratzer et al., 1982).
  • the isolated spectrin was coated onto ELISA plates and investigated for Clec9A binding. Significant binding of mClec9A-ecto was obtained to spectrin isolated if at 4°C, but only poor binding to spectrin isolated at 37°C ( Figure 5A).

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Abstract

The present invention relates to the identification of molecules which bind the dendritic cell marker known as Clec9A. The present invention provides new compounds for targeting therapeutic agents such as antigens to dendritic cells. Also provided are methods of modulating a humoral and/or T-cell mediated immune response to the antigen, methods of delivery of a cytotoxic agent to dendritic cells thereof involved in diseased states, methods of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, and methods of modulating antigen recognition, processing and/or presentation, as well as immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.

Description

MOLECULES WHICH BIND CLEC9A
FIELD OF THE INVENTION
The present invention relates to the identification of molecules which bind the dendritic cell marker known as Clec9A. The present invention provides new compounds for targeting therapeutic agents such as antigens to dendritic cells. Also provided are methods of modulating a humoral and/or T-cell mediated immune response to the antigen, methods of delivery of a cytotoxic agent to dendritic cells thereof involved in diseased states, methods of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, and methods of modulating antigen recognition, processing and/or presentation, as well as immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
BACKGROUND OF THE INVENTION
Dendritic cells (DCs) are an essential link between the adaptive and innate immune systems. DCs initiate adaptive immune responses by processing and presenting antigens to T-cells (Banchereau et al., 2000; Steinman et al., 2003). DCs also monitor the environment using a series of innate receptors for pathogen-associated molecular patterns (PAMPs) and damaged cell-associated molecular patterns (DAMPs). This enables DCs to adjust the balance between tolerance and immunity, and to tailor the immune response to particular pathogens (Shortman and Liu, 2002; Robinson et al., 2006; Cambi and Figdor, 2009; Poon et al., 2010). Particular DC subsets are specialised in detailed aspects of these general functions. In the mouse, the CD8+ DC (Shortman and Heath, 2010) are especially efficient at the uptake and processing of material from dead cells, and in "cross-presenting" such exogenous antigenic material on class I HC (Shortman and Heath, 2010). Human CD141+ (BDCA-3+) DCs have been identified as the lineage and functional equivalents of the mouse CD8+ DCs (Jongbloed et al., 2010; Bachem et al., 2010; Crozat et al., 2010; Poulin et al., 2010), thus they would be expected to have a particular pattern of PAMP and DAMP receptors related to these functions.
One such DAMP receptor on mouse CD8+ DCs and on human CD141+ DCs is Clec9A (also called DNGR-1) (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008; Sancho et al., 2009; WO 2009/026660; WO 2009/137871; WO 2010/108215) which binds to dead cells. Clec9A also regulates the cross-presentation of dead cell-associated antigens in a Syk-dependent manner (Sancho et al., 2009). There is particular interest in Clec9A since it has been found to be an especially effective target for delivery of antigens to DCs, so promoting immune responses (Caminschi et al., 2008; Sancho et al., 2008; Idoyaga et al., 2011; Lahoud et al., 2011). This has great promise as a strategy for enhancing the effectiveness of vaccines.
There is a need for a greater understanding of Clec9A ligands, and Clec9A binding thereto, which can be used to modulate an immune response, or used to design/identify compounds for modulating an immune response. SUMMARY OF THE INVENTION
The present inventors have identified further ligands of Clec9A. These ligands can be used to target therapeutic agents or detectable labels to Clec9A expressing. cells such as dendritic cells.
Thus, in one aspect the present invention provides a polypeptide complex conjugated to a therapeutic agent or a detectable label, wherein the polypeptide complex comprises
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A.
The first polypeptide can be selected from a large variety of proteins which bind actin. Examples include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titin, myosin, tubulin, catenin, keratin, cytokeratin, nestin, larnin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
In an embodiment, the first polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 41 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of
SEQ ID NOs 41 to 89; and/or
c) an actin binding fragment of a) or b).
Preferably, the second polypeptide consists of actin, or a fragment thereof which binds the actin binding domain. In an embodiment, the actin is cytoskeletal actin or muscle actin. In a particularly preferred embodiment, the actin is filamentous actin or a filamentous fragment thereof. In another embodiment, the second polypeptide comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 26 to 40; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 26 to 40; and/or
c) a fragment of a) or b) which binds an actin binding domain.
In a preferred embodidment, the actin is cytoskeletal actin. Thus, in a preferred embodiment the second polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 27, 28, 30, 31, 33, 34, 36, 37, 39 and 40;
r
b) an amino acid sequence which is at least 50% identical to any one or more of
SEQ ID NOs 27, 28, 30, 31, 33, 34, 36, 37, 39 and 40; and/or
c) a fragment of a) or b) which binds an actin binding domain.
In a further embodiment, the Clec9A comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8;
b) an amino acid sequence which is at least 50% identical to any one or more of
SEQ ID NOs 1 to 8.
In another embodiment, the polypeptide complex further comprises Clec9A. In yet another embodiment, when bound to Clec9A, the complex further comprises RNF41 bound to the Clec9A. In an embodiment, the RNF41 comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 21 to 25; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 21 to 25; and/or
c) a fragment of a) or b) which binds Clec9A.
Examples of suitable therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
The antigen can be any molecule that induces an immune response in an animal. Examples include, but are not limited to, a cancer antigen, a self-antigen, an allergen, and or an antigen from a pathogenic and/or infectious organism.
In an embodiment, the antigen from a pathogenic and/or infectious organism can be from, but not limited to, Plasmodium falciparum or Plasmodium vivax.
In one embodiment, the first and second polypeptides are separate polypeptide chains which form the complex at least by the actin binding domain binding the actin.
In an alternate embodiment, first and second polypeptides form part of a single polypeptide chain. In an embodiment, the first and second polypeptides are separated by a peptide linker. In another aspect, the present invention provides a compound that binds a polypeptide complex which comprises:
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A.
In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A alone or a fragment of Clec9A which binds Clec9A alone such as a soluble fragment. n
In a preferred embodiment, the compound is a polypeptide.
In another preferred embodiment of the above aspect the compound is an antibody or antigen-binding fragment thereof. Examples of antibodies or antigen- binding fragment thereof include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
In a particularly preferred embodiment, the actin is filamentous actin or a filamentous fragment thereof.
In a further preferred embodiment, the compound of the above aspect is conjugated to a therapeutic agent such as a cytotoxic agent, drug and/or pharmacological agent.
In a further preferred embodiment, the compound of the above aspect is detectably labelled.
In a further aspect, the present invention provides a composition comprising a polypeptide complex of the invention, or a compound of the invention, and a pharmaceutically acceptable carrier.
In an embodiment, the composition further comprises an adjuvant.
In another embodiment, the compound is encapsulated in, or exposed on the surface of, a liposome.
In a further aspect, the present invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex of the invention,
iii) a compound of the invention, and
iv) a composition of the invention.
In an embodiment, the immune response to an antigen is induced and/or enhanced.
In a particularly preferred embodiment, the immune response is modulated by enhancing a helper T-cell response.
In a further preferred embodiment, the immune response is modulated by the activation of CD4+ and/or CD8+ T-cells.
In another particularly preferred embodiment, the immune response is modulated by enhancing B-cell antibody production. Examples of antibodies produced include, but are not necessarily limited to, IgGl, IgG2b, IgG2c, IgG3, IgG4^ IgM, IgAl, IgA2, IgE and/or IgD antibody isotypes.
In a further preferred embodiment, the immune response is modulated by generating a memory response.
In a particularly preferred embodiment, the subject is administered with a compound comprising the antigen.
In another embodiment, an immune response to a self-antigen or allergen is reduced. In this embodiment, it is preferred that the immune response is modulated by suppressing a T-cell response and/or a B-cell antibody response.
In another aspect, the present invention provides a method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex of the invention,
iii) a compound of the invention, and
iv) a composition of the invention, and administering said cells to the subject.
In an embodiment, the cells have been isolated from the subject.
Preferably, a humoral and/or T-cell mediated response is modulated.
In a further embodiment, naive CD8+ T-cell activation, and/or naive CD4+ T- cell activation, is modulated.
In yet another embodiment, the humoral response comprises the production of IgGl, IgG2b, IgG2c, IgG3, IgG4, IgM, IgAl, IgA2, IgE, and/or IgD antibody isotypes. In another embodiment, the humoral response at least comprises the production of IgGl antibody isptype.
Preferably, the dendritic cell is an animal dendritic cell or precursor of an animal dendritic cell. More preferably, the dendritic cell is a human dendritic cell. Even more preferably, the human dendritic cell is Necl-2+, HLA DR+ and/or BDCA-3+.
In yet another aspect, the present invention provides a method of treating ahd/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, ,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex of the invention,
Hi) a compound of the invention, and
iv) a composition of the invention.
Preferably, the method comprises administering a polypeptide complex or compound of the invention comprising the cytotoxic agent, drug and/or pharmacological agent.
Examples of diseases involving dendritic cells or precursors thereof include, but are not limited to, cancer, an infection, an autoimmune disease or an allergy.
In an embodiment, the autoimmune disease is lupus erythematosus.
In another embodiment, the infection is a Plasmodium sp., such as Plasmodium falciparum or Plasmodium vivax, infection.
In a further aspect, the present invention provides a method of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering to the subject at least one of i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a f agment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec A,
ii) a polypeptide complex of the invention, ^ iii) a compound of the invention, and
iv) a composition of the invention.
In another aspect, the present invention provides a method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding. Clec9A,
ii) a polypeptide complex of the invention,
iii) a compound of the invention, and
iv) a composition of the invention.
In a further aspect, the present invention provides a method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
. ii) a polypeptide complex of the invention,
iii) a compound of the invention, and
iv) a composition of the invention. In one embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is increased. In an alternate embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased.
In another embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is increased. In an alternate embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is decreased.
In another embodiment, the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is increased. In an alternate embodiment, the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is decreased.
In a further preferred embodiment, the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof. Examples of such diseases include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
Also provided is the use of at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex of the invention,
iii) a compound of the invention, and
iv) a composition of the invention,
for the manufacture of a medicament for at least one of
a) modulating an immune response in a subject, b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
Further, provided is a
i) polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex of the invention,
Hi) a compound of the invention, and
iv) a composition of the invention,
for use in at least one of
a) modulating an immune response in a subject,
b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, e) modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
In a further aspect, the present invention provides a method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
i) contacting a cell with a compound that binds a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
- ii) determining whether the polypeptide complex is present or absent, wherein the presence of the complex polypeptide provides a diagnosis, prognosis and/or status of the disease.
In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
In an embodiment, the compound is an antibody or antigen-binding fragment thereof. Examples include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
In a preferred embodiment, the compound is detectably labelled.
In an embodiment, the method is performed in vivo on a subject. In an alternate embodiment, the method is performed in vitro on a sample obtained from a subject.
As noted above, examples of diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury . In a further aspect, the present invention provides a method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
i) exposing a cell to the therapy, and
ii) detecting a cell with a disrupted cell membrane, a dying cell or a dead cell, or a portion thereof, using a method of the above aspect,
wherein the presence of a cell with a disrupted cell membrane, a dying cell or a dead cell indicates that the therapy is effective.
In an embodiment, the cell in step i) is in vivo. In an alternate embodiment, the cell in step i) is in vitro.
Preferably, the therapy is administered to a subject. In an embodiment, the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells. In a preferred embodiment, the subject has cancer or an infection.
In a further embodiment, step ii) is performed on a sample obtained from a subject.
The therapy can be any type of procedure. Examples include, but are not limited to, drug therapy or radiotherapy.
In a further aspect, the present invention provides a method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
i) contacting a cell with a compound that binds a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
ii) determining whether binding of the compound to the polypeptide complex is present or absent,
wherein the compound binding to the polypeptide indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment. In a further aspect, the present invention provides a method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a polypeptide complex of the invention, or a compound of the invention, and
ii) isolating cells bound to the polypeptide complex or compound.
In a preferred embodiment, the cells obtained from step ii) are administered to a subject. In an embodiment, the cells are administered to treat and or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
In another aspect, the present invention provides a method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a polypeptide complex of the invention, or a compound of the invention, and
ii) detecting cells bound to the compound.
In a further aspect, the present invention provides a method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
i) administering to the subject a polypeptide complex of the invention, or a compound of the invention, and
ii) detecting cells bound to the compound.
In an embodiment of the three above aspects, the polypeptide complex or compound is detectably labelled. However, as the skilled addressee will appreciate other procedures could be used, for example, using a detectably labelled secondary antibody that binds the polypeptide complex or compound.
In a preferred embodiment, the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, Clec9A, XCRI and BDCA3.
Preferably, the dendritic cells are human dendritic cells that express one or more of the following markers, Necl-2, HLADR and BDC A3.
In an alternate embodiment, the dendritic cells are murine dendritic cells that express one or more of the following markers, CD24, Necl-2, CD 11c, Clec9A and CD8.
Preferably, the precursor dendritic cells are intermediate or late precursor
, dendritic cells which are capable of differentiating into dendritic cells in culture and/or on transfer into irradiated recipients.
Also provided is a method of detecting a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
i) contacting a cell with a compound that binds a polypeptide complex comprising a) a first polypeptide comprising an actin binding domain, and
b) a second polypeptide comprising actin; or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
ii) determining whether binding of the compound to the polypeptide is present or absent,
wherein the compound binding to the polypeptide complex indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying or is dead.
In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A, Clec9A per se or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
The present inventors have also identified two tryptophan residues which are important for the biological function of Clec9A. Thus, in a further aspect the present invention provides a compound which
i) binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO.l,
ii) binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, or
iii) competes with the one or both tryptophan residues of Clec9A for binding to the polypeptide complex.
In an embodiment, the compound is a polypeptide.
In another preferred embodiment of the above aspect, the compound is an antibody or antigen-binding fragment thereof. Examples of antibodies or antigen- binding fragment thereof include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
In an embodiment, the compound does not affect the binding of RNF41 to Clec9A.
Also provided is a composition comprising a compound of the above aspect, and a pharmaceutically acceptable carrier. In a further aspect, the present invention provides a method of reducing an immune response in a subject, the method comprising administering to the subject a compound of the above aspect and/or a composition of the above aspect.
In another aspect, the present invention provides a method of reducing an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to a compound of the above aspect and or a composition of the above aspect, and administering said cells to the subject.
In a further aspect, the present invention provides a method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject a compound of the above aspect and/or a composition of the above aspect.
In an embodiment, the disease is an autoimmune disease or inflammation.
In yet another aspect, the present invention provides a method of reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
In a further aspect, the present invention provides a method of reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
In another aspect, the present invention provides a method of reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
Also provided is the use of a compound of the above aspect and/or a composition of the above aspect for the manufacture of a medicament for at least one of i) reducing an immune response in a subject,
ii) reducing an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
iii) treating and/or preventing in a subject a disease involving dendritic cells or precursors thereof, iv) reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
v) reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and vi) reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
Further, provided a compound of the above aspect and/or a composition of the above aspect for use in at least one of 3 i) reducing an immune response in a subject,
ii) reducing an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
iii) treating and/or preventing in a subject a disease involving dendritic cells or precursors thereof,
iv) reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
v) reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and vi) reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
In another aspect, the present invention provides an isolated and/or exogenous polynucleotide encoding
i) a polypeptide complex of the invention, wherein the first polypeptide and the second polypeptide form part of a single polypeptide chain,
ii) a compound of the invention, wherein the compound is a polypeptide.
Also provided is a vector comprising a polynucleotide of the invention. Preferably, the vector is an expression vector.
In a further aspect, the present invention provides a host cell comprising a polynucleotide of the invention, and/or a vector of the invention. The cell can be any cell type such as, but not limited to, a bacterial, yeast, animal, insect or plant cell. In a further aspect, the present invention provides an enriched population of dendritic cells and/or precursors thereof, obtained by a method of the invention.
In another aspect, the present invention provides an expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof of the invention.
In a further aspect, provided is a composition comprising a polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or a cell population of the invention, and a pharmaceutically acceptable carrier.
In another aspect, the present invention provides a crystal of the C-type lectin- like domain of human Clec9A.
In a further aspect, the present invention provides a set of atomic coordinates, or subset thereof, provided in Appendix I.
In another aspect, the present invention provides a computer-readable medium having recorded thereon data representing the atomic coordinates, or subset thereof, provided in Appendix I and/or a model produced using the atomic coordinates.
In yet another aspect, the present invention provides a computer-assisted method of identifying a compound that binds Clec9A, the method comprising
i) docking the structure of a candidate compound to a structure defined by the atomic coordinates, or subset thereof, provided in Appendix I, and
ii) identifying a candidate compound which may bind Clec9A.
Preferably, the method further comprises synthesising or obtaining an identified candidate compound and determining if the compound binds Clec9A.
In another aspect, the present invention provides a computer-assisted method for identifying a compound which binds a polypeptide complex which comprises
a) a first polypeptide comprising an actin binding domain, and
b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, the method comprises the steps of:
i) comparing a structure defined by the atomic coordinates, or subset thereof, provided in Appendix I, to a computer database of chemical structures, and
ii) selecting from the database a chemical structure which is complementary or similar to the structure defined by the atomic coordinates, or subset thereof, provided in Appendix I.
Preferably, the method further comprises synthesising or obtaining a selected candidate compound and detennining if the compound binds the polypeptide complex. Preferably, the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
In another aspect, the present invention provides a method of identifying a compound which binds a polypeptide complex comprising
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, the method comprising a) contacting the complex polypeptide with a candidate compound,
b) determining whether the compound binds the polypeptide complex, and c) optionally selecting a compound which binds the polypeptide complex.
In a further aspect, the present invention provides a method of identifying a compound which binds a polypeptide complex comprising
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, the method comprising a) exposing the polypeptide complex to a binding partner which binds the polypeptide complex, and a candidate compound,
b) assessing the ability of the candidate compound to compete with the binding partner for binding to the polypeptide complex, and
c) optionally selecting a compound which competes with the binding partner for binding to the polypeptide complex.
Preferably, the binding partner is Clec9A or a soluble fragment thereof.
In yet a further aspect, the present invention provides a method of identifying a compound which binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO: 1 , the method comprising
a) contacting Clec9A or a fragment thereof comprising one or both of the tryptophan residues with a candidate compound,
b) determining whether the compound binds one or both of the tryptophan residues,
c) determining if the compound reduces or inhibits the binding of Clec9A or a fragment thereof to a polypeptide complex comprising
i) a first polypeptide comprising an actin binding domain, and ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and
d) optionally selecting a compound which binds one or both of the tryptophan residues and which reduces or inhibits the binding of Clec9A or a fragment thereof to the polypeptide complex.
In another aspect, the present invention provides a method of identifying a compound which binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO: 1 , the method comprising
a) exposing Clec9A or a fragment thereof comprising one or both of the tryptophan residues to a binding partner which binds one or both of the tryptophan residues, and a candidate compound,
b) assessing the ability of the candidate compound to compete with the binding partner for binding to one or both of the tryptophan residues,
c) determining if the compound reduces or inhibits the binding of Clec9A or a fragment thereof binding a polypeptide complex comprising
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and
d) optionally selecting a compound which competes with the binding partner for binding to one or both of the tryptophan residues and which reduces or inhibits the binding of Clec9A or a fragment thereof to the polypeptide complex.
Also provided is a compound identified by the method of the invention.
In a further aspect, provided is a kit comprising one or more of a polypeptide complex of the invention, a compound of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, a cell population of the invention, and a composition of the invention.
The present inventors have also identified that Clec9A can bind actin in the absence of a polypeptide which comprises an actin binding domain. Thus, in yet a further aspect the present invention provides an isolated actin or a fragment thereof conjugated to a therapeutic agent or a detectable label, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain. In a preferred embodiment, the actin or fragment thereof is filamentous actin (F- actin) or a filamentous fragment thereof. In an embodiment, the actin is cytoskeletal actin or muscle actin.
In an embodiment, the actin or fragment thereof comprises
5 a) an amino acid sequence as provided in any one of SEQ ID NOs 26 to 40;
b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 26 to 40; and/or
c) a fragment of a) or b) which binds Clec9A.
In a preferred embodidment, the actin is cytoskeletal actin. Thus, in a preferred 0 embodiment the second polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 27, 28, 30, 31, 33, 34, 36, 37, 39 and 40;
b) an amino acid sequence which is at least 50% identical to any one or more of Λ SEQ ID NOs 27, 28, 30, 31, 33, 34, 36, 37, 39 and 40; and/or
5 c) a fragment of a) or b) which binds an actin binding domain.
In another embodiment, the Clec9A comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8;
b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 1 to 8.
0 In another embodiment, the actin or fragment thereof is bound to Clec9A.
In yet another embodiment, when bound to Clec9A, the conjugate further comprises RNF41 bound to the Clec9A. In an embodiment, the RNF41 comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 21 to 25; b) an amino acid sequence which is at least 50% identical to any one or more of5 SEQ ID NOs 21 to 25; and/or
c) a fragment of a) or b) which binds Clec9A.
Examples of polypeptides which comprise an actin binding domain include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titin,0 myosin, tubulin, catenin, keratin, cytokeratin, nestin, lamin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin. Further examples includes polypeptides which comprise
a) an amino acid sequence as provided in any one of SEQ ID NOs 41 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of5 SEQ ID NOs 41 to 89; and/or
c) an actin binding fragment of a) or b). Examples of suitable therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
The antigen can be any molecule that induces an immune response in an animal. Examples include, but are not limited to, a cancer antigen, a self-antigen, an allergen, and/or an antigen from a pathogenic and/or infectious organism.
In an embodiment, the antigen from a pathogenic and/or infectious organism can be from, but not limited to, Plasmodium falciparum or Plasmodium vivax.
In another aspect, the present invention provides a composition comprising conjugated actin or a fragment thereof of the invention, and a pharmaceutically acceptable carrier.
In an embodiment, the composition further comprises an adjuvant.
In another embodiment, the conjugated actin or fragment thereof is encapsulated in, or exposed on the surface of, a liposome.
In another aspect, the present invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof of the invention, and
iv) a composition of the invention. 1
In an embodiment, the immune response to an antigen is induced and/or enhanced.
In a particularly preferred embodiment, the immune response is modulated by enhancing a helper T-cell response.
In a further preferred embodiment, the immune response is modulated by the activation of CD4+ and or CD8+ T-cells.
In another particularly preferred embodiment, the immune response is modulated by enhancing B-cell antibody production. Examples of antibodies produced include, but are not necessarily limited to, IgGl, IgG2b, IgG2c, IgG3, IgG4, IgM, IgAl , IgA2, IgE and or IgD antibody isotypes.
In a further preferred embodiment, the immune response is modulated by generating a memory response. In a particularly preferred embodiment, the subject is administered with a compound comprising the antigen.
In another embodiment, an immune response to a self-antigen or allergen is reduced. In this embodiment, it is preferred that the immune response is modulated by suppressing a T-cell response and/or a B-cell antibody response.
In another aspect, the present invention provides a method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9 A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
Hi) actin or a fragment thereof of the invention, and
iv) a composition of the invention, and
administering said cells to the subject.
In an embodiment, the cells have been isolated from the subject.
Preferably, a humoral and/or T-cell mediated response is modulated.
In a further embodiment, naive CD8+ T-cell activation, and/or naive CD4+ T- cell activation, is modulated.
In yet another embodiment, the humoral response comprises the production of IgGl, IgG2b, IgG2c, IgG3, IgG4, IgM, IgAl, IgA2, IgE, and/or IgD antibody isotypes. In another embodiment, the humoral response at least comprises the production of IgGl antibody isotype.
Preferably, the dendritic cell is an animal dendritic cell or precursor of an animal dendritic cell. More preferably, the dendritic cell is a human dendritic cell. Even more preferably, the human dendritic cell is Necl-2+, HLA DR+ and/or BDCA-3+!
In another aspect, the present invention provides a method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof of the invention, and iv) a composition of the invention.
Preferably, the method comprises administering a conjugated aetin or fragment thereof of the invention comprising the cytotoxic agent, drug and/or pharmacological agent.
Examples of diseases involving dendritic cells or precursors thereof include, but are not limited to, cancer, an infection, an autoimmune disease or an allergy.
In an embodiment, the autoimmune disease is lupus erythematosus.
In another embodiment, the infection is a Plasmodium sp., such as Plasmodium falciparum or Plasmodium vivax, infection.
In another aspect, the present invention provides a method of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected , with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof of the invention, and
iv) a composition of the invention.
In another aspect, the present invention provides a method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof of the invention, and
iv) a composition of the invention.
In yet a further aspect, the present invention provides a method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof of the invention, and
iv) a composition of the invention.
In one embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is increased. In an alternate embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased.
i In another embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is increased. In an alternate embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is decreased.
In another embodiment, the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is increased. In an alternate embodiment, the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is decreased.
In a further preferred embodiment, the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof. Examples of such diseases include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
In a preferred embodiment of the methods of the invention the actin or fragment thereof is filamentous actin or a filamentous fragment thereof.
Also provided is the use of at least one of i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment of the invention, and
/ iv) a composition of the invention,
for the manufacture of a medicament for at least one of
a) modulating an immune response in a subject,
b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
In a further aspect provided is a
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof of the invention, and
iv) a composition of the invention,
for use in at least one of
a) modulating an immune response in a subject, b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) . modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response^ to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
In another aspect, the present invention provides a method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
i) contacting a cell with a compound that binds actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain, and wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
ii) determining whether the actin or fragment thereof is present or absent, wherein the presence of the actin or fragment thereof provides a diagnosis, prognosis and/or status of the disease.
In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
In an embodiment, the compound is an antibody or antigen-binding fragment thereof. Examples include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
In a preferred embodiment, the compound is detectably labelled.
In an embodiment, the method is performed in vivo on a subject. In an alternate embodiment, the method is performed in vitro on a sample obtained from a subject. As noted above, examples of diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
In a further aspect, the present invention provides a method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
i) exposing a cell to the therapy, and
ii) detecting a cell with a disrupted cell membrane, a dying cell or a dead cell, or a portion thereof, using a method of the invention,
wherein the presence of a cell with a disrupted cell membrane, a dying cell or a dead cell indicates that the therapy is effective.
In an embodiment, the cell in step i) is in vivo. In an alternate embodiment, the cell in step i) is in vitro.
Preferably, the therapy is administered to a subject. In an embodiment, the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells. In a preferred embodiment, the subject has cancer or an infection.
In a further embodiment, step ii) is performed on a sample obtained from a subject.
The therapy can be any type of procedure. Examples include, but are not limited to, drug therapy or radiotherapy.
In a further aspect, the present invention provides a method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
i) contacting a cell with a compound that binds actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding- domain, and wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain, and
ii) determining whether binding of the compound to the actin or fragment thereof is present or absent,
wherein the compound binding to the actin or a fragment thereof indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell. In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
Also provided is a method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a conjugated actin or fragment thereof of the invention, and
ii) isolating cells bound to the conjugated actin or fragment.
In a preferred embodiment, the cells obtained from step ii) are administered to a subject. In an embodiment, the cells are administered to treat and/or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
In another aspect, the present invention provides a method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a conjugated actin or fragment of the invention, and
ii) detecting cells bound to the conjugated actin or fragment.
In a further aspect, the present invention provides a method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
i) administering to the subject a conjugated actin or fragment thereof of the invention, and,
ii) detecting cells bound to the conjugated actin or fragment.
In a preferred embodiment, the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, Clec9A, XCRI and BDCA3.
Preferably, the dendritic cells are human dendritic cells that express one or more of the following markers, Necl-2, HLADR and BDCA3.
In an alternate embodiment, the dendritic cells are murine dendritic cells that express one or more of the following markers, CD24, Necl-2, CDl lc, Clec9A and CD8.
Preferably, the precursor dendritic cells are intermediate or late precursor dendritic cells which are capable of differentiating into dendritic cells in culture and/or on transfer into irradiated recipients.
In another aspect, the present invention provides a method of detecting a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
i) contacting a cell with a compound that binds actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain, and wherein the compound is not Clec9A or a polypeptide comprising an actin /
r binding domain, and
ii) determining whether binding of the compound to the actin or fragment thereof is present or absent,
wherein the compound binding to the actin or fragment thereof indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying, or is dead.
In a preferred embodiment of the above aspect, the compound is not an antibody which binds Clec9A, Clec9A per se or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
In a further aspect, the present invention provides an enriched population of dendritic cells and/or precursors thereof, obtained by a method of the invention.
In another aspect, the present invention provides an expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof of the invention.
In a further aspect, provided is a composition comprising a cell population of the invention, and a pharmaceutically acceptable carrier.
In a further aspect, the present invention provides a method of identifying a compound which binds actin or a fragment thereof, the method comprising
a) exposing actin or a fragment thereof to Clec9A or a soluble fragment thereof and a candidate compound, wherein the actin or a fragment thereof is not bound to a polypeptide comprising an actin binding domain,
b) assessing the ability of the candidate compound to compete with the Clec9A or a soluble fragment thereof for binding to actin or a fragment thereof, and
c) optionally selecting a compound which competes with the Clec9A or a soluble fragment thereof for binding to the actin or a fragment thereof.
Also provided is a compound identified by the method of the invention.
In another aspect, the present invention provides a kit comprising one or more of a conjugated actin or fragment thereof of the invention, a cell population of the invention, and a composition of the invention.
Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein. Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1. Generation of Soluble Recombinant Ectodomains of Clec9A.
(A) A schematic representation of the endogenous and recombinant soluble mClec9A proteins. The endogenous protein includes the Clec9A extracellular domains, the transmembrane (TM) and the cytoplasmic (Cyto) domains. Three forms of recombinant soluble mClec9A protein were generated: mClec9A-ecto which consists of the full Clec9A ectodomain, a FLAG tag and a biotinylation consensus sequence (predicted mol wt of 27 kDa); mClec9A-CTLD which consists of the Clec9A-CTLD, FLAG tag and biotinylation consensus sequence (predicted mol wt of 19.7 kDa); mClec9A-stalk which consists of the Clec9A-stalk region, FLAG tag and biotinylation consensus sequence (predicted mol wt of 12 kDa).
(B) Western blot analysis of endogenous mClec9A expression. DCs were produced from cultures of bone marrow with Flt3L (Naik et al., 2005) and DC lysates electrophoresed under non-reducing (N) and reducing (R) conditions. Blots were hybridised using anti-mClec9A Ab (24/04- 10B4) and binding detected using HRP conjugated anti-rat Ig and Enhanced Chemiluminescence-Plus (Amersham). mClec9A was observed to migrate as a dimer under non-reducing conditions.
(C) Western blot analysis of biotinylated recombinant soluble mClec9A protein. Biotinylated mClec9A-ecto and mClec9A-CTLD were electrophoresed under nonreducing (N) and reducing (R) conditions, and proteins detected using SA-HRP and Enhanced Chemiluminescence (Amersham). Recombinant mClec9A-ecto, like endogenous mClec9A, was observed to migrate as a dimer under nonreducing conditions and as a monomer under reducing conditions whereas mClec9A-CTLD migrated as a monomer under all conditions (B, C).
Figure 2. Clec9A Ectodomains Bind to Species Conserved Components of Dead Cells.
(A) Mouse thymocytes were γ-irradiated (5Gy) then cultured for 3.5 h or 16 h, to follow the progress of apoptotic death. Samples were incubated with biotinylated mClec9A-ecto (solid line), or biotinylated Cire-ecto as a background control (dashed line) and binding detected using SA-PE. Thymocytes were stained fwith Annexin V- FITC, analysed by flow cytometry, and gated as Annexin V" (viable), Annexin V+ ΡΓ (early apoptotic) or Annexin V+ PI+ (late apoptotic) cells for analysis of Clec9A binding.
(B) Mouse embryonic fibroblasts (MEFs) overexpressing Noxa to inactivate Mcl-l(van Delft et al., 2006) were induced to undergo apoptosis by treatment with 2.5 μΜ ABT-737 for 16 h. Control untreated MEFs (viable) and ABT-737-treated MEFs (late apoptotic) were harvested and incubated with mClec9A-ecto (solid line), hCLEC9A-ecto (solid line), or Cire-ecto (background control, dashed line). Binding was detected using biotinylated anti-FLAG mAb, SA-PE and flow cytometry. 90% of the untreated MEFs were viable based on normal forward scatter (FSC) and PI exclusion, whereas 95% of the ABT-737 treated MEFs were dead based on reduced FSC and high PI staining.
(C) Viable or freeze-thawed mouse fibroblasts (3T3 cell line) were incubated with biotinylated mClec9A or hCLEC9A ectodomains (solid line), or with biotinylated control (Cire-ecto, dashed line). Binding in (C)-(F) was detected using SA-PE and flow cytometry.
(D) Human 293T-cells were freeze-thawed then incubated with biotinylated mClec9A-ecto, hCLEC9A-ecto (solid line) or Cire-ecto (dashed line) in the absence or presence of 5 mM ethylenediamine tetraacetic acid (EDTA; solid line). . „
(E) Control untreated MEFs (viable) and ABT-737 treated MEFs (2.5 μΜ ABT- 737 for 16 h; late apoptotic) were incubated in PBS alone or in the presence of DNasel, RNaseA, protease K or trypsin. Cells were washed extensively to remove nucleases and proteases, then incubated with biotinylated mClec9A-ecto (solid line), or biotinylated Cire-ecto as a control (dashed line). 80% of the untreated MEFs were viable based on normal FSC and PI exclusion, whereas 97% of the ABT-737 treated MEFs were dead based on reduced FSC and high PI staining.
(F) Mouse 3T3 cells, insect SF21 cells, bacterial JM109 cells and yeast (Pichia pastor is) cells were freeze-thawed twice then incubated with biotinylated mClec9A- ecto (solid line), or biotinylated control (Cire-ecto, dashed line).
(G) Mouse platelets were induced to undergo apoptosis by treatment with 0.5 μΜ ABT-737 for 90 min at 37°C. Control (viable) and ABT-737 treated platelets (apoptotic) were harvested and incubated with mClec9A-ecto (solid line), hCLEC9A- ecto (solid line), or Cire-ecto (background control, dashed line). Binding was detected using FITC-conjugated anti-FLAG mAb, and flow cytometry. Control platelets were confirmed to be Annexin V" and ABT-737 treated platelets apoptotic (AnnexinV+),
(H) Mouse red blood cells (RBC) were isolated, permeabilised with PBS containing 0.15% saponin and 1 x EDTA-free complete protease inhibitors, and sequentially washed using the saponin-containing buffer in order to generate saponin- permeabilised RBC ghosts. Viable RBC and saponin-ghosts were incubated with biotinylated mClec9A-ecto (solid line), or biotinylated Cire-ecto as a background control (dashed line) and binding detected using SA-PE and flow cytometry.
(I) Freeze-thawed human 293F cells and human RBC saponin-permeabilised ghosts were incubated with mClec9A and hCLEC9A-ectodomains (-ecto), C-type lectin like domains (-CTLD) or stalk regions (-stalk) (solid line) or Cire-ecto (dashed line). Binding was detected using a FITC-conjugated anti-FLAG mAb and flow cytometry.
(J) Fixed and permeabilised mouse 3T3 cells were incubated with biotinylated mClec9A-ecto or Cire-ecto and binding detected using SA-Alexa594. Fibroblasts were counterstained with DAPI, and analysed by confocal microscopy.
(K) Fixed and permeabilised mouse embryonic fibroblasts were incubated with biotinylated mClec9A-ecto or Cire-ecto and binding detected using SA-Alexa594. Cells were counterstained with DAPI and with a rabbit anti-actin Ab and binding detected using anti-rabbit Ig-Alexa488, then analysed by confocal microscopy.
(L) Fixed and permeabilised mouse embryonic fibroblasts were incubated with biotinylated mClec9A-ecto and binding detected using SA-Alexa488. Cells were counterstained with DAPI and with phalloidin-Alexa594, then analysed by confocal microscopy. '
Figure 3. Mouse Clec9A Ectodomain Binds to Human Erythrocytic Spectrin.
(A) Mouse saponin-permeabilised RBC ghosts were generated as in Figure 2H, then treated in the presence or absence of spectrin extraction buffer for lh at 37°C. RBC ghost membranes were recovered by ultracentrifugation (26000g 40 min). Biotinylated mClec9A-ecto binding to untreated saponin ghosts (solid thick line), mClec9A-ecto binding to spectrin-extracted saponin ghosts (solid thin line), and Cire- ecto binding (dashed line) were detected using SA-PE and flow cytometry.
(B) Human erythrocytic spectrin (Sigma) and bovine muscle actin (Sigma) were coated onto ELISA plates and binding of mClec9A-ecto and mCire-ecto detected using anti-FLAG-HRP. Figure 4. Analysis of Human Erythrocytic Spectrin and Mouse Apoptotic Thymocyte Spectrin by Size-Exclusion Chromatography and SDS-PAGE.
(A) Analysis of human erythrocyte spectrin (Sigma) by SDS-PAGE and mass spectrometry. Spectrin ^g) was analysed by SDS-PAGE on a NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) under reducing conditions and protein bands visualized by the SimplyBlue staining (Invitrogen). Protein bands were then excised from the gel and subjected to mass spectrometry analysis.
(B) Analysis of human erythrocyte spectrin (Sigma) by size-exclusion chromatography (SEC). Spectrin was chromatographed on a Superose 6 column (300 x 10 mm) at a flowrate of 0.4 mL/min and column fractions at 0.4 mL collected for analysis.
(C) Analysis spectrin fractions by SDS-PAGE. Spectrin fractions from SEC (B) were analysed by SDS-PAGE on a NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) under reducing conditions and protein bands visualized by silver staining. Fraction numbers are indicated above the lanes.
(D) Human Clec9A binding to SEC fractions of human erythrocytic spectrin. Fractions from SEC (C) were coated onto ELISA plates, and incubated with mClec9A- ecto (0.3 g ml). Binding was detected using anti-FLAG-HRP. Clec9A binding to human spectrin is shown as a control.
(E) Analysis of apoptotic thymocyte spectrin by SEC. Mouse thymocytes were γ-irradiated (5Gy) then cultured for 3.5 h. Spectrin was extracted and chromatographed on a Superose 6 column (300 x 10 mm) at a flowrate of 0.4 ml/min and column fractions at 0.4 ml collected for analysis.
(F) Mouse Clec9A binding to SEC fractions of apoptotic thymocyte spectrin. Fractions collected from SEC (E) were coated onto ELISA plates, and incubated with mClec9A-ecto and mClecl2A-ecto (0.3 μg/ml). Binding was detected using anti- FLAG-HRP.
(G) Analysis of apoptotic thymocyte spectrin fractions by SDS-PAGE. Fractions from SEC (E) were analysed by SDS-PAGE on a NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) under reducing conditions and protein bands visualized by silver staining. Fraction numbers are indicated.
Figure 5. Mouse Clec9A Ectodomain Binds to Erythrocytic and Non-erythrocytic Spectrin Cytoskeletal Complexes.
(A) Analysis of mouse Clec9A binding to spectrin by ELISA. Mouse erythrocytic spectrin, isolated at 4°C or at 37°C, was coated onto ELISA plates and
• binding of mClec9A-ecto and the control, Clec 12 A-ecto, to erythrocytic spectrin detected using anti-FLAG-HRP.
(B) Mouse erythrocytic spectrin (5μ ), isolated at 4°C or at 37°C, was analysed by SDS-PAGE under reducing conditions. Protein bands were visualized, and bands A to E subjected to mass spectrometry analysis.
(C) Analysis of mouse spectrin isolated from red blood cells at 4°C and 37°C by size-exclusion chromatography (SEC). Mouse erythrocytic spectrin, isolated at 4°C or at 37°C, was chromatographed on a Superose 6 column and fractions collected for subsequent analysis.
(D) mClec9A binding to SEC fractions of erythrocytic spectrin. Spectrin fractions (Figure 5C) were coated onto ELISA plates, and binding of mClec9A-ecto (0.3 μg/ml) detected.
(E) Analysis of erythrocytic spectrin fractions by SDS-PAGE. Erythrocytic spectrin fractions from SEC (Figure 5C) were analysed by SDS-PAGE under reducing conditions and protein bands visualized by silver staining.
(F) Analysis of human non-erythrocytic spectrin extract isolated from 293F cells at 4°C by SEC on a Superose 6 column .
(G) mClec9A binding to SEC fractions of non-erythrocytic spectrin extracts. Fractions of the non-erythrocytic spectrin extract from SEC (Figure 5F) were coated onto ELISA plates, and binding of mClec9A-ecto (0.3 μg ml) detected.
(H) Analysis of non-erythrocytic spectrin fractions by SDS-PAGE. Non- erythrocytic spectrin extract fractions from SEC (F) were analysed by SDS-PAGE under reducing conditions and protein bands visualized by silver staining.
(I) Analysis of non-erythrocytic spectrin fractions by SDS-PAGE. Non- erythrocytic spectrin fractions 18-22 from SEC (F), corresponding to the Clec9A binding peak (G) were pooled, concentrated and analysed by SDS-PAGE under reducing conditions. Protein bands were visualized, and bands A to C subjected to mass spectrometry analysis. Figure 6. Clec9A Binds to a Spectrin-Actin Complex.
(A) Mouse erythrocytic spectrin was isolated at 4°C, and analysed by SEC (top panel), before dissociation by treatment at 37°C in spectrin extraction buffer for 1 hour and analysis by SEC using a Superose 6 column (bottom panel).
(B) Clec9A binding to SEC fractions of erythrocytic spectrin dissociated at 37°C. Fractions of the dissociated spectrin (A) were coated onto ELISA plates, and binding of mClec9 A-ecto (O^g ml) or the control protein mClecl2A-ecto detected. (C) Fractions of the dissociated spectrin (A) were analysed by SDS-PAGE under reducing conditions and protein bands visualised by silver staining. ^
(D) Re-association of mouse erythrocyte spectrin with actin. Fractions 25-29 of the dissociated spectrin collected from SEC (A,C) and demonstrated to have no binding activity to Clec9A (B) were concentrated in the presence of either bovine serum albumin (BSA) or actin to promote spectrin re-association. Spectrin re-associated in the presence of BSA or actin was then chromatographed on a Superose 6 column, and fractions collected for subsequent analysis for Clec9A binding activity by ELISA (E) and by SDS-PAGE (F).
(E) mClec9A binding to SEC fractions of reassociated spectrin. Fractions of erythrocytic spectrin reassociated in the presence of BSA or actin (D) were coated onto ELISA plates, and binding of mClec9A-ecto (O^g/ml) detected.
(F) Analysis of SEC fractions of reassociated spectrin fractions by SDS-PAGE. Fractions of erythrocytic spectrin reassociated in the presence of BSA or actin (D) were analysed by SDS-PAGE under reducing conditions and protein bands visualized by silver staining.
Figure 7. Clec9A Binds to a Complex of Actin with the Actin binding domain of Spectrin βΐ and Other Cytoskeletal Proteins.
(A) Schematic representation of the spectrin al and βΐ proteins demonstrating the spectrin al repeats 1-21 and the C terminal EF hand domains (EF), and the spectrin βΓ repeats 1-17 and the N-terminal Actin binding domain (ABD). The regions encompassed b the recombinant spectrin fragments investigated are demonstrated below each chain.
(B) Clec9A binding to recombinant GST-tagged erythrocytic spectrin fragments in the presence or absence of actin. Recombinant spectrin was pre-associated with either muscle or platelet actin before coating onto ELISA plates, and incubation with mClec9A-ecto (^g/ml) or the control protein mClecl2A-ecto. Binding was detected using anti-FLAG-HRP. Cumulative data of 4 experiments is shown, demonstrating the mean +/- standard errors of the mean (SEM). Clec9A-ecto binding to the βΐ N-4 + muscle actin was significantly greater than binding to βΐ N-4 alone (*** p<0.0001) or to muscle actin alone (*** p<0.0005). Similarly, Clec9A-ecto binding to βΐ N + muscle actin was significantly greater than binding to βΐ N alone or to muscle actin alone (*** pO.OOOl); Clec9A-ecto binding to βΐ N-4 + platelet actin and to βΐ N + platelet actin was significantly greater than binding to βΐ N or βΐ N-4 alone, respectively, or to platelet actin alone (*** p<0.0001). (C) Clec9A binding to dead cells is inhibited by pre-incubation of Clec9A with GST-tagged erythrocytic spectrin βΐ N plus actin. Pre-associated spectrin βΐ N plus platelet actin, was incubated with mClec9A-ecto (0.5 h, 21°C), before incubation with freeze-thawed 293F cells. Binding of mClec9A-ecto (5 μg/ml; solid line) in the presence or absence of spectrin βΐ N (50 μg/ml) plus actin (50 μg/ml), or the control Cire-ecto (dashed line) was detected using a FITC-conjugated anti-FLAG mAb and flow cytometry.
(D) Clec9A binding to recombinant GST-tagged ABD of erythrocytic spectrin (spectrin βΐ N), non-erythrocytic spectrins (spectrin βΙΙ N) and a-actinin 1 (a-actinin 1 N) in the presence or absence of actin. Pre-associated ABD +/- platelet actin were coated onto ELISA plates, incubated with mClec9A-ecto (1 μg ml) or mClecl2A-ecto, and binding detected using anti-FLAG-HRP. Cumulative data of 3 experiments is shown, demonstrating the mean +/- SEM. Clec9A-ecto binding to the spectrin βΐ N-4 + actin was significantly greater than binding to βΐ N-4 alone (*** pO.0001) or to actin alone (*** p<0.0005). Similarly, Clec9A-ecto binding to the spectrin βΐ N + actin was significantly greater than binding to βΙΙ N or to actin alone (*** p<0.0001); Clec9A- ecto binding to a-actinin- 1 N + actin was significantly greater than binding to a- actinin- 1 N or to actin alone (* * * p<0.0001 ).
(E) Clec9A binding to recombinant GST-tagged spectrin βΐ N, spectrin βΙΙ N and a-actinin- 1 N in the presence or absence of actin. Pre-associated ABD plus platelet actin were coated onto ELISA plates, and binding of mClec9A-ecto or mClecl2A-ecto detected.
(F) Membrane associated Clec9A binding to recombinant GST-tagged spectrin βΙΙ N and actin. CHO- 1 transfectant cells expressing full length mClec9A, or parental CHO-K1 cells, were incubated with pre-associated recombinant GST-tagged spectrin βΙΙ N and actin. Unbound spectrin-actin complexes were washed off, and binding of spectrin-actin complexes detected using a monoclonal mouse anti-GST antibody followed by anti-mouse Ig-PE and flow cytometric analysis. Figure 8. Clec9A Binds to Actin Complexed with Recombinant Actin binding domains.
(A) Mouse Clec9A binding to recombinant GST-tagged domains of erythrocytic spectrin, al and βΐ, in the presence and absence of muscle actin. Spectrin domains were incubated in the presence and absence of actin at 30°C, then coated onto ELISA plates, and incubated with mClec9A-ecto (2 μg ml), or with mClecl2A-ecto (2 μg ml) and mCire-ecto (2 ^πιΐ) as controls. Binding was detected using anti-FLAG-HRP. Clec9A bound only to the βΐ N-4 domains of erythrocytic spectrin that had been pre-incubated with actin.
(B) Clec9A binding to dead cells is inhibited by pre-incubation of CIec9A with GST-tagged erythrocytic spectrin βΐ N plus actin. mClec9A-ecto was incubated with actin, ABD, or pre- associated ABD-actin complexes including spectrin βΐ N plus actin, spectrin βΙΙ N plus actin and a-actinin-1 N plus actin, before incubation with late apoptotic (PI+) thymocytes. Binding of mClec9A-ecto (5 g/ml; solid line) in the presence or absence of ABD (100 μg/ml) and actin (100 g/ml), or the control Cire- ecto (dashed line) was detected using a FITC-conjugated anti-FLAG mAb and flow cytometry,
(C) Clec9A binding to recombinant GST-tagged a-actinin-1 N and Cofilin-1 in the presence or absence of actin. Pre-associated ABD plus muscle actin were coated onto ELISA plates, and binding of mClec9A-ecto (1 pg/ml) or mClec 12 A-ecto detected using anti-FLAG-HRP.
Figure 9. Crystal Structure of the hCLEC9A-C-type Lectin-like Domain.
(A) Crystal structure of the hCLEC9A-CTLD, dimer in the assymetric unit. The conserved tryptophans (W131, W227) are presented. The peptide region to which the anti-CLEC9A Ab were raised (Caminschi et al., 2008) is shown.
(B) Overlay of the CLEC9A CTLD monomer with the CLEC8A dimer.
(C) CLEC9A as a CLEC8A style dimer. Tryptophans (W131, W227) are shown. The peptide region to which the anti-CLEC9A Ab were raised is shown.
Figure 10. Further Structural Observations of hCLEC9A-CTLD.
(A) Validation of the hCLEC9A-CTLD (S225D) used for structural determination. In order to confirm biological activity of the glycosylation modified hCLEC9A-CTLD (S225D), human 293F cells were freeze-thawed to induce membrane disruption then incubated with the original hCLEC9A-CTLD, or hCLEC9A-CTLD (S225D) at 5μg/ml (solid lines). Binding was detected using FITC-conjugated anti- FLAG mAb, and flow cytometry. Staining with secondary reagents alone is demonstrated (dashed line). This confirmed that the hCLEC9A-CTLD (S225D) maintained binding activity to dead cells.
(B) Crystal packing mediated by W131 and W227.
(C) W131 and W227 are spatially located near the native N-linked glycosylation sites on the human CLEC9A CTLD (N223) and the mouse CLEC9A CTLD (N159; equivalent human residue, HI 35). (D) Co-ordination of Ca ion by CLEC9A.
(E) Thermofluor assay of CLEC9A. The melting temperatures (Tm) of hCLEC9A-CTLD (S225D) incubated in the presence of EDTA, Ca2+ or Mg2+ were determined. CLEC9A is less stable in the presence of EDTA (with a negative Tm difference of -3 +/- 2°C) and stabilised in the presence of Ca2+ (2.47mM) (with a positive Tm difference of +15.5 +/- 2.5°C; purple bar). Tm differences were calculated by comparing the Tm of incubated CLEC9A to the Tm of CLEC9A in standard buffer conditions (42°C in Tris buffered saline).
(F) Location of lysines mutated to alanine to test the role of basic residues in ligand binding. Mutated residues are shown on chain A of the CLEC9A-CTLD as a
CLEC8A-like dimer (Top View; related to Figure 9C). Equivalent residues on chain B are identified.
Figure 11. Structural requirements for Clec9A binding to ligands.
(A) CLEC9A binding to dead cells through TRP-mediated interactions. Wild- type and mutated hCLEC9A (K166A, K168A, K215A, W131A and W227A shown as W->A) ectodomains and CTLD were generated and binding examined to freeze-thawed human 293F cells (solid line) Binding of the control Clecl2A is indicated (dashed line). Clec binding was detected using a FITC-conjugated anti-FLAG mAb and flow cytometry.
(B) CLEC9A binding to RBC ghosts through TRP-mediated interactions. Wild- type and mutated hCLEC9A (W131A and W227A shown as W->A) ectodomains and CTLD were examined for binding to saponin-permeabilised mouse RBC ghosts (solid line). Binding of the control Clecl2A is indicated (dashed line). Clec binding was detected using a FITC-conjugated anti-FLAG mAb and flow cytometry.
(C) CLEC9A binding to human erythroid spectrin through TRP-mediated interactions. Erythroid spectrin (Sigma) was coated onto plates, and binding of wild- type and mutated CLEC9A-ecto and controls detected by ELISA using anti-FLAG mAb HRP.
(D) Clec9A binding to spectrin through TRP-mediated interactions. Erythroid spectrin (Sigma) and non-erythroid spectrin isolated from nucleated 293F cells were coated onto plates. Binding of wild-type and mutated CLEC9A-ecto and controls (^g/ml) was detected by ELISA.
(E) Clec9A binding to ABD-actin complexes through TRP-mediated interactions. Pre-associated complexes of the ABD of erythrocytic spectrin (spectrin βΐ N), non-erythrocytic spectrins (spectrin βΙΙ N) and a-actinin-1 (a-actinin-1 N) with platelet actin, were coated onto plates. Binding of wild-type and mutated CLEC9A- ecto and controls (^g/ml) was detected by ELISA using anti-FLAG mAb HRP. Cumulative data of 3 experiments is shown, demonstrating the mean +/- SEM. Binding of the wild-type hCLEC9A-ecto binding to the spectrin βΐ N + actin, spectrin βΙΙ N + actin or a-actinin-l N + actin was significantly greater than binding of the mutated hCLEC9A-ecto (W ->A) (*** p<0.0001) or the binding of the control Clecl2A-ecto (*** p<0.0001) to the ABD-actin complexes.
(F) Clec9A binding to ABD-actin complexes through TRP-mediated interactions. Spectrin βΐ N, spectrin βΙΙ N and a-actinin-l N were associated with platelet actin and coated onto plates. Binding of wild-type and mutated CLEC9A-ecto and controls was detected by ELISA using anti-FLAG mAb HRP.
Figure 12. Schematic representation of RNF41 protein.
Constructs were generated which encoded (1) a full length untagged RNF41 protein, (2) a GST tagged RNF41 C-terminal domain (RNF41-CTD) and (3) a GST-tagged N- terminal domain which included the RING, B-Box, and Coiled-Coil domains (RNF41- RBCC).
Figure 13. Clec9A-RNF41 Interaction.
(A, B) Mouse Clec9A-ecto binding to GST-RNF41. GST-tagged RNF41-C- terminal domain (CTD), RNF41-N -terminal domains (RBCC) and GST control were coated onto ELISA plates at 10μg/ml. FLAG-tagged mClec9A-ecto and mClecl2A- ecto were incubated on the ELISA plates at concentrations from ίθμ^πύ to 0.04 g/ml. Clec binding to RNF41 was detected using anti-FLAG-HRP. Cumulative data of 3 experiments is shown, demonstrating the means of absorbance values.
(A) Analysis of mClec9A-ecto and mClecl2A-ecto binding to GST-RNF41 proteins and the control GST is shown for the highest concentration of Clec proteins investigated tested (^g/ml).
(B) Analysis of mClec9A-ecto and mClecl2A-ecto (^g/ml to 0.04μg nll) binding to RNF41 -CTD and control GST. >
(C, D) Human CLEC9A-ecto binding to GST-RNF41. GST-tagged RNF41-C- terminal domain (CTD), RNF41-N-terminal domains (RBCC) and GST control were coated onto ELISA plates at ^g/ml. FLAG-tagged wildtype CLEC9A (hCLEC9A- ecto) and mutant CLEC9A (W131A, W227A, represented as hCLEC9A-ecto (W->A)) were incubated on the ELISA plates at concentrations from 10μg/ml to 0.12μ§/ιη1. CLEC9A binding to RNF41 was detected using anti-FLAG-HRP. Data is shown for 1 representative experiment.
(C) Analysis of hCLEC9A-ecto and hCLEC9A-ecto (W->A) binding to GST- RNF41 proteins and the control GST is shown for the highest concentration of CLEC9A proteins investigated tested (K^g/ml).
(D) Analysis of hCLEC9A-ecto and hCLEC9A-ecto (W->A) (10μg/ml to 0.(^g/ml) binding to RNF41 -CTD and control GST.
Figure 14. Determining an optimal concentration for mouse Clec9A binding to RNF41 to enable investigation of binding inhibition or synergy.
(A) Mouse Clec9A-ecto binding to GST-R F41. Recombinant GST-tagged RNF41-C-terminat domain (CTD) was coated onto ELISA plates at ΙΟμ πύ. FLAG- tagged mClec9A-ecto was incubated onto ELISA plates at concentrations from ΙΟμξ/πύ to 0.003 μg/ml. mClec9A binding to RNF41 was detected using anti-FLAG- HRP. Arrow denotes the optimal concentration of Clec9A ^g/ml) selected for binding to RNF41.
(B) Human CLEC9A-ecto binding to GST-RNF41. Recombinant GST-tagged RNF41-CTD was coated onto ELISA plates at 10μg ml. FLAG-tagged wildtype CLEC9A (hCLEC9A-ecto) and mutant CLEC9A (W131A, W227A, represented as hCLEC9A-ecto (W->A)) were incubated onto ELISA plates at concentrations from ΙΟμ^αύ to 0.003μg/ml. CLEC9A binding to RNF41 was detected using anti-FLAG- HRP. Arrow denotes the optimal concentration of Clec9A ^g/ml) selected for binding to RNF41. Figure 15. Association of mouse Clec9A with ABD-Actin complexes enhance Clec9A binding to RNF41-CTD.
(A, B) Mouse Clec9A-ecto binding to RNF41 is enhanced by association of CIec9A with ABD-Actin complexes, but not by ABD or actin alone. GST-tagged RNF41-CTD (ίθμ^ταϊ) or the control protein GST (^g/ml) were coated onto ELISA plates. mClec9A-ecto (δμ πύ) was pre-incubated with platelet actin (25μg/ml) complexed with GST-tagged ABD (25μg ml) of eythrocytic spectrin (spectrin βΐ N), non erthyroctic spectrin (spectrin βΙΙ N) and a-actinin-1 (a-actinin-1 N). As a control, mClec9A-ecto (5μg/ml) was pre-incubated with the individual proteins. Clec9A-ABD- Actin complexes and controls were incubated onto ELISA plates and Clec9A binding to RNF41 detected using anti-FLAG-HRP. Cumulative data of 2 experiments is shown, demonstrating the means of absorbance values. (A) Analysis of mClec9A-ecto (5μξ/ηύ) +/- ABD-Actin complexes (25μ^πι1 each) binding to GST-RNF41 -CTD and the control GST.
(B) Analysis of mClec9A-ecto (5μg ml) + ABD-Actin complexes (25μ§/πι1 to 0.^g ml each) binding to R F41-CTD and control GST. Bar denotes mClec9A binding to RNF41 in the absence of ABD-Actin complexes.
(C, D) Mouse Clec9A-ecto binding to RNF41 is enhanced by association of Clec9A with ABD-Actin complexes. The control Clec, mClecl2A, which does not bind to RNF41 is unaffected by the presence of ABD-Actin complexes. GST-tagged RNF41-CTD (ΙΟμ^ιηϊ) was coated onto ELISA plates. mClec9A-ecto (5μg ml) and the control, mClecl2A-ecto (5μg/πll), were pre-incubated with platelet actin (25μg/ml) complexed with GST-tagged ABD (25ug/ml) of eythrocytic spectrin (spectrin βΐ N), non erthyroctic spectrin (spectrin βΙΙ N) and a-actinin-1 (a-actinin-1 N)- Mixtures of Clec and ABD-Actin complexes were incubated onto ELISA plates and Clec binding to RNF41 detected using anti-FLAG-HRP. Cumulative data of 2 experiments is shown, demonstrating the means of absorbance values.
(C) . Analysis of mClec9A-ecto ^g/ml) and mClecl2A-ecto (5μg/ml) + ABD- actin (25 g/ml each) binding to GST-R F41 -CTD.
(D) Analysis of mClec9A-ecto (5μg/ml) and mClecl2A-ecto (5μg/ml) + ABD- Actin complexes (25μg/ml to 0.^g/ml) binding to RNF41-CTD. Bar denotes mClec9A binding to RNF41 in the absence of ABD-actin complex.
Figure 16. Enhancement of Clec9A binding to R F41 requires direct binding between Clec9A and RNF41.
(A, B) The hCLEC9A-ecto mutant (W131A, W227A), which binds to RNF41 but not to ABD-Actin complexes, does not enhance the binding of CLEC9A to RNF41. GST-tagged R F41-CTD was coated onto ELISA plates at ^g/ml. Wildtype hCLEC9A-ecto, mutant hCLEC9A-ecto (W131A, W227A) and control mClecl2A-ecto ^g/ml) were pre-incubated with platelet actin (25μg ml) complexed with GST-tagged ABD (25μg ml) of eythrocytic spectrin (spectrin βΐ N), non erthyroctic spectrin (spectrin βΙΙ N) and a-actinin-1 (a-actinin-1 N). CLEC-ABD-Actin complexes were incubated onto ELISA plates and binding to RNF41 was detected using anti-FLAG- HRP. Cumulative data of 2 experiments is shown, demonstrating the means of absorbance values.
(A) Analysis of wildtype hCLEC9A-ecto, mutant hCLEC9A-ecto (W131A, W227A) and mClecl2A-ecto ^g/ml) +/- ABD-Actin complexes (25μg/ml each) binding to GST-RNF41 -CTD. (B) Analysis of wildtype and mutant hCLEC9A-ecto (W131A, W227A) ^g/ml) + ABD-Actin complexes (25μ^πι1 to O.^g ml) binding to RNF41-CTD. Bar denotes wildtype and mutant hCLEC9A-ecto (W131A, W227A) binding to RNF41 in the absence of ABD-Actin complex.
Figure 17. Clec9A binds to actin filaments.
Ectodomains of mClec9A (5μΜ) were incubated with various concentrations of actin in the form of preformed muscle actin filaments, then centrifuged.
(A) An example of gel electrophoresis of the pellet (P) and supernatant (S) fractions.
(B) Quantitation of the proportion of mClec9A-ecto sedimenting with F-actin, compared to mClecl2A-ecto as a non-specific binding control. Data is mean+/-SEM from 3 experiments. KEY TO THE SEQUENCE LISTING
SEQ ID O: 1 - Human Clec9A.
SEQ ID NO:2 - Murine Clec9A.
SEQ ID NO:3 - Chimpanzee Clec9A.
SEQ ID NO:4 - Rhesus monkey Clec9A.
SEQ ID NO:5 - Dog Clec9A.
SEQ ID NO:6 - Cow Clec9A.
SEQ ID NO:7 - Horse Clec9A.
SEQ ID NO:8 - Rat Clec9A.
SEQ ID NO: 9 - Open reading frame encoding human Clec9A.
SEQ ID NO: 10 - Open reading frame encoding murine Clec9A.
SEQ ID NO:l 1 - Open reading frame encoding chimpanzee Clec9A.
SEQ ID NO: 12 - Open reading frame encoding rhesus monkey Clec9A.
SEQ ID NO: 13 - Open reading frame encoding dog Clec9A.
SEQ ID NO: 14 - Open reading frame encoding cow Clec9A.
SEQ ID NO: 15 - Open reading frame encoding horse Clec9A.
SEQ ID NO: 16 - Open reading frame encoding rat Clec9A.
SEQ ID NO: 17 - Soluble mouse Clec9A including stalk.
SEQ ID NO: 18 - Soluble huma Clec9A including stalk.
SEQ ID NO: 19 - Soluble mouse Clec9A without stalk.
SEQ ID NO:20 - Soluble human Clec9A without stalk. SEQ ID NO:21 - Human RNF41 RING(Really Interesting New Gene) finger protein 41 (isoform 1).
SEQ ID NO:22 - Human RNF41 RING (Really Interesting New Gene) finger protein 41 (isoform 2).
5 SEQ ID NO:23 -Mouse RNF41 RING (Really Interesting New Gene) finger protein 41 (isoform 1). "
SEQ ID NO:24 - Chimpanzee RNF41 RING (Really Interesting New Gene) finger protein 41 (isoform 1).
SEQ ID NO:25 - Horse RNF41 RING (Really Interesting New Gene) finger protein 10 41.
SEQ ID NO:26 - Human muscle alpha actin.
SEQ ID NO:27 - Human cytoplasmic beta actin.
SEQ ID NO:28 - Human cytoplasmic gamma actin.
SEQ ID NO:29 - Mouse muscle alpha actin.
15 SEQ ID NO:30 - Mouse cytoplasmic beta actin. .
SEQ ID NO:31 - Mouse cytoplasmic gamma actin.
SEQ ID NO:32 - Chimpanzee muscle alpha actin.
SEQ ID NO:33 - Chimpanzee cytoplasmic beta actin.
SEQ ID NO:34 - Chimpanzee cytoplasmic gamma actin.
20 SEQ ID NO:35 - Rat muscle alpha actin.
SEQ ID NO:36 - Rat cytoplasmic beta actin.
SEQ ID NO:37 - Rat cytoplasmic gamma actin.
SEQ ID NO:38 - Horse muscle alpha actin.
SEQ ID NO:39 - Horse cytoplasmic beta actin.
25 SEQ ID NO:40 - Horse cytoplasmic gamma actin.
SEQ ID NO:41 - Human erythrocytic spectrin, alpha 1 (elliptocytosis 2 or SPTA1).
SEQ ID NO:42 - Human non-erythrocytic spectrin, alpha 1 (alpha-fodrin or SPTANl)
(isoform 1)
SEQ ID NO:43 - Human non-erythrocytic spectrin, alpha 1 (alpha-fodrin or SPTANl) 30 (isoform 2)
SEQ ID NO:44 - Human erythrocytic beta spectrin, or SPTB (isoform a)
SEQ ID NO:45 - Human erythrocytic beta spectrin, or SPTB (isoform b)
SEQ ID NO:46 - Human non-erythrocytic beta spectrin 1, or SPTBN1 (isoform 1) SEQ ID NO:47 - Human non-erythrocytic beta spectrin 1, or SPTBN1 (isoform 2) 35 SEQ ID NO-.48 - Human non-erythrocytic beta spectrin 2, or SPTBN2.
SEQ ID NO:49 - Mouse spectrin alpha 1 , or SPNA 1. SEQ ID NO:50 - Mouse spectrin alpha 2, or SPNA2.
SEQ ID NO:51 - Mouse spectrin beta 1 , or SPNB 1.
SEQ ID NO:52 - Mouse spectrin beta 2, or SPNB2 (isoform 1)
SEQ ID NO:53 - Mouse spectrin beta 2, or SPNB2 (isoform 2)
SEQ ID NO:54 - Mouse spectrin beta 3, or SPNB3.
SEQ ID NO:55 - Mouse spectrin beta 4, or SPNB4.
SEQ ID NO:56 - Mouse spectrin beta 5, or SPNB5.
SEQ ID NO:57 - Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 1).
SEQ ID NO:58 - Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 2).
SEQ ID NO:59 - Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 3).
SEQ ID NO:60 - Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 1).
SEQ ID NO:61 - Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 2).
SEQ ID NO:62 - Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 3).
SEQ ID NO:63 - Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 4).
SEQ ID NO:64 - Chimpanzee non-erythrocytic beta spectrin 1, or SPTBNl (isoform 1).
SEQ ID NO:65 - Chimpanzee non-erythrocytic beta spectrin 2, or SPTBN2 (isoform 1).
SEQ ID NO:66 - Horse erythrocytic alpha spectrin 1 (elliptocytosis 2 or SPTA1).
SEQ ID NO:67 - Horse erythrocytic beta spectrin, or SPTB.
SEQ ID NO:68 - Horse non-erythrocytic beta spectrin 1 , or SPTBN 1.
SEQ ID NO:69 - Human calponin-1.
SEQ ID NO:70 - Human calponin-2.
SEQ ID NO:71 - Human calponin-3.
SEQ ID NO:72 - Human plectin isoform 1.
SEQ ID NO:73 - Human filamin isoform 1.
SEQ ID NO:74 - Human dystrophin isoform 1.
SEQ ID NO:75 - Human actinin.
SEQ ID NO:76 - Human alpha actinin 2.
SEQ ID NO:77 - Human alpha actinin 1 isoform a.
SEQ ID NO:78 - Human calponin-1 actin binding domain.
SEQ ID NO:79 - Human calponin-2 actin binding domain.
SEQ ID NO:80 - Human calponin-3 actin binding domain. SEQ ID NO:81 - Human plectin isoform 1 actin binding domain.
SEQ ID NO:82 - Human filamin isoform 1 actin binding domain.
SEQ ID NO:83 - Human dystrophin isoform 1 actin binding domain.
SEQ ID NO: 84 - Human actinin actin binding domain.
SEQ ID NO:85 - Human alpha actinin 2 actin binding domain.
SEQ ID NO:86 - Human alpha actinin 1 (isoform a) actin binding domain.
SEQ ID NO:87 - Human spectrin chain brain 1 isoform 1 actin binding domain.
SEQ ID NO:88 - Human spectrin beta chain brain 1 isoform 2 actin binding domain. SEQ ID NO:89 - Human spectrin beta chain erythrocyte isoform b actin binding domain.
SEQ ID NOs 90 to 105 - Oligonucleotide primers.
SEQ ID NO: 106 - Biotinylation consensus sequence.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques and Definitions
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J:E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning. Furthermore, unless stated otherwise the use of the phrase "and or" between the second last and last of a list of elements includes a combination of any two or more of the list elements.
As used herein, the term about, unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The phrase "cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells", and variations thereof, as used herein includes situations where the cell can have, where possible, one or more of these features. For example, the cell could have a disrupted membrane, be infected with a pathogen and be dying.
As used herein, the term "cells with a disrupted cell membrane" or "cell with a disrupted cell membrane" refers to cells where the integrity of the cell membrane has been compromised. This includes cells with pores, as well as damaged or ruptured cells.
As used herein, the term "dying cell" or "dying cells" refers to later stage apoptotic cells or necrotic cells. Preferably, the dying cell(s) are AnnexinV+ or they are propidium iodide (PI)+. For dying cells with a nuclei, it is preferred that they are AnnexinV* and PI+. In a particularly preferred embodiment, the dying cells are at least AnnexinV+. In yet another embodiment, the necrotic cells are secondary necrotic cells.
As used herein, "early stage apoptotic cell" or "early stage apoptotic cells" includes cells that are AnnexinV+ and ΡΓ.
As used herein, the term "dead cell" or "dead cells" refers to cell(s) that has passed a point of no return in the death prbcess and which changes cannot be reversed. The cell(s) may have died through apoptosis or necrosis.
With regard to the phrase "cells infected with a pathogen", the term pathogen includes any organism which can infect a cell. Examples include, but are not limited to, viruses, protozoa and bacteria.
As used herein, the term "uptake and/or clearance" of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, refers to the removal of cellular material, such a proteins or fragments thereof, of the cells. In an embodiment, dendritic cells are responsible, at least in part, for the uptake and/or clearance of the cells. Preferably, the dendritic cells are Clec9A+. As used herein, the term "surrounding cells" refers to cells in close proximity to one or more of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of a molecule of the invention (such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein) useful for the invention sufficient to reduce or eliminate at least one symptom of the specified condition.
As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of a molecule of the invention (such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein) useful for the invention sufficient to stop or hinder the development of at least one symptom of the specified condition.
As used herein, the term "diagnosing" or variations thereof refers to the detection of a disease.
As used herein, the term "prognosing" or variations thereof refers to an assessment of the future outcome of a disease.
As used herein, the term "monitoring the status" or variations thereof refers to determining the stage of a disease. The status can be determined before, during and/or after a subject has been administered with a treatment for the disease.
As used herein, the term "Clec9A" refers to a polypeptide which comprises; i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8;
ii) an amino acid sequence which is at least 50%, more preferably at least 90% and even more preferably at least 95%, identical to any one or more of SEQ ID NOs 1 to 8; and/or
iii) a biologically active and/or soluble fragment of i) or ii). In one embodiment, the polypeptide is at least expressed on a subset of dendritic cells. Preferably, the dendritic cells also express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, XCR1, Clec9A and BDCA3. Examples of soluble fragments include those with the CTLD domain (for instances fragments comprising sequences as provided in SEQ ID NOs 17 to 20). Clec9A has also been referred to in the art as 5B6 and HEEE9341.
As used herein, the phrase "tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l" refer to the relative position of the amino acid compared to surrounding amino acids. For example, in some embodiments a polypeptide of the invention may have deletional or substitutional mutations which alters the relative positioning, of the amino acid when aligned against, for example, SEQ ID NO:l. For instance, tryptophan residues 131 and 227 of full length human Clec9A correspond to residues 65 and 161 respectively of the fragment provided as SEQ ID NO: 18. Furthermore, the position of the tryptophan residues may vary between orthologs of Clec9A. For instance, with respect to murine Clec9A the phrase "tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l" encompasses tryptophan residues 155 and 250 respectively of SEQ ID NO:2. In an embodiment, the polypeptide comprises the defined amino acid at the nominated residue number.
As used herein, in some embodiments the "sample" can be any biological material suspected of having cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof. In other embodiments, the "sample" can be any biological material suspected of having Clec9A+ dendritic cells Examples include, but are not limited to, blood, for example, whole peripheral blood, cord blood, foetus blood, bone marrow, plasma, serum, urine, cultured cells, saliva or urethral swab, lymphoid tissues, for example tonsils, peyers patches, appendix, thymus, spleen and lymph nodes, and any biopsy samples taken for routine screening, diagnostic or surgical reason such as tumour biopsy or bioposy of inflamed organs/ tissues. The sample may be tested directly or may require some form of treatment prior to testing. For example, a biopsy sample may require homogenization to produce a cell suspension prior to testing. Furthermore, to the extent that the biological sample is not in liquid form (for example, it may be a solid, semi-solid or a dehydrated liquid sample), it may require the addition of a reagent, such as a buffer, to mobilize the sample. The mobilizing reagent may be mixed with the sample prior to placing the sample in contact with a compound as defined herein.
As used herein, the terms "conjugate", "conjugated" or variations thereof are used broadly to refer to any form to covalent or non-covalent association between a compound useful for the invention and a therapeutic agent or a detectable label, or to placing a compound useful for the invention and a therapeutic agent or detectable label in close proximity to each other such as in a liposome.
As used herein, the term "immune response" refers to an alteration in the reactivity of the immune system of a subject in response to an antigen and may involve antibody production, induction of cell-mediated immunity, complement activation and/or development of immunological tolerance. As used herein, the term "subject" preferably relates to an animal. More preferably, the subject is a mammal such as a human, dog, cat, horse, cow, or sheep. Most preferably, the subject is a human.
Molecules
The present inventors have identified protein complexes which bind Clec9A. The protein complexes comprise
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain. The present inventors have also identified that when bound to the protein complex, Clec9A is still capable of binding RNF41.
In addition, the present inventors have identified that Clec9A binds actin without the need for an actin binding protein.
Proteins with an Actin binding domain
Hundreds of proteins have been identified which comprise an actin binding domain (ABD) (Dos Remedios et al., 2003; Winder and Ayscough, 2005; Uribe and Jay, 2009). Examples include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titiii, myosin, tubulin, catenin, keratin, cytokeratin, nestin, lamin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
Actin binding domains relevant to the invention comprise a calponin homology domain (CHD). Thus, the terms "actin binding domain" and "calponin homology domain" are generally used herein interchangelably. The ABD is typically about 100 residues. Three major groups of ABD proteins have been recognized on the basis of sequence analysis, namely i) proteins containing a single N-terminal ABD including calponin, Vav, IQGAP and Cdc24, ii) proteins with an F-actin binding domain composed of two ABDs in tandem including spectrins, dystrophin, filamins and plakins, and iii) proteins of the fimbrin plastin family which also contain two ABDs in tandem (Gimona et al., 2002; Korenbaum and Rivero, 2002; Banuelos et al., 1998; Stradal et al., 1998). Thus, a first polypeptide as defined herein can comprise one, two or possibily more calponin homology domains. In one embodiment, an actin binding domain relevant to the invention comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 78 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 78 to 89.
In a further embodiment, the first polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 41 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 41 to 89; and/or
c) an actin binding fragment of a) or b).
In yet a further embodiment, the first polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 69 to 86; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 69 to 86; and/or
c) an actin binding fragment of a) or b).
As used herein, the phrase "variant thereof which binds actin" generally refers to naturally occurring proteins, or fragments thereof comprising an actin binding domain, which bind actin and which have been altered at one or more amino acids but still maintain the ability to bind actin. Preferably, variants are at least 75%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to a naturally occurring actin binding protein, preferably a mammalian actin binding protein such as those described herein.
In some embodiments, the first polypeptide is itself a protein complex of two or more subunits (for instance spectrin may be present as a complex), in other embodiments the first polypeptide is a single polypeptide chain.
The term "spectrin" as used herein refers to membrane-associated cytoskeletal proteins involved in the crosslinking of filamentous actin which act as molecular scaffold proteins to link the actin cytoskeleton to the plasma membrane, and function in the determination of cell shape, arrangement of transmembrane proteins, and organization of organelles (Broderick and Winder, 2005).
The spectrins are traditionally divided into erythrocytic and non-erythrocytic forms, the former being exclusive to red blood cells and being responsible for the elasticity of BCs. Spectrins are ubiquitous in cells and different isoforms may be expressed in different tissues in different organisms. Spectrins are highly modular proteins, containing many repeating alpha-helical 106-amino acid units (or 'spectrin repeats'). Alpha forms generally contain 20 spectrin repeats and, in contrast to the beta forms, generally lack an actin binding domain (ABD). Most alpha forms contain an SH3 (Src homology 3 domains) for binding polyproline-containing proteins. Non- erythrocytic alpha isoforms generally contain an EF-hand motif for binding calcium. Examples of erythrocytic alpha forms of spectrin are given as SEQ ID NOs: 41, 57-59 and 66. Examples of non-erythrocytic alpha forms of spectrin are given as SEQ ID NOs: 42-43. Mutations in the human SPTA1 gene (encoding erythrocytic spectrin alpha 1) are the cause of elliptocytosis type 2 (EL2), an autosomal dominant hematological disorder characterised by hemolytic anemia and elliptical or oval RBC shape. SPTA1 mutations also cause hereditary pyropoikilocytosis (HPP) and spherocytosis type III (SPH3), both being hemolytic disorders. Mutations in the non- erythrocytic alpha 1 gene (SPTANl) cause Sjogrens syndrome, autoimmune diseaeses, rheumatoid arthritis, multiple sclerosis, neurodegenerative diseases and xerostomia. Non-erythrocytic forms of alpha 1 spectrin (encoded by the SPTANl gene) are also known as alpha-fodrin.
Beta forms generally contain 17 spectrin repeats and an actin binding domain (ABD). ABDs generally contain two CH (calponin homology) domains, which enable beta forms of spectrin to interact with F-actin. Non-erythrocytic forms of beta spectrin contain a PH (pleckstrin homology) domain for interaction with membrane phospholipids. Beta forms of spectrin generally lack EF-hand motifs. Examples of erythrocytic beta forms of spectrin are given as SEQ ID NOs: 44-45, 60-63 and 67. , Examples of non-erythrocytic beta forms of spectrin are given as SEQ ID NOs: 46-48, 64-65 and 68. Mutations in the human SPTB gene (encoding erythrocytic spectrin beta) are the cause of RBC disorders including elliptocytosis type 3 (EL3), spherocytosis type I (SPH1), muscular dystrophy, various anemic disease and pyropoikilocytosis. Mutations in the non-erythrocytic beta 1 gene (SPTBN1) cause neurofibromatosis type 2 and leukemia. Non-erythrocytic forms of beta 1 spectrin (encoded by the SPTBN1 gene) are also known as beta-fodrin.
Spectrin functions as a tetramer of alpha and beta dimers linked in a head-to- head arrangement. Alpha and beta spectrin interact to form a dimer and two heterodimers form the functional tetramer. Tetramers bind via their tail ends to a junctional complex consisting of filamentous actin and band 4.1 protein. Spectrin also binds to integral membrane proteins via ankyrin and band 3 protein (especially in RBCs) and also via protein 4.1 and glycophorin C. Interactions also occur with phospholipids via the PH domains of beta spectrin. In one embodiment, the first polypeptide is not spectrin. Thus, in an embodiment, the polypeptide does not comprise
a) an amino acid sequence as provided in any one of SEQ ID NOs 41 to 68 or 87 to 89;
b) an amino acid sequence which is at least 50% identical to any one or more of
SEQ ID NOs 41 to 68 or 87 to 89; and/or
c) an actin binding fragment of a) or b).
Actin
Actin is a well known globular protein about 42 kDa in size found in close to all eukaryotic cells (Rohn and Baum et al., 2010). Actin is one of the most highly- conserved proteins, differing by as little as 20% in primary amino acid sequence in species as diverse as algae and humans. Actin is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Actin participates in many important cellular processes including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes. In vertebrates, three main groups of actin isoforms, alpha, beta, and gamma have been identified. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus (examples provided as SEQ ID NOs 26, 29, 32, 35 and 38). The beta and gamma actins co-exist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility (examples provided as SEQ ID NOs 27, 28, 30, 31 , 33, 34, 36, 37, 39 and 40).
G-actin is the monomeric form of the globular protein which assembles into actin filaments. F-actin is the polymeric form of the globular, protein assembled into actin filaments. In a particularly preferred embodiment, the actin is filamentous actin or a filamentous fragment thereof. In another embodiment, the actin is a polymer comprising at least two actin monomer units or fragments thereof.
Determining whether a compound will bind acti can be determined using standard techniques such as using a simple actin binding assay (Corrado et al., 1994). Briefly, the candidate protein is expressed as fusion proteins with a tag, F-actin is absorbed on to microtiter plates, the candidate protein is incubated on the F-actin coated microtiter plates, the plates are washed, anti-tag rabbit antibody is added, anti- rabbit antibody conjugated to a detectable marker is added, and the plates screened for the detectable marker. As the skilled person would readily appreciate, a similar method can be used to identify fragments of actin which bind Clec9A.
RNF-41 protein
RNF-41 protein is also known as RING (Really Interesting New Gene) finger protein, neuregulin receptor degradation protein- 1 (NRDP1), or fetal liver RING protein (FLRF), refers to a protein which acts as an E3-ubiquitin ligase and regulates the degradation of target proteins. Target proteins for RNF-41 include members of the EGF (epidermal growth factor) receptor family, for example ErbB3 (or Her3). Other targets of RNF-41 include ErbB4, ubiquitin-specific protease 8 (Usp8), Birc6 and reticulon 4 (Rtn4, also known as NogoA). Mutations in RNF-41 have been linked to tumour diseases. Overexpression of RNF-41 has been shown to decrease ErbB3 and inhibition of breast cancer growth. Decreased levels of RNF-41 are inversely correlated with ErbB3 levels in primary human breast cancer tissue.
In humans, three transcript variants encode 2 isoforms of RNF-41, namely isoform 1 and 2, given in SEQ ID NOs: 21 and 22, respectively. Examples of RNF-41 proteins from other organisms are given in SEQ ID NOs: 23-25.
Compounds
It has previously shown that Clec9A is expressed by a subset of dendritic cells, can be targetted to modulate an immune response, and binds a ligand on cells with a disrupted cell membrane, cells infected with a pathogen, dying cells and dead cells (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008; Sancho et al., 2009; WO 2009/026660; WO 2009/137871; WO 2010/108215). This enabled compounds which modulate the binding of Clec9A to the ligand, and/or compounds which modulate the production of the ligand to be used in a wide variety of diagnostic, prognostic and therapeutic procedures. The present inventors have now identified further molecules which bind Clec9A which can be used to, inter alia, target therapeutic molecules to dendritic cells.
In one embodiment, the invention relates to a polypeptide complex comprising a first polypeptide comprising an actin binding domain and a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and which has been modified to deliver a therapeutic agent. In another embodiment, the invention relates to a compound that binds a polypeptide complex which comprises a first polypeptide comprising an actin binding domain and a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A. In yet a further embodiment, the present invention provides a compound which
i) binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l,
ii) binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, or
iii) competes with the one or both tryptophan residues of CIec9A for binding to the polypeptide complex.
In another embodiment, the invention relates to an isolated actin or a fragment thereof conjugated to a therapeutic agent or a detectable label, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain. In another embodiment, the invention relates to the use of actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain. In a further embodiment, the present invention relates to the use of a compound which binds actin or a fragment thereof of the previous embodiment, wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain.
The binding may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.. When the interaction produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of hydrophilic/lipophilic interactions. In a preferred embodiment, the compound is a purified and/or recombinant polypeptide (which can be a polypeptide complex).
Although not essential, the polypeptide complex, conjugate or compound may bind specifically a target molecule. The phrase "specifically binds", means that under particular conditions, the polypeptide complex, conjugate or compound binds the target and does not bind to a significant amount to other, for example, proteins or carbohydrates. In one embodiment, the polypeptide complex, conjugate or compound specifically binds the target and not other molecules in a sample obtained from a subject comprising, for example, cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof. In another embodiment, a polypeptide complex, conjugate or compound is considered to "specifically binds" if there is a greater than 2-fold difference, and preferably a 5, 25, 50 or 100 fold greater difference between the binding of the polypeptide complex, conjugate or compound when compared to another protein.
Antibodies
In one embodiment, a compound of, and/or useful for, the invention is an antibody or antigen-binding fragment thereof. The terms "antibodies" and "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized, see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region typically is comprised of three domains, CHI, Cm, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically is comprised of one domain, CL- The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
Each VH and VL is typically composed of three CDRs and four FRs, arranged from amirio-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, 1987). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (phrases such as variable domain residue numbering as in Kabat or according to Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable domains). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
The term "humanized antibody", as used herein, refers to herein an antibody derived from a non-human antibody, typically murine, that retains or substantially retains the antigen-binding properties of the parent antibody but which is less immunogenic in humans.
The term complementarity determining region (CDR), as used herein, refers to amino acid sequences which together define the binding affinity and specificity of a variable fragment (Fv) region of a immunoglobulin binding site.
The term framework region (FR), as used herein, refers to amino acid sequences interposed between CDRs. These portions of the antibody serve to hold the CDRs in appropriate orientation (allows for CDRs to bind antigen). A variable region, either light or heavy, comprises a framework and typically three CDRs.
The term constant region (CR) as used herein, refers to the portion of the antibody molecule which confers effector functions. The constant regions of the subject humanized antibodies are derived from human immunoglobulins. The heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, antibodies with desired effector function can be produced. Preferred heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3) and gamma 4 (IgG4), more preferably gamma 4 (IgG4). The light chain constant region can be of the kappa or lambda type, preferably of the kappa type.
Antibodies may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CHI domain. A scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody". Also encompassed are fragments of antibodies such as Fab, (Fab')2 and FabFc2 fragments which contain the variable regions and parts of the constant regions. CDR-grafted antibody fragments and oligomers of antibody fragments are also encompassed. The heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (for example mouse, rabbit or rat) or human origin or may be chimeric (Morrison et al., 1984) or humanized (Jones et al., 1986; UK 8707252). As used herein the term "antibody" includes these various forms. Using the guidelines provided herein and those methods well known to those skilled in the art which are described in the references cited above and in such publications as Harlow & Lane {supra) the antibodies for use in the methods of the present invention can be readily made.
As used herein, an "antigen-binding fragment" refers to a portion of an antibody as defined herein that is capable of binding the same antigen as the full length molecule.
Antibodies or antigen-binding fragments of, and/or useful for, the invention which are not from a natural source, such as a humanized antibody, preferably retain a significant proportion of the binding properties of the parent antibody. In particular, such antibodies or fragments of, and/or useful for, the invention retain the ability to specifically bind the antigen recognized by the parent antibody used to produce the antibody or fragment such as a humanized antibody. Preferably, the antibody or fragment exhibits the same or substantially the same antigen-binding affinity and avidity as the parent antibody. Ideally, the affinity of the antibody or fragment will not be less than 10% of the parent antibody affinity, more preferably not less than about 30%, and most preferably the affinity will not be less than 50% of the parent antibody. Methods for assaying antigen-binding affinity are well known in the art and include half-maximal binding assays, competition assays, and Scatchard analysis.
A variety of immunoassay formats may be used to select antibodies or fragments that are specifically immunoreactive with the ligand. For example, surface labelling and flow cytometric analysis or solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See Harlow & Lane (supra) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
The antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain. The light and heavy chains may be joined directly or through a linker. As used herein a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed. Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.
In another embodiment, recombinantly produced single chain scFv antibody, preferably a humanized scFv, is used in the methods of the invention.
Monoclonal Antibodies
Monoclonal antibodies of, and/or useful for, the invention can be readily produced by one skilled in the art.
The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against target epitopes can be screened for various properties; i.e. for isotype and epitope affinity.
Animal-derived monoclonal antibodies can be used for both direct in vivo and extracorporeal immunotherapy. However, it has been observed that when, for example, mouse-derived monoclonal antibodies are used in humans as therapeutic agents, the patient produces human anti-mouse antibodies. Thus, animal-derived monoclonal antibodies are not preferred for therapy, especially for long term use. With established genetic engineering techniques it is possible, however, to create chimeric or humanized antibodies that have animal-derived and human-derived portions. The animal can be, for example, a mouse or other rodent such as a rat.
If the variable region of the chimeric antibody is, for example, mouse-derived while the constant region is human-derived, the chimeric antibody will generally be less immunogenic than a "pure" mouse-derived monoclonal antibody. These chimeric antibodies would likely be more suited for therapeutic use, should it turn out that "pure" mouse-derived antibodies are unsuitable.
Methodologies for generating chimeric antibodies are available to those in the art. For example, the light and heavy chains can be expressed separately, using, for example, immunoglobulin light chain and immunoglobulin heavy chains in separate plasmids. These can then be purified and assembled in vitro into complete antibodies; methodologies for accomplishing such assembly have been described (see, for example, Sun et al., 1986). Such a DNA construct may comprise DNA encoding functionally rearranged genes for the variable region of a light or heavy chain of an antibody linked to DNA encoding a human constant region. Lymphoid cells such as myelomas or hybridomas transfected with the DNA constructs for light and heavy chain can express and assemble the antibody chains.
In vitro reaction parameters for the formation of IgG antibodies from reduced isolated light and heavy chains have also been described. Co-expression of light and heavy chains in the same cells to achieve intracellular association and linkage of heavy and light chains into complete H2L2 IgG antibodies is also possible. Such co- expression can be accomplished using either the same or different plasmids in the same host cell.
In another preferred embodiment of the present invention the antibody is humanized, that is, an antibody produced by molecular modeling techniques wherein the human content of the antibody is maximised while causing little or no loss of binding affinity attributable to the variable region of, for example, a parental rat, rabbit or murine antibody.
There are several factors to consider in deciding which human antibody sequence to use during the humanisation. The humanisation of light and heavy chains are considered independently of one another, but the reasoning is basically similar for each.
This selection process is based on the following rationale: A given antibody's antigen specificity and affinity is primarily determined by the amino acid sequence of the variable region CDRs. Variable domain framework residues have little or no direct contribution. The primary function of the framework regions is to hold the CDRs in their proper spatial orientation to recognize antigen. Thus the substitution of animal, for example, rodent CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework is highly homologous to the animal variable domain from which they originated. A human variable domain should preferably be chosen therefore that is highly homologous to the animal variable domain(s). A suitable human antibody variable domain sequence can be selected as follow.
Step 1. Using a computer program, search all available protein (and DNA) databases for those human antibody variable domain sequences that are most homologous to the animal-derived antibody variable domains. The output of a suitable program is a list of sequences most homologous to the animal-derived antibody, the percent homology to each sequence, and an alignment of each sequence to the animal- derived sequence. This is done independently for both the heavy and light chain variable domain sequences. The above analyses are more easily accomplished if only human immunoglobulin sequences are included. Step 2. List the human antibody variable domain sequences and compare for J homology. Primarily the comparison is performed on length of CDRs, except CDR3 of the heavy chain which is quite variable. Human heavy chains and Kappa and Lambda light chains are divided into subgroups; Heavy chain 3 subgroups, Kappa chain 4 subgroups, Lambda chain 6 subgroups. The CDR sizes within each subgroup are similar but vary between subgroups. It is usually possible to match an animal-derived antibody CDR to one of the human subgroups as a first approximation of homology. Antibodies bearing CDRs of similar length are then compared for amino acid sequence homology, especially within the CDRs, but also in the surrounding framework regions. The human variable domain which is most homologous is chosen as the framework for humanisation.
The Actual Humanising Methodologies/Techniques
An antibody may be humanized by grafting the desired CDRs onto a human framework according to standard procedures such as those decribed in EP 0239400. A DNA sequence encoding the desired reshaped antibody can therefore be made beginning with the human DNA whose CDRs it is wished to reshape. The animal- derived variable domain amino acid sequence containing the desired CDRs is compared to that of the chosen human antibody variable domain sequence. The residues in the human variable domain are marked that need to be changed to the corresponding residue in the animal to make the human variable region incorporate the animal-derived CDRs. There may also be residues that need substituting in, adding to or deleting from the human sequence.
Oligonucleotides are synthesized that can be used to mutagenize the human variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.
Synthetic gene sequences, such as those encoding humanized antibodies or fragments thereof, can be commercially ordered through any of a number of service companies, including DNA 2.0 (Menlo Park, Calif.), Geneart (Regensburg, Germany), CODA Genomics (Irvine, Calif.), and GenScript, Corporation (Piscataway, N.J.).
Alternatively, humanisation may be achieved using the recombinant polymerase chain reaction (PCR) methodology of WO 92/07075. Using this methodology, a CDR may be spliced between the framework regions of a human antibody. In general, the technique of WO 92/07075 can be performed using a template comprising two human framework regions, AB and CD, and between them, the CDR which is to be replaced by a donor CDR. Primers A and B are used to amplify the framework region AB, and primers C and D used to amplify the framework region CD. However, the primers B and C each also contain, at their 5' ends, an additional sequence corresponding to all or at least part of the donor CDR sequence. Primers B and C overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a PCR to be performed. Thus, the amplified regions AB and CD may undergo gene splicing by overlap extension to produce the humanized product in a single reaction.
Following the mutagenesis reactions to reshape the antibody, the mutagenised DNAs can be linked to an appropriate DNA encoding a light or heavy chain constant region, cloned into an expression vector, and transfected into host cells, preferably mammalian cells. These steps can be carried out in routine fashion. A reshaped antibody may therefore be prepared by a process comprising:
(a) preparing a first replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least a variable domain of an Ig heavy or light chain, the variable domain comprising framework regions from a human antibody and the CDRs required for the humanized antibody of the invention;
(b) preparing a second replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least the variable domain of a complementary Ig light or heavy chain respectively;
(c) transforming a cell line with the first or both prepared vectors; and
(d) culturing said transformed cell line to produce said altered antibody.
Preferably the DNA sequence in step (a) encodes both the variable domain and each constant domain of the human antibody chain. The humanized antibody can be prepared using any suitable recombinant expression system. The cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus. Most preferably, the immortalised cell line is a myeloma cell line or a derivative thereof.
The CHO cells used for expression of the antibodies may be dihydrofolate reductase (DHFR) deficient and so dependent on thymidine and hypoxanthine for growth. The parental DHFR* CHO cell line is transfected with the DNA encoding the antibody and DHFR gene which enables selection of CHO cell transformants of DHFR positive phenotype. Selection is carried out by culturing the colonies on media devoid of thymidine and hypoxanthine, the absence of which prevents untransformed cells from growing and transformed cells from resalvaging the folate pathway and thus bypassing the selection system. These transformants usually express low levels of the DNA of interest by virtue of co-integration of transfected DNA of interest and DNA encoding DHFR. The expression levels of the DNA encoding the antibody may be increased by amplification using methotrexate (MTX). This drug is a direct inhibitor of the enzyme DHFR and allows isolation of resistant colonies which amplify their DHFR gene copy number sufficiently to survive under these conditions. Since the DNA sequences encoding DHFR and the antibody are closely linked in the original transformants, there is usually concomitant amplification, and therefore increased expression of the desired antibody.
Another preferred expression system for use with CHO or myeloma cells is the glutamine synthetase (GS) amplification system described in WO 87/04462. This system involves the transfection of a cell with DNA encoding the enzyme GS and with DNA encoding the desired antibody. Cells are then selected which grow in glutamine free medium and can thus be assumed to have integrated the DNA encoding GS. These selected clones are then subjected to inhibition of the enzyme GS using methionine sulphoximine (Msx). The cells, in order to survive, will amplify the DNA encoding GS with concomitant amplification of the DNA encoding the antibody.
Although the cell line used to produce the humanized antibody is preferably a mammalian cell line, any other suitable cell line, such as a bacterial cell line or a yeast cell line, may alternatively be used. In particular, it is envisaged that E. co/i'-derived bacterial strains could be used. The antibody obtained is checked for functionality. If functionality is lost, it is necessary to return to step (2) and alter the framework of the antibody.
Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be recovered and purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (See, generally, Scopes, R., Protein Purification, Springer- Verlag, N.Y. (1982)). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, a humanized antibody may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (See, generally, Lefkovits and Pernis (editors), Immunological Methods, Vols. I and II, Academic Press, ( 1979 and 1981 )). Studies carried out by Greenwood et al. (1993) have demonstrated that recognition of the Fc region of an antibody by human effector cells can be optimised by engineering the constant region of the immunoglobulin molecule. This could be achieved by fusing the variable region genes of the antibody, with the desired specificity, to human constant region genes encoding immunoglobulin isotypes that have demonstrated effective antigen dependent cellular cytotoxicity (ADCC) in human subjects, for example the IgGl and IgG3 isotypes (Greenwood and Clark, Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man. Mike Clark (editor), Academic Titles, Section II, p.85-l l3, (1993)). The resulting chimeric or humanized antibodies should be particularly effective in modulating humoral immunity and/or T-cell mediated immunity.
Antibodies with fully human variable regions can also be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Various subsequent manipulations can be performed to obtain either antibodies per se or analogs thereof (see, for example, US 6,075,181).
Preparation of Genes Encoding Antibodies or Fragments Thereof
Genes encoding antibodies, both light and heavy chain genes or portions thereof, e.g., single chain Fv regions, may be cloned from a hybridoma cell line. They may all be cloned using the same general strategy such as RACE using a commercially available kit, for example as produced by Clontech. Typically, for example, poly(A)+mRNA extracted from the hybridoma cells is reverse transcribed using random hexamers as primers. For Fv regions, the VH and VL domains are amplified separately by two polymerase chain reactions (PCR). Heavy chain sequences may be amplified using 5' end primers which are designed according to the amino-terminal protein sequences of the heavy chains respectively and 3' end primers according to consensus immunoglobulin constant region sequences (Kabat et al., Sequences of Proteins of Immunological Interest. 5th edition. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Light chain Fv regions are amplified using 5' end primers designed according to the amino- terminal protein sequences of light chains and in combination with the primer C-kappa. One of skill in the art would recognize that many suitable primers may be employed to obtain Fv regions.
The PCR products are subcloned into a suitable cloning vector. Clones containing the correct size insert by DNA restriction are identified. The nucleotide sequence of the heavy or light chain coding regions may then be determined from double stranded plasmid DNA using sequencing primers adjacent to the cloning site. Commercially available kits (e.g., the Sequenase™ kit, United States Biochemical Corp., Cleveland, Ohio, USA) may be used to facilitate sequencing the DNA. DNA encoding the Fv regions may be prepared by any suitable method, including, for example, amplification techniques such as PCR and LCR.
Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. While it is possible to chemically synthesize an entire single chain Fv region, it is preferable to synthesize a number of shorter sequences (about 100 to 150 bases) that are later ligated together.
Alternatively, sub-sequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.
Once the Fv variable light and heavy chain DNA is, obtained, the sequences may be ligated together, either directly or through a DNA sequence encoding a peptide linker, using techniques well known to those of skill in the art. In one embodiment, heavy and light chain regions are connected by a flexible peptide linker (e.g., (Gh/4Ser)3) which starts at the carboxyl end of the heavy chain Fv domain and ends at the amino terminus of the light chain Fv domain. The entire sequence encodes the Fv domain in the form of a single-chain antigen-binding protein.
Therapeutic Agents
Polypeptide complexes, conjugates and compounds defined herein can be used to deliver a therapeutic agent. Examples of therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
In some embodiments, the therapeutic agent may be a polypeptide fused to the polypeptide complex, actin or a fragment thereof, or compound as defined herein. Fusion polypeptides comprising the polypeptide complex or compound may be prepared by methods known to one of skill in the art. For example, a gene encoding a polypeptide chain is fused to a gene encoding a therapeutic agent. Optionally, the gene is linked to a segment encoding a peptide connector. The peptide connector may be present simply to provide space between the compound and the therapeutic agent or to facilitate mobility between these regions to enable them to each attain their optimum conformation. The DNA sequence comprising the connector may also provide sequences (such as primer sites or restriction sites) to facilitate cloning or may preserve the reading frame between the sequence encoding the binding moiety and the sequence encoding the therapeutic agent. The design of such connector peptides is well known to those of skill in the art.
Polypeptide complexes, actins or compounds useful for the invention may be fused to, or otherwise bound to the therapeutic agent by any method known and available to those in the art. The two components may be chemically bonded together by any of a variety of well-known chemical procedures. For example, the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like. Production of various immunotoxins, as well as chemical conjugation methods, are well-known within the art (see, for example, "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, p. 168-190 (1982); Waldmann, 1991 ; Vitetta et al., 1987; Pastan et al., 1986; and Thorpe et al., 1987).
Examples of drugs and/or pharmacological agents include, but are not limited to, agents that promote DC activation (e.g. TLR ligands), agents that suppress DC activation or function (e.g. specific inhibitors or promotors of DC signalling molecules such as kinases and phosphatases), and agents that modulate DC death (e.g. promotors or suppressors of apoptosis). Such drugs and/or pharmacological agents are well known to those skilled in the art.
The skilled person will appreciate that there are a number of bacterial or plant polypeptide toxins that are suitable for use as cytotoxic agents in the methods of the invention. These polypeptides include, but are not limited to, polypeptides such as native or modified Pseudomonas exotoxin (PE), diphtheria toxin (DT), ricin, abrin, gelonin, momordin II, bacterial RIPs such as shiga and shiga-Iike toxin a-chains, luffm, atrichosanthin, momordin I, Mirabilis anti- viral protein, pokeweed antiviral protein, byodin 2 (U.S. 5,597,569), gaporin, as well as genetically engineered variants thereof. Native PE and DT are highly toxic compounds that typically bring about death through liver toxicity. Preferably, Pseudomonas exotoxin and DT are modified into a form that removes the native targeting component of the toxin, e.g., domain la of Pseudomonas exotoxin and the B chain of DT. One of skill in the art will appreciate that the invention is not limited to a particular cytotoxic agent.
Other suitable cytotoxic agents for use in the present invention include, but are not limited to, agents such as bacterial or plant toxins, drugs, e.g., cyclophosphamide (CTX; Cytoxan), chlorambucil (CHL; leukeran), cisplatin (CisP; CDDP; platinol), busulfan (myleran), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and other alkylating agents; methotrexate (MTX), etoposide (VP- 16; vepesid), 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5FU), dacarbazine (DTIC), 2-chlorodeoxyadenosine (2-CdA), and other antimetabolites; antibiotics including actinomycin D, doxorubicin (DXR; adriamycin), daunorubicin (daunomycin), bleomycin, mithramycin as well as other antibiotics; alkaloids such as vincristin (VCR), vinblastine, and the like; as well as other anti-cancer agents including the cytostatic agents glucocorticoids such as dexamethasone (DEX; decadron) and corticosteroids such as prednisone, nucleotide enzyme inhibitors such as hydroxyurea, and the like.
Those skilled in the art will realize that there are numerous other radioisotopes and chemocytotoxic agents that can be coupled to compounds of the invention by well known techniques, and delivered to specifically destroy dendritic cells (see, e.g., U.S. 4,542,225). Examples of photo-activated toxins include dihydropyridine-and omega- conotoxin. Examples of cytotoxic reagents that can be used include ,25I, ,31I, mIn, ml, "mTc, and 32P. The antibody can be labeled with such reagents using techniques known in the art. For example, see Wenzel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. (1983) for techniques relating to the radiolabeling of antibodies (see also, Colcher et al., 1986; "Order, Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (editors), Academic Press, p. 303-16, (1985)).
In one example, the linker-chelator tiuexutan is conjugated to the compound by a stable thiourea covalent bond to provide a high-affinity chelation site for Indium- 111 or Yttrium-90.
In one embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent. In another embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent. In a further embodiment, the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
In one embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein. In another embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein. In a further embodiment, the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
Antigens ,
Polypeptide complexes, conjugates or compounds useful for the methods of the invention may also be conjugated to an "antigen".
The term "antigen" is further intended to encompass peptide or. protein analogs of known or wild-type antigens such as those described above. The analogs may be more soluble or more stable than wild type antigen, and may also contain mutations or modifications rendering the antigen more immunologically active. Also useful in the present invention are peptides or proteins which have amino acid sequences homologous with a desired antigen's amino acid sequence, where the homologous antigen induces an immune response to the respective tumor or organism.
A "cancer antigen," as used herein is a molecule or compound (e.g., a protein, peptide, polypeptide, lipid, glycolipid, carbohydrate and/or DNA) associated with a tumor or cancer cell and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Cancer antigens include self-antigens, as well as other antigens that may not be specifically associated with a cancer, but nonetheless induce and/or enhance an immune response to and/or reduce the growth of a tumor or cancer cell when administered to an animal.
An "antigen from a pathogenic and/or infectious organism" as used herein, is an antigen of any organism and includes, but is not limited to, infectious virus, infectious bacteria, infectious parasites including protozoa (such as Plasmodium sp.) and worms and infectious fungi. Typically, for use in the invention the antigen is a protein or antigenic fragment thereof from the organism, or a synthetic compound which is identical to or similar to naturally-occurring antigen which induces an immune response specific for the corresponding organism. Compounds or antigens that are similar to a naturally-occurring organism antigens are well known to those of ordinary skill in the art. A non-limiting example of a compound that is similar to a naturally- occurring organism antigen is a peptide mimic of a polysaccharide antigen.
Specific embodiments of cancer antigens include, e.g., mutated antigens such as the protein products of the Ras p21 protooncogenes, tumor suppressor p53 and HER- 2/neu and BCR-abl oncogenes, as well as CD 4, MUM1, Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, Aldolase A, FRAME, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionic gonadotropin (hCG); self-antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens such as Mart 1 Melan A, gplOO, gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene products such as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and other cancer testis antigens such as NY-ESOl, SSX2 and SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutral glycolipids and glycoproteins such as Lewis (y) and globo-H; and glycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn.
Cancer antigens and their respective tumor cell targets include, e.g., cytokeratins, particularly cytokeratin 8, 18 and 19, as antigens for carcinoma. Epithelial membrane antigen (EM A), human embryonic antigen (HEA-125), human milk fat globules, MBrl, MBr8, Ber-EP4, 17-1 A, C26 and T16 are also known carcinoma antigens. Desmin and muscle-specific actin are antigens of myogenic sarcomas. Placental alkaline phosphatase, beta-human chorionic, gonadotropin, and alpha- fetoprotein are antigens of trophoblastic and germ cell tumors. Prostate specific antigen is an antigen of prostatic carcinomas, carcinoembryonic antigen of colon adenocarcinomas. HMB-45 is an antigen of melanomas. In cervical cancer, useful antigens could be encoded by human papilloma virus. Chromagranin-A and synaptophysin are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid tumor masses having necrotic areas. Antigens derived from pathogens known to predispose to certain cancers may also be advantageously used in the present invention. Pathogens of particular interest for use in the cancer vaccines provided herein include the hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EBV) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), HTLVL (adult T-cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B-cell gastric lymphoma). Other medically relevant microorganisms that may serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g., C. G. A Thomas, Medical Microbiology, Bailliere Tindall, (1983).
Exemplary viral pathogens include, but are not limited to, infectious virus that infect mammals, and more particularly humans. Examples of infectious virus include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-IH, LAV or HTLV-III LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses such as the SARS coronavirus); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parairifluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).
Also, gram negative and gram-positive bacteria may be targeted by the subject compositions and methods in vertebrate animals. Such gram-positive bacteria include, but are not limited to Pasteurella sp, Staphylococci sp., and Streptococcus sp. Gram- negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas sp., and Salmonella sp. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borella burgdorferi, Legionella pneumophilia, Mycobacteria sp. (e.g. M. tuberculosis, M. avium, M: intracellular , M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus infuenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii.
Polypeptides of bacterial pathogens which may find use as sources of antigen in the subject compositions include but are not limited to an iron-regulated outer membrane protein ("IROMP"), an outer membrane protein ("O P"), and an A-protein of Aeromonis sdlmonicida which causes furunculosis, p57 protein of Renibacterium salmoninarum which causes bacterial kidney disease ("BKD"), major surface associated antigen ("msa"), a surface expressed cytotoxin ("mpr"), a surface expressed hemolysin ("ish"), and a flagellar antigen of Yersiniosis; an extracellular protein ("ECP"), an iron-regulated outer membrane protein ("IROMP"), and a structural protein of Pasteur ellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V. ordalii; a flagellar protein, an OMP protein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; and surface antigen of Ichthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and regulatory protein of Rickettsia. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
Examples of pathogens further include, but are not limited to, infectious fungi and parasites that infect mammals, and more particularly humans. Examples of infectious fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
Examples of parasites include intracellular parasites and obligate intracellular parasites. Examples of parasites include but are not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense, Wuchereria bancrofti, Brugia malayi, Brugia timori, Ascaris lumbricoides, Onchocerca volvulus and Schistosoma mansoni.
Other medically relevant microorganisms that serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, (1983). In addition to the treatment of infectious human diseases and human pathogens, the compositions and methods of the present invention are useful for treating infections of nonhuman mammals. Exemplary non-human pathogens include, but are not limited to, mouse mammary tumor virus ("MMTV"), Rous sarcoma virus ("RSV"), avian leukemia virus ("ALV"), avian myeloblastosis virus ("AMV"), murine leukemia virus ("MLV"), feline leukemia virus ("FeLV"), murine sarcoma virus ("MSV"), gibbon ape leukemia virus ("GALV"), spleen necrosis virus ("SNV"), reticuloendotheliosis virus ("RV"), simian sarcoma virus ("SSV"), Mason-Pfizer monkey virus ("MPMV"), simian retrovirus type 1 ("SRV-l"), lentiviruses such as HIV-1, HIV-2, SIV, Visna virus, feline immunodeficiency virus ("FIV"), and equine infectious anemia virus ("EIAV"), T-cell leukemia viruses such as HTLV-1, HTLV-II, simian T-cell leukemia virus ("STLV"), and bovine leukemia virus ("BLV"), and foamy viruses such as human foamy virus ("HFV"), simian foamy virus ("SFV") and bovine foamy virus ("BFV").
In one embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen. In another embodiment, antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells, is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen. In a further embodiment, the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen. Detectable Labels
Polypeptide complexes, conjugates or compounds useful for the invention may be employed in a range of detection systems. For example, the polypeptide complex, conjugate or compound may be used in methods for imaging an internal region of a subject and/or diagnosing the presence or absence of a disease in a subject.
It will be apparent to those skilled in the art that the diagnostic, prognostic and/or monitoring methods of the present invention involve a degree of quantification to determine levels of Clec9A, Clec9A expressing cells, ligand and/or ligand expressing cells present in patient samples. Such quantification is readily provided by the inclusion of appropriate control samples.
Preferably, internal controls are included in the methods of the present invention. A preferred internal control is one or more samples taken from one or more healthy individuals.
Polypeptide complexes, conjugates or compounds useful for the present invention when used diagnosticall may be linked to a diagnostic reagent such as a detectable label to allow easy detection of binding events in vitro or in vivo. Suitable labels include radioisotopes, or non-radioactive labels such as biotin, enzymes, chemiluminescent molecules, fluorophores, dye markers or other imaging reagents for detection and or localisation of target molecules. Alternatively, a second labelled molecule (such as an antibody) which binds the polypeptide complex, actin or fragment thereof, conjugate or compound can be used for detection.
In the case of an enzyme immunoassay, an enzyme can be conjugated to the second molecule, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above.
In another example, fluorescent compounds, such as but not limited to fluorecein and rhodamine amongst others, may be chemically coupled to, for examples, antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
By further way of non-limiting example, the compounds coupled to imaging agents can be used in the detection of Clec9A in histochemical tissue sections. The polypeptie complexes, actin, or compound (for example) may be covalently or non- covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, echogenic, radioactive, or non-radioactive labels such as biotin or avidin.
Labelled Cell Detection and Isolation
Cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells can be detected in a sample by a variety of techniques well known in the art, including cell sorting, especially fluorescence-activated cell sorting (FACS), by using an affinity reagent bound to a substrate (e.g., a plastic surface, as in panning), or by using an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles). Naturally, the procedure used to detect the cells will depend upon how the cells have been labelled.
In one example, any detectable substance which has the appropriate characteristics for the cell sorter may be used (e.g., in the case of a fluorescent dye, a dye which can be excited by the sorter's light source, and an emission spectra which can be detected by the cell sorter's detectors). In flow cytometry, a beam of laser light is projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties. Cells labelled with a suitable dye are excited by the laser beam, and emit light at characteristic wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital signals, allowing for their storage, analysis and display.
Many larger flow cytometers are also "cell sorters", such as fluorescence- activated cell sorters (FACS), and are instruments which have the ability to selectively deposit cells from particular populations into tubes, or other collection vessels. In a particularly preferred embodiment, the cells are isolated using FACS. This procedure is well known in the art and described by, for example, Melamed et al., Flow Cytometry and Sorting, Wiley-Liss, Inc., (1990); Shapiro, Practical Flow Cytometry, 4th Edition, Wiley-Liss, Inc., (2003); and Robinson et al., Handbook of Flow Cytometry Methods, Wiley-Liss, Inc. (1993). In order to sort cells, the instruments electronics interprets the signals collected for each cell as it is interrogated by the laser beam and compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination.
The cells can automatically be deposited in collection vessels as single cells or as a plurality of cells, e.g. using a laser, e.g. an argon laser (488 run) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA). Other examples of suitable FACS machines useful for the methods of the invention include, but are not limited to, MoFlo™ High-speed cell sorter (Dako-Cytomation ltd), FACS Aria™ (Becton Dickinson), FACS Diva (Becton Dickinson), ALTRA Hyper sort (Beckman Coulter) and CyFlow sorting system (Partec GmbH).
For the detection of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells from a sample using solid-phase particles, any particle with the desired properties may be utilized. For example, large particles (e.g., greater than about 90-100 μιη in diameter) may be used to facilitate sedimentation. Preferably, the particles are "magnetic particles" (i.e., particles which can be collected using a magnetic field). Labelled cells are retained in the column (held by the magnetic field), whilst unlabelled cells pass straight through and are eluted at the other end. Magnetic particles are now commonly available from a variety of manufacturers including Dynal Biotech (Oslo, Norway) and Milteni Biotech GmbH (Germany). An example of magnetic cell sorting (MACS) is provided by Al-Mufti et al. (1999).
Laser-capture microdissection can also be used to selectively detect labelled cells on a slide using methods of the invention. Methods of using laser-capture microdissection are known in the art (see, for example, U.S. 20030227611 and Bauer et al., 2002).
Labelled Dendritic Cell or Precursor Thereof Detection and Isolation
As used herein, the terms "enriching" and "enriched" are used in their broadest sense to encompass the isolation of dendritic cells or precursors thereof such that the relative concentration of dendritic cells or precursors thereof to non-dendritic cells or precursors thereof in the treated sample is greater than a comparable untreated sample. Preferably, the enriched dendritic cells and/or precursors thereof are separated from at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% of the non-dendritic cells or precursors thereof in the sample obtained from the original sample. Most preferably, the enriched cell population contains no non-dendritic cells or precursors thereof (namely, pure). The terms "enrich" and variations thereof are used interchangeably herein with the term "isolate" and variations thereof. Furthermore, a population of cells enriched using a method of the invention may only comprise a single dendritic cell or precursor thereof. In addition, the enrichment methods of the invention may be used to isolate a single dendritic cell or precursor thereof.
Dendritic cells or precursors thereof can be enriched from the sample by a variety of techniques well known in the art, including cell sorting, especially fluorescence-activated cell sorting (FACS), by using an affinity reagent bound to a substrate (e.g., a plastic surface, as in panning), or by using an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles). Naturally, the procedure used to enrich the dendritic cells 'and/or precursors thereof will depend upon how the cells have been labelled.
In one example, any detectable substance which has the appropriate characteristics for the cell sorter may be used (e.g., in the case of a fluorescent dye, a dye which can be excited by the sorter's light source, and an emission spectra which can be detected by the cell sorter's detectors). In flow cytometry, a beam of laser light is projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties. Cells labelled with a suitable dye are excited by the laser beam, and emit light at characteristic wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital signals, allowing for their storage, analysis and display.
Many larger flow cytometers are also "cell sorters", such as fluorescence- activated cell sorters (FACS), and are instruments which have the ability to selectively deposit cells from particular populations into tubes, or other collection vessels. In a particularly preferred embodiment, the cells are isolated using FACS. This procedure is well known in the art and described by, for example, Melamed et al., Flow Cytometry and Sorting, Wiley-Liss, Inc., (1990); Shapiro, Practical Flow Cytometry, 4th Edition, Wiley-Liss, Inc., (2003); and Robinson et al., Handbook of Flow Cytometry Methods, Wiley-Liss, Inc. (1993).
In order to sort cells, the instruments electronics interprets the signals collected for each cell as it is interrogated by the laser beam and compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to "the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination.
The cells can automatically be deposited in collection vessels as single cells or as a plurality of cells, e.g. using a laser, e.g. an argon laser (488 nm) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA). Other examples of suitable FACS machines useful for the methods of the invention include, but are not limited to, MoFlo™ High-speed cell sorter (Dako-Cytomation ltd), FACS Aria™ (Becton Dickinson), FACS Diva (Becton Dickinson), ALTRA™ Hyper sort (Beckman Coulter) and CyFlow™ sorting system (Partec GmbH).
The enrichment of dendritic cells and/or or precursors thereof from a sample using solid-phase particles, any particle with the desired properties may be utilized. For example, large particles (e.g., greater than about 90-100 μπι in diameter) may be used to facilitate sedimentation. Preferably, the particles are "magnetic particles" (i.e., particles which can be collected using a magnetic field). Labelled cells are retained in the column (held by the magnetic field), whilst unlabelled cells pass straight through and are eluted at the other end. Magnetic particles are now commonly available from a variety of manufacturers including Dynal Biotech (Oslo, Norway) and Milteni Biotech GmbH (Germany). An example of magnetic cell sorting (MACS) is provided by Al- Mufti et al. (1999).
Laser-capture microdissection can also be used to selectively enrich labelled dendritic cells or precursors thereof on a slide using methods of the invention. Methods of using laser-capture microdissection are known in the art (see, for example, U.S. 20030227611 and Bauer et al., 2002).
Following enrichment, the cells can be used immediately or cultured in vitro to expand dendritic cells and/or precursors thereof numbers using techniques known in the art. Furthermore, dendritic cell precursors can be cultured to produce mature dendritic cells. Identification of Compounds
Methods of screening test compounds are described which can identify a compound, for example, that binds to a polypeptide complex defined herein, that binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l, that binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex defined herein, or which competes with the one or both tryptophan residues of Clec9A for binding to the polypeptide complex, and are thus useful in a method of the invention.
Compounds may be screened by resort to assays and techniques useful in identifying molecules capable of binding to the ligand (such as a polypeptide complex of the invention) and thereby inhibiting its biological activity by blocking Clec9A binding. Such assays include the use of mammalian cell lines (for example, CHO cells or 293T-cells) for phage display system for expressing the polypeptide complex and using a culture of transfected mammalian or E. coli or other microorganism to produce proteins for binding studies of potential binding compounds.
As another example, a method for identifying compounds which specifically bind to the polypeptide complex can include simply the steps of contacting a selected cell expressing the complex with a test compound to permit binding of the test compound to the complex, and determining the amount of test compound, if any, which is bound to the complex. Such a method involves the incubation of the test compound and the complex immobilized on a solid support. Typically, the surface containing the immobilized compound is permitted to come into contact with a solution containing the complex and binding is measured using an appropriate detection system. Suitable detection systems are known in the art, some of which are described herein.
Similar methods to those described above can be used to identify compounds which bind one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of human Clec9A, which binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex of the invention, or which competes with the one or both tryptophan residues of Clec9A for binding to the polypeptide complex.
Three Dimensional Structure of Clec9A
As used herein, the term "crystal" means a structure (such as a three dimensional
(3D) solid aggregate) in which the plane faces intersect at definite angles and in which there is a regular structure (such as internal structure) of the constituent chemical species. The term "crystal" refers in particular to a solid physical crystal form such as an experimentally prepared crystal.
It will be understood that any reference herein to the atomic coordinates or subset of the atomic coordinates shown in Appendix I shall include, unless specified otherwise, atomic coordinates having a root mean square deviation of backbone atoms of not more than 1.5 A, preferably not more than 1 A, when superimposed on the corresponding backbone atoms described.by the atomic coordinates shown in Appendix I. The following defines what is intended by the term "root mean square deviation (RMSD)" between two data sets. For each element in the first data set, its deviation from the corresponding item in the second data set is computed. The squared deviation is the square of that deviation, and the mean squared deviation is the mean of all these squared deviations. The root mean square deviation is the square root of the mean squared deviation. Preferred variants are those in which the RMSD of the x, y and z coordinates for all backbone atoms other than hydrogen is less than 1.5 A (preferably less than 1 A, 0.7 A or less than 0.3 A) compared with the coordinates given in Appendix I. It will be readily appreciated by those skilled in the art that a 3D rigid body rotation and/or translation of the atomic coordinates does not alter the structure of the molecule concerned.
As used herein, a "subset" of the atomic coordinates provided in Appendix I refers to a group of the co-ordinates which can be used in a method of the invention such as a computer-assisted method of identifying a compound that binds Clec9A, or a computer-assisted method for identifying a compound which binds a polypeptide complex as defined herein. In an example, the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
The three-dimensional structure of Clec9A defined, herein can be used to identify antagonists or agonists through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., 1997). Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of a candidate compound to the polypeptide. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential agonist or antagonist will be since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential agonist or antagonist the more likely that it will not interfere with other proteins.
Initially a potential compound could be obtained, for example, using methods of the invention such as by screening a random peptide library produced by a recombinant bacteriophage or a chemical library. A compound selected in this manner could then be systematically modified by computer modeling programs until one or more promising potential compounds are identified.
Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful agonist or antagonist. Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized. Thus through the use of the three-dimensional structure and computer modeling, a large number of these compounds can be rapidly screened on the computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.
For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. Exemplary forcefields that are known in the art and can be used in such methods include, but are not limited to, the Constant Valence Force Field (CVFF), the AMBER force field and the CHARM force field. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
Further examples of molecular modeling systems are the CHARMm and QUANTA programs (Polygen Corporation, Waltham, MA). CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behaviour of molecules with each other. c Diseases Associated with Cells with a Disrupted Cell Membrane. Cells Infected with a Pathogen. Dying Cells or Dead Cells
Examples of the diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not necessarily limited to, the following:
1) Diseases in which apoptosis is induced in response to a signal generated by a cell of an immune system responsible for biophylaxis, for example, graft versus host disease (GVHD) and autoimmune diseases (systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, Sjogren's syndrome, multiple sclerosis, insulin dependent diabetes mellitus, ulcerative colitis).
2) Diseases in which cell death is induced by viral infection or apoptosis is induced by reaction of a cell of an immune system with a cell infected by a virus or parasitic infection, for example, virus associated hemophagocytic syndrome (VAHS) and other viral infections (HCV, HIV, influenza virus).
3) Diseases in which cell death is induced by an abnormal apoptosis signal, for example, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease).
4) Leukemia, for example, acute lymphatic leukemia.
5) Diseases in which apoptosis is artificially induced by, for example, radiation exposure or medication (anticancer drug etc.)
6) Systemic inflammatory reaction syndrome (SIRS), diseases in which organ disorder occurs because the immune system is nonspecifically activated in response to invasion to a living body and thus control of cytokine production becomes impossible (HPS, severe pancreatitis).
7) Diseases where there is a lack of cell death such as cancer.
8) Injury, particularly post-injury recovery.
Cell death progressing in a living body can be detennined using the present invention, and hence progress of these diseases can be monitored. In particular, the invention is useful for GVHD, human immunodeficiency virus (HIV), hemophagocytic syndrome (HPS), especially virus associated hemophagocytic syndrome (VAHS), acute lymphatic leukemia, influenzal encephalitis, encephalopathy, and malaria.
Polypeptides
The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms "proteins" and "polypeptides" as used herein may also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 200 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 200 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
As used herein a "biologically active fragment" is a portion of a polypeptide as described herein which maintains a defined activity of the full-length polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, biologically active fragments are at least 100 amino acids in length.
As used herein, a "soluble fragment" refers to a portion of Clec9A which lacks the membrane spanning region. In a preferred embodiment, the soluble fragment does not comprise at least about 40, at least about 50, at least about 55, or at least about 100, N-terminal residues of any one of SEQ ID NOs 1 to 8. In a further preferred embodiment, the soluble fragment comprises the C-type lectin-like domain of a polypeptide which comprises:
i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8; or ii) an amino acid sequence which is at least 50% identical to any one or more of
SEQ ID NOs 1 to 8. In a further embodiment, the soluble fragment comprises:
i) an amino acid sequence as provided in any one of SEQ ID NOs 17 to 20; or ii) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 17 to 20,
wherein the soluble fragment does not comprise at least the about 40 N-terminal residues of any one of SEQ ID NOs 1 to 8.
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments.
Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Amino acid sequence mutants of a polypeptide described herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, a polynucleotide described herein can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques may include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides defined herein are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they are able to confer the desired phenotype.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical, and/or those in which particular residues obtained from related proteins are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1.
Table 1 - Exem lar substitutions.
Figure imgf000083_0001
Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into a polypeptides described herein. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2- aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β- methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogues in general.
Also included within the scope of the invention are the use of polypeptides which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide.
Polypeptides described herein can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, tissue culture flasks, shake flasks, test tubes, microtiter dishes, and Petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Polynucleotides and Recombinant Vectors
The term "polynucleotide" is used interchangeably herein with the term "nucleic acid". Usually, monomers of a polynucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate and phosphoramidate.
Recombinant vectors useful for the invention can include at least one polynucleotide molecule described herein, and/or a polynucleotide encoding a polypeptide as described herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Such a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.
One type of recombinant vector comprises the polynucleotide(s) operably linked to an expression vector. The phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Vectors can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
"Operably linked" as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cw-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance .
In particular, expression vectors contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or animal cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells.
Host cells
Also useful for certain embodiments of the invention is a recombinant cell comprising a host cell transformed with one or more recombinant molecules described herein or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides described herein or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as described herein. Host cells of the present invention can be any cell capable of producing at least one protein defined herein, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, animal and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, CHO cells, 293 cells, EL4 cells, MDCK cells, CRFK cells,. CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are. translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
Pharmaceutical Compositions. Dosages, and Routes of Administration
Compositions comprising the polypeptide complex, actin or fragment thereof, conjugate or compound together with an acceptable carrier or diluent are useful in the methods of the present invention. Therapeutic compositions can be prepared by mixing the desired component having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions. Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions such as sodium; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.
Therapeutic compositions to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The composition may be stored in lyophilized form or in solution if administered systemically. If in lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use. An example of a liquid formulation is a sterile, clear, colorless unpreserved solution filled in a single-dose vial for subcutaneous injection.
Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The compositions are preferably administered subcutaneously, intramuscularly or parenterally, for example, as intravenous injections or infusions or administered into a body cavity.
The polypeptide complex, actin or fragment thereof, conjugate or compound may be administered in an amount of about 0.001 to 2000 mg kg body weight per dose, and more preferably about O.pi to 500 mg/kg body weight per dose. Repeated doses may be administered as prescribed by the treating physician.
Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. The dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease or immune response required, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference, 56th ed. (2002). Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.,
In another example, a polypeptide complex, actin or fragment thereof, conjugate or compound useful for the methods of the invention comprises an antigen, such as a cancer antigen or an antigen of a pathogen or infectious organism, and can be delivered by intramuscular, subcutaneous or intravenous injection, or orally, as a vaccine to enhance humoral and/or T-cell mediated immune responses. In another example, the antigen is a self-antigen or allergenic antigen which can used to diminish immune responses similar to that described for 33D1 and DEC-205 (Bonifaz et al., 2002; Finkelman et al., 1996).
In another example of the present invention, a radiolabeled form of the polypeptide complex, actin or fragment thereof, conjugate or compound is delivered by intravenous injection as a therapeutic agent to target cells that express Clec9A. Previous examples of radiolabeled antibodies and the methods for their administration to patients as therapeutics are known to those skilled in the art. Examples include Iodine131 labeled Lym-1, against the β subunit of HLA-DR and the anti-CD20 Indium111 and Yttrium90 labeled Ibritumomab Tiuxetan (IDEC-Y2B8, ZEVALIN®) and Iodine 1 131 Tositumomab (BEXXAR®).
In one embodiment, the composition does not comprise an adjuvant. In another embodiment, the composition does comprise an adjuvant. Examples of adjuvants include, but are not limited to, aluminium hydroxide, aluminium phosphate, aluminium potassium sulphate (alum), muramyl dipeptide, bacterial endotoxin, lipid X, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.).
In an embodiment, the composition comprises liposomes or membrane vesicles. Examples of such liposomes are described in US 2007/0026057, Leserman (2004) and van Broekhoven et al. (2004). In these instances the polypeptide complex, conjugate or compound can be used to target the liposome to enhance the delivery of an agent of interest. As outlined in US 2007/0026047, processes for the preparation of membrane vesicles for use in the invention are described in WO 00/64471.
Compositions for detection of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, modulating an immune response, and/or antigen recognition, processing and/or presentation, are conventionally administered parenterally, by injection, for example, subcutaneously, intramuscularly or intravenously. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer. Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
In any treatment regimen, the therapeutic composition may be administered to a patient either singly or in a cocktail containing other therapeutic agents, compositions, or the like.
In an embodiment, the immune response is modulated by using a DNA vaccine encoding a polypeptide complex, actin or fragment thereof, conjugate or compound of the invention conjugated to an antigen. DNA vaccination involves the direct in vivo introduction of DNA encoding the antigen into tissues of a subject for expression of the antigen by the cells of the subject's tissue. Such vaccines are termed herein "DNA vaccines" or "nucleic acid-based vaccines". DNA vaccines are described in US 5,939,400, US 6,110,898, WO 95/20660, WO 93/19183, Demangel et al. (2005) and Nchinda et al. (2008).
To date, most DNA vaccines in mammalian systems have relied upon viral promoters derived from cytomegalovirus (CMV). These have had good efficiency in both muscle and skin inoculation in a number of mammalian species. A factor known to affect the immune response elicited by DNA immunization is the method of DNA delivery, for example, parenteral routes can yield low rates of gene transfer and produce considerable variability of gene expression. High-velocity inoculation of plasmids, using a gene-gun, enhanced the immune responses of mice, presumably because of a greater efficiency of DNA transfection and more effective antigen presentation by dendritic cells. Vectors containing the nucleic acid-based vaccine of the invention may also be introduced into the desired host by other methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter.
Transgenic plants producing an antigenic polypeptide can be constructed using procedures well known in the art. A number of plant-derived edible vaccines are currently being developed for both animal and human pathogens. Immune responses have also resulted from oral immunization with transgenic plants producing virus-like particles (VLPs), or chimeric plant viruses displaying antigenic epitopes. It has been suggested that the particulate form of these VLPs or chimeric viruses may result in greater stability of the antigen in the stomach, effectively increasing the amount of antigen available for uptake in the gut.
EXAMPLES
Example 1 - Materials and Methods
Mice
Female C57BL/6J WEHI mice, 8-12 weeks of age were bred under specific pathogen free conditions at The Walter and Eliza Hall Institute (WEHI). Animals were handled according to the guidelines of the National Health and Medical Research Council of Australia. Experimental procedures were approved by the Animal Ethics Committee, WEHI.
Recombinant Expression of Soluble Clec9A
Three versions of soluble Clec9A were generated, a full Clec9A ectodomain
(Clec9A-ecto; stalk and CTLD), a Clec9A CTLD only (Clec9A-CTLD) and a Clec9A stalk only (Clec9A-stalk). cDNA containing the required ectodomain region was amplified from the original Clec9A cDNA using Advantage high fidelity 2 polymerase (Clontech, MountainView, CA) or HotStar HiFidelity polymerase (Qiagen, Victoria, Australia) and the listed primers (Table 2).
The amplified cDNA was subcloned into a pEF-Bos vector modified to contain the biotinylation consensus sequence (a peptide consensus sequence NSGLHHILDAQKMVWNHR (SEQ ID NO: 106) recognised specifically by E coli bioti holoenzyme synthetase BirA) and the FLAG epitope. The resulting fusion constructs thus included (in order of N-terminus): the IL-3 signal sequence (to ensure secretion), the biotinylation consensus peptide sequence, a FLAG-tag, and Clec9A cDNA fragment. As controls, expression constructs encoding the ectodomains of mouse Clecl2A and Cire were similarly generated. Recombinant proteins were expressed in mammalian 293T or FreeStyle 293F cells by transient transfection, followed by culture in protein-free/serum-free media: X- Vivo- 10 (BioWhittaker, Walkersville, MD) or FreeStyle Expression Media (Invitrogen, Victoria, Australia) respectively. Media containing the secreted recombinant protein was assayed for the presence of soluble mClec9A by reactivity with anti-mClec9A mAb (24/04- 10B4), concentrated 100-fold using a 10,000 molecular weight cutoff centrifugal device (Millipore, Billerica, MA) and either used directly or en2ymatically biotinylated using BirA enzyme (Avidity). Where required, Clec9A soluble proteins were purified by affinity chromomatography using an anti-FLAG M2 agarose resin (Sigma, Castle Hill, Australia) and elution with 100 g/ml FLAG peptide (Auspep, Victoria, Australia), and further purified by size-exclusion chromatography using a pre-packed Superdex 200 column (GE Healthcare, Rydalmere, Australia). Table 2 - Construct s nthesis.
Figure imgf000093_0001
Figure imgf000094_0001
Recombinant Expression of Mutated hCLEC9A
Expression constructs encoding hCLEC9A-ecto or hCLEC9A-CTLD with specific mutations, and fused to either the FLAG epitope or the FLAG epitope and a biotinylation consensus sequence, were synthesised (GeneArt, Germany) and sublconed into pcDNA3.1+ (Invitrogen). The resulting fusion constructs for hCLEC9A-CTLD (S225D) thus included (in order of N-terminus): the IL-3 signal sequence (to ensure secretion), a FLAG-tag, and hCLEC9A-CTLD (S225D). The resulting fusion constructs for hCLEC9A-ecto (W131A; W227A) included the IL-3 signal sequence, the biotinylation consensus peptide sequence, a FLAG-tag, and hCLEC9A-ecto (WT31A; W227A). Recombinant proteins were expressed in FreeStyle 293F cells and hCLEC9A soluble proteins purified from the secreted media by affinity chromomatography using an anti-FLAG M2 agarose resin, and further purified by size- exclusion chromatography using a pre-packed Superdex 200 column.
Circular Dichroism was performed to compare the structural profiles of the
(glycosylation-null) (original) hCLEC9A-CTLD (S225D) and the mutated hCLEC9A- CTLD (S225D; W131A; W227A). The Far-UV CD spectra, averaged over five accumulation scans from 260 to 190 nm, of the wild-type and mutated proteins were recorded at 20°C in 10 mM phosphate buffer, pH 7.0, containing 100 mM NaCl in a 0.1 cm path length quartz cuvette on a Jasco J815 CD spectropolarimeter equipped with a thermostated cell holder and interfaced with a Peltier unit. Mutation of hCLEC9A (W131A; W227A) did not result in any significant changes in the secondary structure of the protein. Analysis of the spectra for contributions of particular secondary structural elements, performed using an online CD analyzing program (Whitmore and Wallace, 2004 and 2008), also resulted in similar secondary structures with 7% alpha- Helix, 25% beta-Sheet, 19% Turns, and 48% unordered estimated for both proteins.
Crystallization. Data Collection and Structure Determination
Crystals of purified, glycosylation null, human CLEC9A CTLD were grown in hanging drops at 4°C (reservoir solution; 30 % PEG8000, 0.2 M MgCl2, 0.1 M Tris pH 8.0, drop supplemented with a 1 :50 molar ratio of Enterokinase). Crystals were equilibrated in cryo-protectant consisting of reservoir solution supplemented with 15% (v/v) ethylene glycol and then flash-frozen in liquid N2. X-ray data were collected at the Australian Synchrotron on beamline PX2 at 100 K. The data were processed using XDS (Kabsch, 2010). The structure was solved by molecular replacement with PHASER (McCoy, Acta Crys D 2005) using a Chainsaw (Stein, 2008) modified model of CD69 as the search model (Protein Data Bank (PDB) 1FM5 (Natarajan et al., 2000). Several rounds of building were performed in COOT (Emsley and Cowtan, 2004) and refinement in PHENIX (Adams et al., 2010) incorporating simulated annealing, TLS, individual site refinement and individual ADP refinement. The final refinement statistics for the model are given in Table 8.
Clec Ectodomain Binding to Platelets and Erythrocyte Ghosts
Platelets and red blood cells were purified from mouse blood. Binding studies were performed by incubating cells with purified or biotinylated Clec ectodomains and binding detected using fluorescently conjugated secondary reagents and flow cytometric analysis.
Isolation of Spectrin Proteins
Isolation of cellular spectrin complexes was performed essentially as in Ungewickell and Gratzer (1978) and Gratzer (1982), and further purified by SEC. In brief, erythrocytes were lysed for 30 min at 4°C in a hypotonic lysis buffer (5mM sodium phosphate, pH7.6, lmM PMSF, 1 x EDTA-free protease cocktail (Roche)) and erythrocyte membranes washed repeatedly using the lysis buffer. Spectrin was extracted by resuspension of erythrocyte ghosts in a spectrin extraction buffer (0.3mM sodium phosphate, 0.1 mM EDTA, 0.1 mM PMSF) and overnight dialysis at 4°C against spectrin extraction buffer. Membrane fragments were removed by ultracentrifugation (90000g, 30 min, 4°C) and the supernatant containing spectrin harvested for further analysis. Isolation of non-erythroid spectrin from frozen pellets of human 293F cells (1 x 108 cells) was performed as for erythrocytic spectrin, with the addition of 50U/ml benzonase (Novagen) in the lysis buffer. Spectrin samples were further purified by size exclusion chromatography using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min in PBS containing 10% glycerol, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF. Column fractions of 0.4 mL were collected for analysis.
Reassociation of cellular spectrins with actin was performed by collecting fractions of dissociated spectrin following SEC, and concentrating them in the presence of ΙΟμ^ιχύ of either bovine muscle actin or a control protein BSA, in PBS containing 10% glycerol, 0.1 mM PMSF, 1 mM EDTA, 1 mM DTT. The spectrin-actin BSA samples were then incubated at 30°C for 2.5 h, then further purified by SEC using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min as above, and 0.4 ml fractions collected for analysis. Generation of Recombinant Domains of Spectrin and Other Cytoskeletal Proteins
Recombinant GST-tagged fragments of erythrocytic spectrin (spectrin βΐ) were expressed as previously described (Pei et al., 2005). The Actin binding domains (ABD) of human Spectrin beta II (spectrin beta chain, brain 1 isoform 1; beta-fodrin, NM_003128.2) and of human alpha-actinin I (alpha-actinin-1 isoform a; alpha-actinin cytoskeletal isoform, NM_001130004.1) were expressed as glutathione S-transferase (GST) fusion proteins using E. coli BL21 (DE3). In brief, cDNA containing the ABD regions were amplified from 293F cell cDNA using HotStar HiFidelity polymerase (Qiagen) using the primers listed below (Table 3), and subcloned into a modified pGex2T vector (GE Healthcare).
Protein expression was induced using 0.1 mM isopropyl-β- thiogalactopyranoside (IPTG) for 5 hours at 19°C, and IPTG-induced cells lysed with lysis buffer (phosphate-buffered saline containing 0.2 mg/mL lysoz me, 1% Triton-X 100, 30 μg/mL DNase Γ, 1 mM PMSF). GST-tagged proteins were purified by affinity chromatography using Glutathione-Sepharose 4B resin (GE Healthcare) and elution with 50mM Tris-HCl, pH8.0, 150mM NaCl containing 50mM reduced glutathione (Sigma), and further purified by size-exclusion chromatography using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min in 50mM Tris-HCl, pH 8.0 containing 150 mM NaCl.
Reassociation studies were performed by incubating GST-tagged spectrin and actinin proteins in the presence or absence of bovine muscle actin (Sigma) or platelet actin (>99% purity, Cytoskeleton) in PBS, for 2.5 h (30°C) at a concentration of 0.2mg/ml per protein. Proteins were diluted to ΙΟ ^ηιΙ in PBS for coating onto ELISA plates.
Table 3 - Construct synthesis for recombinant domains of spectrin and other cytoskeletal proteins.
Figure imgf000098_0001
ELISA and Statistical Analysis
ELISA plates (Costar) were coated overnight at 4°C with either commercially available erythrocytic spectrin (Sigma), bovine muscle actin (Sigma) or platelet actin (>99% pure, Cytoskeleton) at ΙΟμ^ιηΙ, or with cellular spectrins (K^g/ml) or complexes containing GST-tagged spectrin fragments, (l(^g spectrin fragment/ml) or with SEC fractions of spectrin (50μ1). Bovine muscle actin and platelet actin appeared to have comparable purity on SDS-PAGE analysis. Unbound proteins were then washed away (PBS-0.05%Tween 20). ELISA plates were blocked (1% BSA in PBS), then incubated with FLAG-tagged Clec9A ectodomain fragments or controls (mClecl2A-ecto , mCire DC-Sign-ecto). Binding was detected by incubation with an anti-FLAG antibody M2-conjugated to HRP (Sigma-Aldrich), and visualized by the addition of the HRP substrate ABTS, followed by measurement of absorbance at 405- 490 nm (Vmax Kinetic Microplate Reader, Molecular Devices).
Statistical analysis of ELISA data was performed using an unpaired two-tailed /- test on log-transformed data. The significance of difference is indicated as *** pO.0001. Analysis was performed in Prism (GraphPad Software).
Thermofluor Assays
Thermofluor assays were performed to determine protein stability (Phillips and de la Pena, 2011). hCLEC9A-CTLD (S225D) was incubated in Tris buffered saline in the presence or absence of 6mM EDTA, 1.24mM MgS04 or 2.47mM CaCl2 for lh at 4°C. Thermofluor assays were performed using 18.4μΜ hCLEC9A-CTLD and Sypro Orange (1 :1250 dilution, Invitrogen) in a total volume of 25μί. Thermal denaturation curves were measured on a CI 000 thermocycler equipped with a CFX384 Real-Time System fluorescence reader (BIORAD). A temperature gradient of l°C/min from 25°C to 95 °C was applied to the samples, and fluorescence measured every degree step using the FRET channel. The melting temperature (Tm) of each sample was calculated by determining the inflexion point of each thermal denaturation curves (BIORAD). Tm differences were calculated by subtracting the Tm of the incubated CLEC9A from the Tm of CLEC9A in the Tris buffered saline condition
Cellular Binding Assays using Soluble Clec9A
Binding assays were performed in binding buffer (PBS containing 0.2%BSA and 0.02% sodium azide), on ice. Cells were washed 3 times with PBS to remove serum proteins, then resuspended in binding buffer. Cells were incubated with either (1) biotinylated soluble Clec9A and controls, and detected with Streptavidin (SA)-PE, or (2) soluble FLAG-tagged Clec9A and detected either with FITC conjugated rat anti- FLAG mAb 9H1, or with biotinylated rat anti-FLAG mAb 9H1 and SA-PE. Live cells were gated on forward and side scatter, or by PI exclusion, whereas dead cells were gated on forward and side scatter, or by PI inclusion. Analysis of soluble Clec9 A binding was performed by flow cytometry using a FACScan (Becton Dickinson). The specificity of the binding was demonstrated by comparison to binding to other soluble FLAG-tagged C-type lectins, mouse Cire/mDCSign (Caminschi et al., 2001) and Clecl2A (Pyz et al., 2008). Mouse Platelets
Venous blood was obtained from C57B16 mice by cardiac puncture into 0.1 volume of Aster Jandl citrate-based anticoagulant (Aster and Jandl, 1964) (85 mM sodium citrate, 69 mM citric acid, 20 mg/ml glucose, pH 4.6). Platelet rich plasma was obtained by centrifugation of the murine blood at 125 x g for 8 min, followed by centrifugation of the supernatant buffy coat at 125 x g for 8 min. Platelets were washed by two sequential centrifugations at 860 x g for 5 min in 140 mM NaCl, 5 mM C1, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM sucrose, pH 6.0 the platelet pellet resuspended in 10 mM HEPES, 140 Mm NaCl, 3 mM KC1, 0.5 mM MgCl2, 10 mM glucose and 0.5 mM NaHCC>3, pH 7.4. Washed platelets were treated with 0.5 μΜ ABT-737 for 90 min at 37°C. Control or ABT-737 treated platelets were incubated with 5 μg/ml of Flag-tagged recombinant proteins mClec9A or hClec9A for 30 min at room temperature. Anti-Flag-FITC secondary antibodies were added to the platelets and incubated for 30 min at room temperature. Samples were diluted out in PBS containing 0.2% BSA and analyzed on a FACSCalibur flow cytometer. ABT-737 treated platelets were confirmed to be AnnexinV-APC positive, and resting control platelets AnnexinV-APC negative.
Confocal Microscopy
Mouse fibroblasts (NIH 3T3) cells were cultured on glass coverslips, fixed, permeabilised using PBS containing 0.3% Triton X-100 and incubated either with biotinylated mClec9A-ecto, or biotinylated Cire-ecto as a background control and binding detected using SA-Alexa594. Fibroblasts were counterstained with DAPI, and analysed by confocal microscopy using a Leica SP2 (Wilson et al., 2003). Mass Spectrometry Analysis
Protein bands were excised from Simply blue (Invitrogen) stained gels and subjected to manual in-gel digestion. The gel bands were reduced with lOmM DTT (Calbiochem) for 30 min, alkykated for 30 min with 50mM iodoacetic acid (Fluka) and digested with 375ng trypsin (Worthington) for 16hrs at 37°C. The extracted peptide solutions (0.1% formic acid) were then concentrated to approximately ΙΟμΙ by centrifugal lyophilisation using a SpeedVac AES 1010 (Savant). Digests were then subjected to MS/MS analysis on the LTQ-Orbitrap (Thermo Fischer Scientific) mass spectrometer.
Extracted peptides were injected and fractionated by nanoflow reversed-phase liquid chromatography on a nano LC system (1200 series, Agilent, USA) using a nano flow reversed-phase-HPLC (Model 1200, Agilent). Fractionation was performed using a nano-Acquity (CI 8) 150 mm x 0.15 mm I.D. RP-UPLC column (Waters) developed with a linear 60 min gradient from 0-100% Buffer B (0.1% (v/v) aqueous formic acid / 60% (v/v) acetonitrile) with a flow rate of 0.8 μΙ7ηιίη at 45°C, where Buffer A was 0.1% (v/v) aqueous formic acid. The capillary HPLC was coupled on-line to the LTQ- Orbitrap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific). Positive ion mode was used for data-dependent acquisition. Survey MS scans were acquired with the resolution set to 30,000. Each scan was recalibrated in real time by co-injecting an internal standard from ambient air into the C-trap (Olsen et al., 2005). The five most intense ions per cycle were fragmented and analysed in the linear trap. Target ions already selected for MS/MS were dynamically excluded for 180s.
Acquired MS/MS spectra for each instrument RAW file were extracted to DTA files (peak lists) using extract-msn as part of Bio Works 3.3.1 (Thermo Fisher Scientific). The parameters used to create the peak lists were as follows: minimum mass 700; maximum mass 7000; no merging of scans; 10 peaks minimum and Total Ion Current (TIC) of 100. The PeakListExto actor program (JPSF in-house software) was used for generating Mascot generic files (MGF format) from the DTA files. Peak lists were searched against the LudwigNR protein sequence database (10753 sequence entries) (version Q409ml - www.ludwig.edu.au/archiveAudwigNR/ludwigNR.pdf) using the Mascot search algorithm (v2.2.04, Matrix Science, U.K.) (Perkins et al., 1 99). The search parameters were as follows: carboxymethylation of cysteine as a . fixed modification (+58 Da) as well as variable modifications consisting of N¾- terminal acetylation (+42 Da) and oxidation of methionine (+16 Da), and the allowance for up to three missed tryptic cleavage sites (trypsin P). Precursor and fragment ion mass tolerance values were +/- 20ppm and 0.8Da respectively.
Expression, purification and ELISA of RNF41 proteins
In order to express GST-fusion recombinant proteins, bacterial BL21 E. coli were electroporated with plasmid DNA and incubated at 37°C overnight on LB Agar plates containing Ampicillin (50μg/ml). Bacterial culture broth containing ampicillin (100μg ml) was inoculated with a single colony and incubated with shaking overnight at 37°C. Overnight bacterial cultures were diluted into Superbroth containing ampicillin (100μg/ml), at 1:20 dilution and incubated in a shaking incubator at 30°C degrees until the cell density reached an ODioo of 0.8. Isopropyl-P-D- thiogalactopyranoside (ΙΟΟμΜ) was added to cultures to induce expression of proteins and cultures incubated at 19°C for 5 hours. Cells were harvested by centrifugation and stored at -80°C.
Bacterial cell pellets were lysed with lysozyme lysis buffer (phosphate buffered saline (PBS) containing 0.2mg/ml lysozyme, 30μg/ml DNase I, 1 mM PMSF) and incubated on ice for one hour. Total lysates were subjected to sonication using the Bandelin sonoplus (50% duty cycle, 10 second pulse, 22 second cycle x5) and subsequently clarified by centrifugation. The supernatant was incubated with 50% Giutathione-Sepharose resin slurry for 1 hour at 4°C on a rotating wheel and resin recovered in column (Bio-Rad Laboratories). Resin was washed extensively with PBS, and proteins eluted with 20mM reduced glutathione pH 7.0 in PBS.
ELISA plates (Costar) were coated with GST-RNF41 fusion proteins (^g/ml) in tris buffered saline or phosphate buffered saline overnight at 4°C. Plates were washed with wash buffer (PBS/ 0.05% Tween20) to remove unbound proteins and then blocked for 1 hour in 5% skim milk in PBS at room temperature. Dilutions of Clec proteins in 5% skim milk in PBS were added and the plates incubated at RT for 2 hours. Plates were washed with wash buffer to remove unbound Clec proteins, and plates incubated with anti-FLAG 2 antibody conjugated to Horseradish Peroxidase (Sigma) for 2 hours at RT. Plates were washed again to remove unbound M2-HRP and bound proteins visualized using the HRP substrate 2,2'-Azino-bis(3- ethylbenzothiazoline-6-sufonic acid) diammonium salt (ABTS) in 0.1 M citric acid (pH 4.0). Absorbance was measured at 405-490nm (Vmax Kinetic Microplate Reader, Molecular Devices). Data was analysed using GraphPad Prism.
ELISA to measure enhancement of Clec9A binding to RNF41 by ABD-actin complexes were performed as follows. GST-tagged RNF41 C-terminal domain and control GST were coated on ELISA plates at ΙΟμ^πιΙ overnight at 4°C. Unbound proteins were washed away and plates blocked with 1% BSA in PBS. ABD-actin complexes of platelet actin with eythrocytic spectrin (spectrin βΐ N), non-erthyroctic spectrin (spectrin βΙΙ N) and a-actinin-1 (a-actinin-1 N) were pre-associated at 50μg ml for 2.5 hours at 30°C, before incubation with Clec proteins for 1 hour at room temperature. Clec-ABD-actin complexes were incubated on the ELISA plates for 2 hours at room temperature. Clec binding was detected using anti-FLAG M2-HRP and visualised using ABTS as described above. Example 2 - Mouse and Human Clec9A Bind to Damaged Cells
As reagents to identify Clec9A ligands, the inventors generated recombinant tagged soluble forms of the ectodomains of mouse Clec9A (mClec9A) and human CLEC9A (hCLEC9A). These consisted of a FLAG-tag, a biotinylation consensus sequence and either the full ectodomain (Clec9A-ecto; stalk and CTLD), or the Clec9A-CTLD, or the Clec9A-stalk (Figure 1). To determine if Clec9A recognised normal self-components, a range of mouse and human cells were stained with the soluble Clec9A ectodomains. No binding was observed to the surface of viable cells, but binding was observed to dead cells identified by propidium iodide (PI) staining, as previously observed (Sancho et al., 2009).
The inventors then investigated various stages of cell death, by following thymocytes undergoing apoptosis induced by γ-irradiation or mouse embryonic fibroblasts (MEFs) undergoing apoptosis induced by BH3-only ligands (van Delft et al., 2006). Cells were stained with Annexin V, an early marker of apoptosis, and with PI, a late marker, which stains nuclei once the cell membrane is damaged. mClec9A- ecto strongly bound to late stage apoptotic/secondary necrotic cells (Annexin V+ PI ) but not to early stage apoptotic cells where cell membranes are still intact (Annexin V+ ΡΓ) (Figure 2A). Soluble ectodomains of both mClec9A and hCLEC9A strongly bound to the late stage apoptotic MEFs, but not to live cells (Figure 2B). In all cases the level of binding to dead cells was much higher than any "non-specific" binding seen with soluble forms of the other C-type lectins tested, namely mouse Cire (Figures 2A to 2J) and Clecl2A (data not shown).
The possibility that membrane rupture was sufficient to reveal the ligand was tested by staining with Clec9A immediately after freezing and thawing cells. This served as a model of primary necrosis. mClec9A and hCLEC9A ectodomains bound strongly to freeze/thawed mouse 3T3 cells (Figure 2C) or human 293 cells (Figure 2D), as well as to fixed and permeabilised cells (data not shown). Example 3 - Some Characteristics of the Clec9A Ligand
The binding of mClec9A-ecto or hCLEC9A-ecto to dead cells occurred in the presence of EDTA (Figure 2D). Thus, ligand recognition was not dependent on readily chelated divalent metal ions at physiological temperatures, as would be expected of a classical C-type lectin binding to a carbohydrate ligand. Treatment of late stage apoptotic cells with proteases (trypsin, protease K), but not with nucleases, reduced the Clec9A binding in a dose dependent manner, suggesting the ligand was a protein (Figure 2E). However, pre-treatment of viable cells with trypsin prior to freeze thawing did not eliminate Clec9A binding after freeze thawing (data not shown), emphasising that the ligand was intracellular and only revealed on membrane disruption.
Example 4 - Cell and Species Distribution of the Clec9A Ligand
Both mClec9A and hCLEC9A ectodomains bound to all dead mouse or human nucleated cells tested, including cultured cell lines and primary cells (Figure 2). They also bound to freeze/thawed Chinese hamster (CHO) and African green monkey (Vero) cells (data not shown), and to freeze/thawed insect (SF21) cells, but not to freeze/thawed bacteria or yeast (Figure 2F). Thus, recognition of the dead cell ligand was conserved across a wide evolutionary range. A clue to the nature of the ligand was the finding that mClec9A-ecto and hCLEC9A-ecto also bound to disrupted non- nucleated cells, including apoptotic mouse platelets (Figure 2G) and mouse and human erythrocyte (RBC) ghosts prepared by saponin treatment (Figures 2H and 21). Example 5 - Binding of Clec9A to Dead Cells is via the CTLD
The requirements for Clec9A binding were investigated by comparing the binding of the Clec9A CTLD or the Clec9A stalk to the binding of the full ectodomain (stalk plus CTLD). At saturating concentrations, both mClec9A-CTLD and hCLEC9A-CTLD showed similar levels of binding to both dead cells and erythrocyte saponin as the full ectodomains (Figure 21). In contrast, the Clec9A stalk region showed no detectable binding (Figure 21).
Example 6 - Location of Clec9A Ligand within Cells
Examination of mClec9A-ecto binding to permeabilised 3T3 cells by immunofluorescence microscopy revealed staining over the cytoplasmic area, in a pattern indicative of membrane or cytosolic localisation (Figure 2J), Colocalisation studies with the cytoskeletal protein actin were performed using an anti-actin antibody which labels all cellular actin, and using phalloidin which labels filaments of actin (F- actin). This revealed that Clec9A binding localised with actin filaments, but not with all of the cellular actin (Figure 2K and 2L).
Example 7 - Clec9A Binds to Cytoskeletal Components including Spectrin
Several preliminary approaches suggested the intracellular ligand for Clec9A could be a cytoskeletal component. Mass spectrometry analysis of Clec9A complexes isolated by co-immunoprecipitation from the lysates of mouse thymocytes and of a suspension-adapted subline of human embryonic kidney cells (Freestyle 293F) revealed a complex of cytoskeletal proteins that migrated at 250-300 kDa including spectrin, filamin, myosin (Table 4) and smaller proteins including actin and actin-related proteins. However the extraction of these poorly soluble complexes was inefficient and identification of a single protein ligand was challenging.
Table 4 - Proteomic analysis of Clec9A interacting proteins isolated from mouse thymocytes and human 293 F cells. Clec9A interacting proteins were isolated by incubation of FLAG-tagged Clec9A-ecto with lysates of mouse thymocytes or a suspension adapted subline of human 293 cells (Freestyle human 293 F) and affinity purification using anti-FLAG resin. Protein complexes were analysed by SDS-PAGE under reducing conditions, and a protein band of 220-300 kDa was excised and subjected to mass spectrometry analysis. The five most abundant proteins are described below.
Protein name Accession Mol wt (kDa) Unique
number peptides
Mouse
Thymocytes
Cation-independent mannose-6- Q07113 281 kDa 26 phosphate receptor precursor
Spectrin alpha chain, brain PI 6546 285 kDa 12
Spectrin beta chain, brain 1 062261 275 10
Myosin-XVTJla Q9JMH9 234 kDa 8
Filamin, alpha A2AM91 281 7
Human 293F
cells
Spectrin beta chain, brain 1 Q01082 275 25
Translational activator GCN1 Q92616 293 25
(SPTANDlsofonn 2 of Q13813 Ql 3813-2 285 24
Filamin A Q60FE5 278 24
Myosin-9 P35579 227 9 Selective extraction of spectrin from erythrocyte ghosts resulted in a marked reduction of binding of mClec9A-ecto to the ghosts (Figure 3A), suggesting spectrin was a likely Clec9A ligand. Erythroid spectrin is composed of an al (246kDa) βΐ (280kDa) heterodimer which self-associates to form a tetramer, and binds to cytoskeletal proteins including actin and band4.1 to form the membrane skeleton (Baines, 2010; An et al., 2011). The binding of mClec9A-ecto to a commercial preparation of human erythrocytic spectrin was tested by ELISA (Figure 3B): High affinity binding was observed, compared to very low binding of the controls mCire- ecto and mClecl2A-ecto. This binding was stable over the pH range 7.4 to 4.4. Analysis of this spectrin preparation by SDS-PAGE and protein staining (Figure 4A) indicated the preparation was relatively pure as judged by the expected 246kDa and 280kDa sized protein bands, but minor protein bands were also evident. Mass spectroscopic analysis revealed a majority of peptides corresponding to the expected spectrin al and spectrin βΐ, but also peptides corresponding to spectrin-associated proteins including actin, band 4.1, adducins and tropomodulins (Table 5).
Table 5 - Proteomic analysis of human erythrocytic spectrin, related to Figure 3 and
Protein name Accession . Mol wt Unique number (kDa) peptides
Band A
Spectrin alpha chain, P02549; 280 146 erythrocyte P08032
Spectrin beta chain, PI 1277; 246; 268 140 erythrocyte B2RX08 kDa
Clathrin heavy chain 1 Q00610 192 25
Band B
Spectrin alpha chain, P02549; 280 160 erythrocyte P08032
Spectrin beta chain, PI 1277; 246; 268 154 erythrocyte B2RX08 kDa
Clathrin heavy chain 1 000610 192 20
Transitional endoplasmic Q01853 89 10 reticulum ATPase
Band C
Spectrin beta chain, PI 1277; 246; 268 82 erythrocyte B2RX08 kDa
Spectrin alpha chain, P02549; 280 78 erythrocyte P08032
Transitional endoplasmic Q01853 89 32 reticulum ATPase
Beta-adducin Q9QYB8 81 16
26S proteasome non-ATPase Q99460 106 16 regulatory subunit 1
26S proteasome non-ATPase Q8VDM4 100 13 regulatory subunit 2
AP-2 complex subunit 095782 108 13 alpha- 1
Ankyriri-l Q02357 204 10
Band D
Spectrin beta chain, PI 1277; 246; 268 50 erythrocyte B2RX08 kDa
Spectrin alpha chain, P02549; 280 37 erythrocyte P08032
Erythrocyte protein band 4.1 A2A839; 72; 97 kDa 28
P11171
Heat shock cognate 71 kDa P63017 71 14 protein
Band E
Actin, cytoplasmic 1 P60710 42 20
Spectrin alpha chain, P02549; 280 18 erythrocyte P08032
Tropomodulin-l P28289 41 16
Spectrin beta chain, PI 1277; 246; 268 15 erythrocyte B2RX08 kDa
26S proteasome non-ATPase 000231 47 15 regulatory subunit 11
26S proteasome non-ATPase Q 15008 46 15 regulatory subunit 6
26S protease regulatory P62334 44 11 subunit 10B Example 8 - Clec9A binds to a higher order complex of erythrocyte spectrin
Spectrin extractions from mouse erythrocyte ghosts prepared by osmotic lysis were performed at 4°C, which enables the extraction of spectrin in the form of tetramers and higher order complexes, or at 37°C, which produces mainly spectrin dimers (Ungewickell and Gratzer, 1978; Gratzer et al., 1982). The isolated spectrin was coated onto ELISA plates and investigated for Clec9A binding. Significant binding of mClec9A-ecto was obtained to spectrin isolated if at 4°C, but only poor binding to spectrin isolated at 37°C (Figure 5A). The spectrin preparations were then examined by polyacrylmide gel electrophoresis under reducing conditions. The spectrin protein bands appeared similar under the two extraction conditions (Figure 5B). Mass spectrometric analysis of the excised protein bands showed there were no differences at mis level in the protein content of these spectrin extracts, the major components in both cases being spectrin a and spectrin β together with actin and band 4.1 (Table 6).
Table 6 - Proteomic analysis of mouse erythrocytic spectrin, related to Figure 5.
Figure imgf000108_0001
Beta-adducin Q9QYB8 81 28 23
Transitional endoplasmic Q01853 89 .27 25 reticulum ATPase
Erythrocyte protein band 4.1 A2A839 72 17 19
Endoplasmin P08113 92 15 16
Ankyrin-1 002357 204 15 18
Alpha-adducin 09QYC0 81 11 8
Band 3 anion transport protein P04919 103 10 7
Band D
Spectrin beta 1 B2RX08 268 46 40
Spectrin alpha chain, P08032 280 41 36 erythrocyte
Erythrocyte protein band 4.1 A2A839 72 38 37
78 kDa glucose-regulated P20029 72 22 26 protein
Heat shock cognate 71 kDa P63017 71 21 20 protein
Alpha-adducin Q9QYC0 81 18 15
Ankyrin-1 002357 204 16 10
Beta-adducin Q9QYB8 81 12 9
Acylamino-acid-releasing Q8R146 82 12 10 enzyme
Putative uncharacterized protein Q3TH46 77 12 12
(Fragment)
Erythrocyte membrane protein P49222 77 4 11 band 4.2
Band E
Actin, cytoplasmic 1 P60710 42 23 21
Spectrin beta 1 B2RX08 268 11 10
Spectrin . alpha chain, P08032 280 10 13 erythrocyte
Tropomodulin-1 P28289 41 9 10
Erythrocyte protein band 4.1 A2A839 72 8 18
Fructose-bisphosphate aldolase P05064 39 3 10
A This suggested the differences in Clec9A binding between the spectrin preparations might be due to differences in the incidence of higher order complexes. Accordingly the two purified spectrin samples were analysed by size-exclusion chromatography. Mouse eythrocyte spectrin gave one major peak of protein when isolated at 4°C (Peak 1, Figure 5C), whereas isolation at 37°C yielded two peaks, a minor peak corresponding to Peak 1 and a major Peak 2 of smaller molecular size (Figure 5C). Fractions spanning these regions were coated onto ELISA plates and tested for their ability to bind mClec9A-ecto. The higher molecular size samples from Peakl of both samples bound Clec9A, whereas material from Peak 2 did not bind (Figure 5D). Analysis by SDS-PAGE revealed that while both Peak 1 and Peak 2 were primarily composed of spectrin, Peak 1 also contained two additional bands at approximately 75 and 45 kDa, corresponding to band 4.1 and actin, respectively (Figure 5E). The commercial preparation of human erythrocyte spectrin also displayed a principle peak of protein eluting at the position of Peak 1, and this retained all the ability to bind mClec9A-ecto (Figure 4). Overall these experiments indicated that Clec9A binds to a higher order complex of spectrin with associated proteins, but does not bind spectrin alone. Example 9 - Clec9A Binds to a Higher Order Complex of Nucleated Cell Spectrin
Spectrin complexes are found in both erythrocytic and non-erythrocytic cells, although different subunits of spectrin are expressed in different cell types (Baines et al., 2010; Rotter et al., 2004; Uribe and Jay, 2009). To determine if a spectrin complex represented a Clec9A ligand within nucleated cells, the inventors isolated non- erythrocyte spectrin from 293F cells, a subline of human embryonic kidney cells. A modification of the erythrocyte spectrin isolation was used, and the extract further purified and analysed by size-exclusion chromatography. A protein peak appeared in the same elution position as erythrocytic spectrin Peak 1 (Figure 5F), separated from a spread of proteins of lower molecular sizes. When the fractions were coated on ELISA plates, mClec9A-ecto bound predominantly to fractions corresponding to Peak 1 (Figure 5G). SDS-PAGE and mass spectroscopic analysis revealed the major constituent of Peak 1 from these nucleated cells was spectrin a and β chains, together with a range of other protein components including actin (Figure 5H, I and Table 7). Furthermore, a spectrin complex isolated from apoptotic cells was similarly bound by Clec9A (Figure 4E-G). Thus Clec9A recognition of spectrin complexes is maintained, even following apoptotic cell death and associated proteolysis. Table 7 - Proteomic analysis of non-erythrocytic spectrin isolated from human 293F cells, related to Figure 5. Proteins included if samples had greater or equal to 10 peptides.
Protein name Accession Mol wt Unique number (kDa) Peptides
Band A
Spectrin alpha chain, brain Q13813 285 263
Spectrin beta chain, brain 1 Q01082 275 217
Filamin-A P21333 281 96
Spectrin beta chain, brain 2 015020 271 47
Filamin-B 075369 278 36
Lamin-Bl P20700 66 11
Hemoglobin subunit beta P68871 16 10
Band B
Lamin-Bl P20700 66 79
Lamin-B2 Q03252 68 65
Prelamin-A/C P02545-2 65 45
Heat shock 70 kDa protein P08107 70 38
1A/1B
Dihydrolipoyllysine-residue P10515 69 24 acetyltransferase component
of pyruvate dehydrogenase
complex, mitochondrial
X-ray repair cross- P12956 70 17 complementing protein 6
Spectrin beta chain, brain 1 Q01082 275 10
Insulin-like growth factor 2 Q9NZI8 63 10 mRNA-binding protein 1
Plastin-3 P13797 71 10
Band C
Actin, cytoplasmic 1 P60709 42 28
Heterogeneous nuclear P07910 34 15 ribonucleoproteins C1/C2
Interleukin enhancer-binding 012905 43 14 factor 2
Tubulin beta chain P07437 50 11
Lamin-Bl P20700 66 10
Sterol-4-alpha-carboxylate 3- Q15738 42 10 dehydrogenase,
decarboxylating
Example 10 - Spectrin- Actin Interaction Required for Clec9A Binding
The inventors sought to determine what features of the erythrocytic spectrin complexes were essential for recognition by Clec9A, by dissociation then reassociation of the structures. Spectrin complexes were extracted from mouse erythrocyte ghosts at 4°C, then treated for 1 hour at 37°C to dissociate the complex into its individual components. Size-exclusion chromatography revealed that most of the complex had been dissociated, shifting from the previous Peak 1 to the lower molecular size Peak 2 (Figure 6 A). Binding of mClec9A-ecto to the dissociated spectrin preparation was greatly reduced and size-exclusion chromatography showed that the residual binding was restricted to the remaining undissociated complex in Peak 1 (Figure 6B). Importantly, the dissociated spectrin at Peak 2, now free of most other proteins (Figure 6C), showed no binding of Clec9A (Figure 6B). No significant binding was observed to fractions containing other dissociated protein components of the complex, nor was there binding to pure actin alone. The purified, dissociated and non-binding spectrin fractions of Peak 2 were then concentrated and allowed to reassociate in the presence of either bovine muscle actin (Sigma), or bovine serum albumin (BSA) as a control, for 2 hr at 30°C. Both samples showed partial re-association, as judged by regeneration of the larger molecular size Peak 1 on size-exclusion chromatography (Figure 6D). However only the Peak 1 complex reconstituted with actin showed pronounced binding to mClec9A-ecto (Figure 6E). Even the slight binding of the complex reconstituted with BSA could be attributed to traces of actin remaining in the preparation (Figure 6D, F). This indicated that Clec9A binds to a higher order complex consisting of both spectrin and actin.
Example 11 - Minimal Requirements for a Spectrin-Actin Complex that Binds Clec9A
Erythroid and non-erythroid spectrins contain multiple spectrin repeat elements, and an Actin binding domain (ABD) in the N-terminus of β-spectrin, and a calmodulin- like domain at the C-terminus of the a-spectrin (Baines, 2010; An et al., 2011). The inventors tested recombinant fragments of human erythrocytic spectrin (Figure 7A) for ability to bind mClec9A-ecto in the presence or absence of bovine muscle actin (Signia) (Figure 8A). None of the individual fragments of spectrin showed binding, nor did the actin alone obtained from Sigma. However, the complex of actin with the N- terminal domains of the β chain of spectrin (βΐ N-4), which encompasses the spectrin βΐ actin binding domain (ABD) and repeats 1-4, showed significant binding to mClec9A-ecto. The inventors then compared the binding of CIec9A to a complex of muscle actin with either the N-terminal domains of spectrin βΐ (βΐ N-4) or with the ABD only (βΐ N). It was found Clec9A binding activity was directed against the complex of spectrin βΐ ABD with actin (Figure 7B).
As the binding of Clec9A to this reconstructed fragment of the complex was lower than the binding to the intact commercial human erythrocyte spectrin sample, the inventors compared the effects of different forms of actin on Clec9A binding. There are three classes of actin, a-actins are found in muscle, β- and γ-actins found in most cell types associated with the cytoskeleton (Bergeron et al., 2010). Therefore, the inventors compared Clec9A binding to complexes of the spectrin βΐ ABD with muscle actin (Sigma) or with human platelet actin (85% β-actin, 15% γ-actin) (>99% purity, (Cytoskeleton)). Clec9A binding to spectrin βΐ ABD with platelet actin was higher, and comparable to that of commercial human erythrocytic spectrin (Figure 7B), indicating that the main determinant of Clec9A binding to the erythrocytic spectrin complex is a complex of the spectrin βΐ ABD with actins.
To test if this binding of Clec9A to the spectrin-actin complex was related to the recognition of dead cells, Clec9A-ecto was pre-incubated with spectrin βΐ ABD and platelet actin. This inhibited the ability of Clec9A-ecto to bind to dead cells, both freeze-thawed and apoptotic cells, (Figure 7C), indicating that Clec9A recognises spectrin βΐ ABD-actin complexes using similar determinants to the recognition of dead cells.
Example 12 - Other Actin-Compiexed Cvtoskeletal Proteins Bind Clec9A
The ABD of spectrin β chains is composed of 2 calponin homology domains of approximately 110 residues. This domain is also found in other proteins of the spectrin family and several other actin binding proteins, including a-actinin, dystrophin and filamin ( orenbaum and Rivero, 2002; Uribe and Jay* 2009). Accordingly, the inventors compared the binding of Clec9A to a complex of the ABD of erythrocytic spectrin (spectrin βΐ N), non-erythrocytic spectrin (spectrin βΙΙ N) and human a- actinin-1 (a-actinin- 1 N), in the presence and absence of platelet actin. The mClec9A- ecto showed significant and comparable binding to the complex of the ABD of erythrocytic and non-erythrocytic spectrins and a-actinin, with platelet actin (Figure 7D, E), but not to the individual components of these complexes. Furthermore, preincubation of Clec9A with actin complexed with the ABD of erythrocytic spectrin, 5 non-erythrocytic spectrin and a-actinin 1 inhibited the ability of Clec9A-ecto to bind to dead cells (Figure 8B). In contrast, Clec9A had much less binding when the actin was complexed with an unrelated actin binding protein, Cofilin-l (Figure 8C). Thus Clec9A recognises a widespread form of actin complexed with ABDs with the 2 calponin homology domain motif. Importantly, Clec9A expressed in its native state on the 10 surface of transfected CHO- 1 cells, showed binding to actin complexed with the ABD of non-erythrocytic spectrin (Figure 7F).
Example 13 - Structure of the Clec9A C-Type Lectin-Like Domain
Because of the role of Clec9A as a DAMP receptor, the inventors determined
15 the crystal structure of the CTLD and compared it to other C-type lectin receptors. The CTLD of human CLEC9A (hCLEC9A) was expressed in 293F cells with an N-terminal FLAG tag. Additionally, protein glycosylation was eliminated through mutation of the sole glycosylation site (223-NCS-225) to the equivalent murine sequence (NCD). The resulting protein was confirmed to maintain binding activity to dead cells as compared
20 to the native hCLEC9A-CTLD (Figure 10A). Crystals diffracting to high resolution were obtained by including enterokinase in the crystallisation drops to remove the extraneous N-terminal sequence including the FLAG epitope.
The CLEC9A CTLD displayed the canonical fold of C-type lectin domains. A DALI search (Holm and Rosenstrom, 2010) revealed the CTLD of the Low Density
25 Lipoprotein Receptor, CLEC8A (LOX-1) (PDB: 1YPU, (Park et al., 2005) to have the strongest structural homology to the CLEC9A CTLD. Crystals contained two copies of the CLEC9A CTLD within the asymmetric unit related by 2-fold symmetry (Figure 9A; Table 8). This dimer interface is unlike canonical dimers of previously characterised CTLD domains (eg. LOX-1: (Park et al., 2005), CD69: (Natarajan et al.,
30 2000)). As it is highly fenestrated and contains little buried surface area, this dimer is likely an artefact of crystallisation. Furthermore, antibodies targeting a loop region within this dimer interface (position 185-211, Figure 9A,C) are biologically active and highly effective, for targeting antigen to CLEC9A, indicating this loop is exposed on cell surface CLEC9A. Full length Clec9A exists as a disulphide-linked dimer on the
35 cell surface (Figure IB; Sancho et al., 2008), most likely mediated by cysteine residues in CLEC9A N-terminal stalk regions of the ecto-domain. Orientation of the CLEC9A CTLD in a CLEC8A style dimer revealed that the CLEC9A structure is compatible with such a dimer interface with only small refolding of a loop present at the top of the interface (166-KIKG-169) being required to remove backbone clashes (Figure 9B,C). Table 8 - Crystallographic statistics for the CLEC9A CTLD structure. Values in brackets represent highest resolution shell.
Figure imgf000115_0001
Crystal packing is mediated by burial of two exposed tryptophan residues (W131, W227 - Figure 9A,C) into the edge of the crystallographic dimer groove of adjacent asymmetric units (Figure 10B). These tryptophans are conserved between the human and mouse. Whilst the tryptophans are unlikely to adopt these exposed conformations in solution, they are both located close to the human and murine glycosylation sites (223-NCS-225, 159-NIS-161 respectively) (Figure IOC). Within a monomer of the CLEC9A CTLD a Ca^ ion was observed co-ordinated by El 52, El 56, E223, the peptide carbonyl of Q150 and two waters (Figure 10D). This site is analogous to a previously described Ca^ binding site on CTLDs (site 4, (Zelensky and Gready, 2005)), where it's role is to stabilize the structure of the protein rather than being directly involved in receptor/ligand interactions. Thermofluor assays confirmed that Ca^ stabilises, and EDTA destabilises, the tertiary structure of hCLEC9A-CTLD (Figure 10E; Table 9).
Table 9 - Analysis of thermofluor assays of hCLEC9A-CTLD (S225D) in Tris Buffered Saline.
Figure imgf000116_0001
Example 14 - Regions of the Clec9A CTLD Involved in Ligand Recognition
In order to identify regions of CLEC9A involved in ligand recognition, constructs of the hCLEC9A ectodomain were prepared with a range of mutations. Like CLEC8A (Ohki et al., 2005), CLEC9A has a stretch of basic residues on or near the surface of the CLEC8A-style dimer (K166, K168, K215, K228; Figure 10F). Whilst CLEC9A mutant K228A did not express, mutants K166A, K168A and K215A were expressed and exhibited binding to dead cells comparable to that of wild-type CLEC9A (Figure 11 A). The most striking effects were obtained with the double mutant Wl 31 A, W227A (W131 and W227 highlighted in Figure IOC). This mutant had comparable secondary structure to the original CLEC9A-CTLD based on circular dichroism analysis (data not shown), but showed functional differences in binding Clec9A ligands. This mutant did not bind freeze-thawed human nucleated cells, mouse erythrocyte saponin ghosts (Figure 11A, B), human erythrocytic spectrin (Figure 11C, D), or non-erythrocytic spectrin (Figure 11D). Furthermore, it did not bind recombinant spectrin βΐ, spectrin βΙΙ or alpha-actinin-I ABD associated with actin (Figure HE, F). As all binding readouts were abrogated by the same mutation, this further supports the hypothesis that actin in the form obtained when complexed with actin binding proteins containing calponin homology-based ABD, represent dead cell ligands recognised by Clec9A.
Example 15 - Characterisation of the Clec9A-RNF41 interaction
In order to investigate the interaction between Clec9A and RNF41, an enzyme- linked immunosorbent assay (ELISA) was established. A panel of GST-tagged RNF41 proteins were expressed including the full length human RNF41 protein, the N-terminal domains of RNF41, the RING, B-Box, Coiled-Coil (RNF41-RBCC), and the C- terminal domains (RNF41 -CTD) (Figure 12).
GST-tagged RNF41 proteins were coated onto ELISA plates and tested for binding against FLAG-tagged Clec9A-ectodomains and control protein Clecl2A. Clec9A bound to the C-terminal domain of RNF41 (RNF41-CTD) but not to the N- terminal domains (RNF41-RBCC) or to GST controls (Figure 13 A, B). In contrast, the independent C-type lectin-like protein Clecl2A did not bind GST-tagged RNF41 proteins (Figure 13A, B). Human CLEC9A-ecto similarly bound to the C-terminal domain of RNF41 alone and not to the N-terminal domains and GST control (Figure 13C, D). This data indicates that the interaction between Clec9A and RNF41 is conserved across mouse and human species.
As described in Example 14, tryptophan residues W131 and W227 were required for binding to dead cells and ABD-actin complexes. The present inventors investigated the binding of the wildtype and hCLEC9A mutant (W131A, W227A) to determine if these residues were similarly required for binding interactions with RNF41. It was found that the binding of wild-type hCLEC9A-ecto and mutant hCLEC9A-ecto (W131A, W227A) to RNF41 was comparable (Figure 13C, D). This indicated that the Clec9A residues required for recognition of both dead cells and ABD-actin complexes differed from those required to bind RNF41.
As the residues required for Clec9A binding to RNF41 differ to those required for ABD-actin complexes, the inventors explored whether the two ligands compete for binding to Clec9A, or whether they synergise to enhance binding. Before commencement of assays, they determined an optimal concentration of mouse and human Clec9A to be incubated with RNF41 -C-terminal domain (CTD) that would allow the detection of binding inhibition or synergy for the reagents and protein stocks used in subsequent experiments (Figure 14A, B). Mouse Clec9A-ecto and control Clecl2A were pre-incubated either with actin that had been pre-associated with the ABD of spectrin βΐ (spectrin βΐ N), spectrin βΙΙ (spectrin βΙΙ N) or a-actinin-1 (a-actinin-1 N), or with ABD only controls. Clec proteins and Clec-ABD-actin complexes were then compared for their ability to bind GST-tagged R F41-CTD and the control GST by ELISA. Pre-incubation of Clec9A with ABD-actin complexes enhanced the binding of Clec9A to RNF41 (Figure 15 A, B). In contrast, incubation of Clec9A with ABD or with actin alone had no effect on binding to RNF41. There was no binding to the control protein GST. Enhancement of RNF41 binding by association with ABD-actin complexes was specific to the Clec9A- ecto, as the control FLAG-tagged Clecl2A-ecto did not bind RNF41 neither in the presence nor the absence of ABD-actin complexes (Figure 15C, D).
The hCLEC9A-ecto mutant (W131A, W227A) binds to RNF41 but not to ABD- actin complexes. Pre-incubation of wild-type hCLEC9A-ecto with ABD-actin complexes enhanced binding of hCLEC9A-ecto to RNF41, whereas pre-incubation of the mutant hCLEC9A-ecto (W131A, W227A) with ABD-actin complexes did not affect binding to RNF41 (Figure 16A, B). This suggests that enhancement of CLEC9A binding to RNF41 is dependent on the formation of a protein complex between CLEC9A and ABD-actin. Example 16 - Clec9A recognises F-actin
From the preceding results, Clec9A might recognise a pattern formed by the complex between actin and the ABDs, or a conformational form of actin induced by the interaction with ABDs. Focus shifted to actin itself when 2 of 7 commercial preparations of actin showed direct binding to Clec9A in ELISA assays after preincubation at room temperature. To examine this further the preparations were first incubated under conditions promoting the G-actin form then the G-actin purified by size-exclusion chromatography to eliminate contaminating actin binding proteins. G- actin was then converted to F-actin, incubated with mClec9A-ecto or Clecl2A-ecto as control, then centrifuged, Clec9A was found to sediment with F-actin to a much greater extent than Clecl2A, showing it had a capacity to recognise and bind to the filamentous conformation of actin itself (Figure 17A, B).
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
The present application claims priority from US 61/547,632 filed 14 October 2011, US 61/596,083 filed 7 February 2012 and US 61/596,081 filed 7 February 2012, the entire contents of each of which are incorporated herein by reference.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
Any discussion of documents, acts, materials, devices, articles or the like which "has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
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APPENDIX I
REMARK Date 2011-08-09 Time 17:28:58 EST +1000 (1312874938.55 s)
REMARK PHENIX refinement
REMARK REMARK ****************** INPUT FILES AND LABELS ****************************** REMARK Reflections:
REMARK file name : /export/share/disk502/labll01/czabotar/b8p39_Clec9A_Ho REMARK labels : [TP, SIGFPj DANO, SIGDANO' ]
REMARK R-free flags:
REMARK file name : /export/share/disk502/labll01/czabotar/b8p39_Clec9A_Ho REMARK label : FreeRflag
REMARK test_flag_value : 0
REMARK Model file name(s) : '
REMARK /export/share/disk502/labl101/czabotar/b8p39_Clec9A_Home/phenix/model4p REMARK REMARK ******************** REFINEMENT SUMMARY: QUICK FACTS ******************* REMARK Start: r_work = 0.1897 r_free = 0.2271 bonds = 0.006 angles = 1. σΐ8
REMARK Final: r_work = 0.1866 r_free = 0.2254 bonds = 0.006 angles = 1.012 REMARK ************************************************************************ REMARK REMARK ****************** REFINEMENT STATISTICS STEP BY STEP ****************** REMARK leading digit, like 1_, means number of macro-cycle
REMARK 0 : statistics at the very beginning when nothing is done yet
REMARK 1 s: bulk solvent correction and/or (anisotropic) scaling
REMARK 1 z: refinement of coordinates
REMARK 1 p: refinement of ADPs (Atomic Displacement Parameters)
REMARK REMARK R-factors , x-ray target values ,and norm of gradient of x -ray target REMARK stage r-work r-free xray target w xray target t
REMARK 0 0.1876 0.2272 3.014677e+00 3.160669e+00
REMARK l_bss : 0.1897 0.2271 3.025237e+00 3.156405e+00
REMARK l_xyz : 0.1899 0.2272 3.028272e+00 3.161691e+00
REMARK l_adp: 0.1885 0.2270 3.020062e+00 3.156934e+00
REMARK 2_bss : 0.1885 0.2266 3.019905e+00 3.156338e+00
REMARK 2_xyz : 0.1887 0.2262 3.020407e+00 3.156676e+00
REMARK 2_adp: 0.1873 0.2257 3.014882e+00 3.154386e+00
REMARK 3_bss : 0.1872 0.2256 3.014802e+00 3.154380e+00
REMARK 3 xyz : 0.1870 0.2254 3.013591e+00 3.153038e+00
REMARK 3_adp: 0.1867 0.2255 3.012684e+00 3.153866e+00
REMARK 3_bss : 0.1866 0.2254 3.012738e+00 3.153705e+00
REMARK REMARK stage k sol b sol bll b22 b33 bl2 bl3 b23 REMARK 0 : 0.381 50.102 5.644 -0.682 -4.962 0.000 -5. 590 -0 .000 REMARK l_bss : 0.381 50.102 5.644 -0.682 -4.962 0.000 -5. 590 -0 .000 REMARK l_xyz : 0.381 50.102 5.644 -0.682 -4.962 0.000 -5. 590 -0 .000 REMARK l_adp : 0.381 50.102 5.644 -0.682 -4.962 0.000 -5. 590 -0 .000 REMARK 2_bss: 0.381 50.102 5.644 -0.682 -4.-962 0.000 -5. 590 -0 .000 REMARK 2 xyz : 0.381 50.102 5.644 -0.682 -4.962 0.000 -5. 590 -0 .000 REMARK 2_adp : 0.381 50.102 5.644 -0.682 -4.962 0.000 -5. 590 -0 .000 REMARK 3_bss : 0.381 50.736 5.633 -0.660 -4.973 -0.000 -5. 623 -0 .000 REMARK 3_xyz : 0.381 50.736 5.633 -0.660 -4.973 -0.000 -5. 623 -0 .000 REMARK 3_adp : 0.381 50.736 5.633 -0.660 -4.973 -0.000 -5. 623 -0 .000 REMARK 3_bss: 0.381 50.828 5.619 -0.617 -5.002 -0.000 -5. 638 -0 .000 REMARK REMARK stage <pher> fom alpha beta 9
REMARK 0 : 24.030 0.8336 0.2153 121 .631
REMARK l_bss : 24.071 0.8329 0.2219 120 .699
REMARK l_xyz : 24.268 0.8308 0.2215 121 .308'
REMARK l_adp: 24.025 0.8335 0.2157 121 .330
REMARK 2 bss: 24.013 0.8336 0.2157 120 .959 REMARK 2_xyz : 24.024 0.8335 0.2157 120.668
REMARK 2_adp : 23.920 0.8346 0.2147 120 .040
REMARK 3_bss : 23.926 0.8345 0.2160 119 .973
REMARK 3_xyz : 23.854 0.8353 0.2160 119 .541
REMARK 3_adp : 23.825 0.8356 0.2153 119 .350
REMARK 3_bss : 23.832 0.8355 0.2163 119 .275
REMARK
REMARK stage angl bond chir dihe plan repu geom_target
REMARK 0 : 1.018 0.006 0.065 12.699 0 .005 4 .114 6.9283e-02
REMARK l_bss : 1.018 0.006 0.065 12.699 0 .005 4 .114 6.9283e-02
REMARK l_xyz : 1.011 0.006 0.065 12.669 0 .005 4 .114 6.8258e-02
REMARK l_adp : 1.011 0.006 0.065 12.669 0 .005 4 .114 6.8258e-02
REMARK 2_bss : 1.011 0.006 0.065 12.669 0 .005 4 .114 6.8258e-02
REMARK 2 xyz : 1.005 0.006 0.065 12.564 0 .005 4 .114 6.7617e-02
REMARK 2_adp : 1.005 0.006 0.065 12.564 0 .005 4 .114 6.7617e-02
REMARK 3_bss : 1.005 0.006 0.065 12.564 0 .005 4 .114 6.7617e-02
REMARK 3_xyz : · 1.012 0.006 0.065 12.556 0 .005 4 .114 6.8022e-02
REMARK 3_adp: 1.012 0.006 0.065 12.556 0 .005 4 .114 6.8022e-02
REMARK 3_bss : 1.012 0.006 0.065 12.556 0 .005 4 .114 6.8022e-02
REMARK
REMARK Maximal deviations:
REMARK stage angl bond chir dihe plan repu Igradl
REMARK 0 : 6.498 0.041 0.219 76.371 0 .043 2 .297 5.9078e-02
REMARK l_bss : 6.498 0.041 0.219 76.371 0 .043 2 .297 5.9078e-02
REMARK l_xyz : 6.549 0.035 0.210 76.835 0 .043 2 .299 5.7251e-02
REMARK l_adp: 6.549 0.035 0.210 76.835 0 .043 2 .299 5.7251e-02
REMARK 2_bss : 6.549 0.035 0.210 76.835 0 .043 2 .299 5.7251e-02
REMARK 2 xyz : 6.493 0.034 0.210 76.804 0 .042 2 .312 5.6517e-02
REMARK 2_adp: 6.493 0.034 0.210 76.804 0 .042 2 .312 5.6517e-02
REMARK 3_bss : 6.493 0.034 0.210 76.804 0 .042 2 .312 5.6517e-02
REMARK 3_xyz : 6.445 0.036 0.219 76.996 0 .043 2 .308 5.7350e-02
REMARK 3_adp : 6.445 0.036 0.219 76.996 0 .043 2 .308 5.7350e-02
REMARK 3_bss : 6.445 0.036 0.219 76.996 0 .043 2 .308 5.7350e-02
REMARK
REMARK — — 1 1 — 1 1 1
REMARK stage b max b min b ave b max b min b ave b max b min b ,ave
REMARK 0 : 92.69 4.48 24.72 92.69 4 .48 22 .90 63 .27· 14.94 35 .48
REMARK 1 bss : 95.87 4.92 25.24 95.87 4 .92 23 .50 63 .27 14.94 35 .48
REMARK l_xyz : 95.87 4.92 25.24 95.87 4 .92 23 .50 63 .27 14.94 35 .48
REMARK l_adp : 92.30 4.37 24.58 92.30 4 .37 22 .75 63 .37 14.86 35 .44
REMARK 2_bss: 92.30 4.37 24.58 92.30 4 .37 22 .75 63 .37 14.86 35 .44
REMARK 2_xyz : 92.30 4.37 24.58 92.30 4 .37 22 .75 63 .37 14.86 35 .44
REMARK 2_adp : 95.77 4.13 24.66 95.77 4 .13 22 .85 63 .40 14.85 35 .38
REMARK 3_bss : 95.89 4.25 24.77 95.89 4 .25 22 .96 63 .51 14.97 35 .49
REMARK 3_xyz : 95.89 4.25 24.77 95.89 4 .25 22 .96 63 .51 14.97 35 .49
REMARK 3_adp : 97.26 4.28 24.79 97.26 4 .28 23 .00 63 .50 14.77 35 .44
REMARK 3_bss : 97.36 4.38 24.89 97.36 4 .38 23 .09 63 .59 14.86 35 .53
REMARK
REMARK stage Deviation <of refined
REMARK model from start model
REMARK max min mean
REMARK 0 : 0.000 0. 000 0 .000
REMARK l_bss : 0.000 0. 000 0 .000
REMARK 1 xyz : 2.371 0. 000 0 .022
REMARK l_adp: 2.371 0. 000 0 .022
REMARK 2_bss : 2.371 0. 000 0 .022
REMARK 2_xyz : 0.129 0. 000 0 .012
REMARK 2_adp : 0.129 0. 000 0 .012
REMARK 3 bss: 0.129 0. 000 0 .012
REMARK 3_xyz : 0.164 0. 000 0 .009
REMARK 3_adp : 0.164 0. 000 0 .009 REMARK 3 bss: 0.164 0.000 0.009!
REMARK REMARK MODEL CONTENT.
REMARK ELEMENT ATOM RECORD COUNT OCCUPANCY SUM REMARK S 14 14.00
REMARK C 1255 1239.00
REMARK Ca 2 2.00
REMARK O 718 716.00
REMARK N 322 320.00
REMARK TOTAL 2311 2291.00
REMARK REMARK r^free_flags .md5.hexdigest dca49c313fe591111e7b8e7ddalcdb58
REMARK REMARK IF THIS FILE IS FOR PDB DEPOSITION: REMOVE ALL FROM THIS LINE UP.
REMARK 3
REMARK 3 REFINEMENT.
REMARK 3 PROGRAM PHENIX (phenix. refine: 1.7_650)
REMARK 3 AUTHORS Adams, Afonine, Chen, Davis, Echols, Gopal,
REMARK 3 Grosse-Kunstleve, Headd, Hung, Immormino, loerger, McCoy, REMARK 3 McKee, Moriarty, Pai , Read-, Richardson, Richardson, Romo, REMARK 3 Sacchettini, Sauter, Smith, Storoni, Terwilliger, Zwart REMARK 3
REMARK 3 REFINEMENT TARGET ML
REMARK 3
REMARK 3 DATA USED IN REFINEMENT.
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1.642
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 19.894
REMARK 3 MIN (FOBS/SIGMA_FOBS) 1.24
REMARK 3 COMPLETENESS FOR RANGE (%) 97.39
REMARK 3 NUMBER OF REFLECTIONS 56633
REMARK ' 3
REMARK 3 FIT TO DATA USED IN REFINEMENT.
REMARK 3 R VALUE (WORKING + TEST SET) 0.1886
REMARK 3 R VALUE (WORKING SET) 0.1866
REMARK 3 FREE R VALUE 0.2254
REMARK 3 FREE R VALUE TEST SET SIZE (%) 5.02
REMARK 3 FREE R VALUE TEST SET COUNT 2843
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT (IN BINS)
REMARK 3 BIN RESOLUTION : RANGE COMPL. NWORK NFREE RWORK RFREE REMARK 3 1 19. 8952 - 4 .4421 1. 00 2752 146 0 .1958 0 .2002 REMARK 3 2 4. 4421 - 3 .5323 1. 00 2749 148 0 .1466 0 .1991 REMARK 3 3 3. 5323 - 3 .0877 0. 99 2745 146 0 .1618 0 .1905 REMARK 3 4 3. 0877 - 2 .8062 0. 99 2706 145 0 .1772 0 .1907 REMARK 3 5 2. 8062 - 2 .6055 0. 99 2760 147 0 .1846 0 .2326 REMARK 3 6 2. 6055 - 2 .4522 , 0. 99 . 2721 142 0 .1778 0 .2322 REMARK 3 7 2. 4522 - 2 .3296 0. 98 2733 147 0 .1855 0 .2003 REMARK 3 8 2. 3296 - 2 .2283 0. 98 2690 141 0 .1803 0 .2405 REMARK 3 9 2. 2283 - 2 .1426 0. 98 2715 139 0 .1774 0 .2373 REMARK 3 10 2. 1426 - 2 .0688 0. 98 2727 143 0 .1896 0 .2387 REMARK 3 11 2. 0688 - 2 .0042 0. 97 2670 141 0 .1850 0 .2393 REMARK 3 12 2. 0042 - 1 .9469 0. 97 2711 144 0 .1710 0 .2323 REMARK 3 13 1. 9469 - 1 .8957 0. 97 2661 141 0 .1863 0 .2483 REMARK 3 14 1. 8957 - 1 .8495 0. 98 2685 143 0 .1960 0 .2605 REMARK 3 15 1. 8495 - 1 .8075 0. 97 2677 140 0 .2094 0 .2575 REMARK 3 16 1. 8075 - 1 .7690 0. 97 2683 140 0 .2179 0 .2579 REMARK 3 17 1. 7690 - 1 .7337 0. 97 2628 140 0 .2458 0 .2659 REMARK 3 18 1. 7337 - 1 .7010 0. 96 2695 140 0 .2776 0 .3484 REMARK 3 19 1. 7010 - 1 .6706 0. 96 2636 141 0 .3062 0 .3426 REMARK 3 20 1. 6706 - 1 .6423 0. 88 2446 129 0 .3252 0 .3280 REMARK 3 REMARK 3 BULK SOLVENT MODELLING.
REMARK 3 METHOD USED FLAT BULK SOLVENT MODEL
REMARK 3 SOLVENT RADIUS 0.90
REMARK 3 SHRINKAGE RADIUS 0.61
REMARK 3 GRID STEP FACTOR 4.00
REMARK 3 K_SOL 0.381
REMARK 3 B SOL 50.828
REMARK 3
REMARK 3 ERROR ESTIMATES.
REMARK 3 COORDINATE ERROR (MAXIMUM"-LIKELIHOOD BASED) 0.20 REMARK 3 PHASE ERROR (DEGREES, MAXIMUM-LIKELIHOOD BASED) 23.83 REMARK 3
REMARK 3 OVERALL SCALE FACTORS.
REMARK 3 SCALE = SUM ( I F OBS | * I F MODEL | ) /SUM ( I F MODEL | <*2 ) 0.2343 REMARK 3 ANISOTROPIC SCALE MATRIX ELEMENTS (IN CARTESIAN BASIS) REMARK 3 Bll 5. 6189
REMARK 3 B22 -0 .6171
REMARK 3 B33 -5 .0017
REMARK 3 B12 -0 .0000
REMARK 3 B13 -5 .6379
REMARK 3 B23 -0 .0000
REMARK 3
REMARK 3 R FACTOR FORMULA.
REMARK 3 R = SUM (I |F OBS | -SCALE* |F MODEL | ) /SUM( |F OBSI )
REMARK 3
REMARK 3 TOTAL MODEL STRUCTURE FACTOR (F_MODEL).
REMARK 3 F_MODEL = FB_CART * ( F_CALC_ATOMS + F_BULK)
REMARK 3 F_BULK = K_SOL * EXP(-B_SOL * S**2 / 4) * F_MASK
REMARK 3 F_CALC_ATOMS = ATOMIC MODEL STRUCTURE FACTORS
REMARK 3 FB_CART = EXP(-H(t) * A(-l) * B * A(-lt) * H)
REMARK 3 A = orthogonalization matrix, H = MILLER INDEX
REMARK 3 (t) = TRANSPOSE, (-1) = INVERSE
REMARK 3
REMARK 3 STRUCTURE FACTORS CALCULATION ALGORITHM : FFT
REMARK 3
REMARK 3 DEVIATIONS FROM IDEAL VALUES.
REMARK 3 RMSD MAX COUNT
REMARK 3 BOND 0.006 0 .036 2040
REMARK 3 ANGLE 1.012 6 .445 2767
REMARK 3 CHIRALITY 0.065 0 .219 283
REMARK 3 PLANARITY 0.005 0 .043 349
REMARK 3 DIHEDRAL 12.556 76 .996 733
REMARK 3 MIN NONBONDED DISTANCE : 2. 308
REMARK 3
REMARK 3 ATOMIC DISPLACEMENT PARAMETERS.
REMARK 3 WILSON B : 14.51
REMARK 3 RMS (B ISO OR EQUIVALENT BONDED) 4.54
REMARK 3 ATOMS NUMBER OF ATOMS
REMARK 3 ISO. ANISO.
REMARK 3 ALL 2311 1975
REMARK 3 ALL (NO H) 2311 1975
REMARK 3 SOLVENT 334 0
REMARK 3 NON-SOLVENT 1977 1975
REMARK 3 HYDROGENS 0 0
REMARK 3
REMARK 3 TLS DETAILS.
REMARK 3 NUMBER OF TLS GROUPS: 16
REMARK 3 ORIGIN: CENTER OF MASS
REMARK 3 TLS GROUP : 1
REMARK 3 SELECTION: chain *A* and (resseq 111:133)
REMARK 3 ORIGIN FOR THE GROUP (A): -10.1340 6.4102 6.4706 REMARK 3 T TENSOR
REMARK 3 Til 0. 1371 T22 : 0. 1551
REMARK ,3 T33 0. 0711 T12 : -0. 0597
REMARK 3 T13 : 0. 0229 T23 : -0. 0065
REMARK 3 L TENSOR
REMARK 3 Lll 0. 3088 L22 : 0. 3362
REMARK 3 L33 0. 1144 L12 : 0. 1165
REMARK 3 L13 0. 1630 L23 0. 1256
REMARK 3 S TENSOR
REMARK 3 Sll -0. 0284 S12 . -0. 0370 S13 -0. 0419
REMARK 3 S21 0. 0297 S22 0. 0515 S23 -0. 0282
REMARK 3 S31 0. 1609 S32 ■ -°- 1780 S33 -0. 0106
REMARK 3 TLS GROUP : 2
REMARK 3 SELECTION: chain Ά and (resseq 134 :153)
REMARK 3 ORIGIN FOR THE SROUP (A) : -o. 3671. 3. 6537 3.4892
REMARK 3 T TENSOR
REMARK 3 Til 0. 1585 T22 0. 0688
REMARK 3 T33 0. 11 5 T12 0. 0084
REMARK 3 T13 -0. 0310 T23 -0. 0042
REMARK 3 L TENSOR
REMARK 3 Lll 0. 4596 L22 0. 1688
REMARK 3 L33 0. 4986 L12 0. 0760
REMARK 3 L13 0. 1793 L23 0. 0326
REMARK 3 S TENSOR •
REMARK 3 Sll 0. 0957 S12 0. 0787 S13 -0. 1815
REMARK 3 S21 0. 1974 S22 0. 0871 S23 -0. 0885
REMARK 3 S31 0. 2950 S32 0. 0541 S33 -0. 0549
REMARK 3 TLS GROUP : 3
REMARK■ 3 SELECTION: chain Ά and (resseq 154 :166)
REMARK 3 ORIGIN FOR THE GROUP (A) : -13. 5033 12. 1754 -4.3796
REMARK 3 T TENSOR
REMARK 3 Til 0. 1125 T22 0. 1526
REMARK 3 T33 0. 1813 T12 -0. 0004
REMARK. 3 T13 -0. 0086 T23 -0. 0053
REMARK 3 ' L TENSOR
REMARK 3 Lll 0. 3786 L22 0. 2006
REMARK 3 L33 0. 1814 L12 -0. 1240
REMARK 3 L13 0. 0153 L23 -0. 0068
REMARK 3 S TENSOR -
REMARK 3 Sll:/ -0. 0012 S12: -0. 1244 S13 0. 2539
REMARK 3 S21 0. 0044 S22: -0. 0107' S23 -0. 0592
REMARK 3 S31: 0. 0009 S32 -0. 0850 S33 0. 0059
REMARK 3 TLS GROUP : 4
REMARK 3 SELECTION: chain Ά and (resseq 167 :179)
REMARK 3 ORIGIN FOR THE" GROUP (A) : -2. 0082 14. 8530 -0.1150
REMARK 3 T TENSOR
REMARK 3 Til 0. 0622 T22 0. 0190
REMARK 3 T33 0. 0699 T12 0. 0324
REMARK 3 T13 -0. 0060 T23 -o. 0439
REMARK 3 L TENSOR
REMARK 3 Lll 0. 1161 L22 0. 1338
REMARK 3 L33 0. 0838 L12 -0. 0630
REMARK 3 L13 0. 0990 L23 0. 0036
REMARK 3 S TENSOR
REMARK 3 Sll 0. 0376 S12 -0. 0087 S13 0. 0117
REMARK 3 S21 0. 0230 S22 -0. 1096 S23 -0. 1058
REMARK 3 S31 : -0. 1240 S32 -o. 0048 S33 -0. 0384
REMARK 3 TLS GROUP : 5
REMARK 3 , SELECTION: chain Ά and (resseq 180 :188)
REMARK 3 ORIGIN FOR THE GROUP (A) : 7. 5896 4. 4472 -11.2785
REMARK 3 T TENSOR REMARK 3 Til : 0.1148 T22 : 0.1839
REMARK 3 T33 : 0. 1530 T12 : 0. 0236
REMARK 3 T13 : 0. 0175 T23 : -0. 0536
REMARK 3 L TENSOR
REMARK 3 Lll : 0. 3232 L22 : 0. 1805
REMARK 3 L33 : 0. 1816 L12 : 0. 1751
REMARK 3 L13 : -0. 2030 L23 : -0. 1772
REMARK 3 S TENSOR
REMARK 3 Sll : 0. 1755 S12 : 0. 0764 S13 0. 0273
REMARK 3 S21 : 0. 0160 S22 : 0. 1093 S23 -0. 0923
REMARK 3 S31 : 0. 0513 S32 : 0. 2198 S33 -0. 1260
REMAR 3 TLS GROUP : 6
REMARK 3 SELECTION: chain ' A and (resseq 189 :209)
REMARK 3 ORIGIN FOR THE GROUP (A) : 1. 0894 6. 2672 -10. 5341
REMARK 3 T TENSOR
REMARK 3 Til 0. 1178 T22 0. 1042
REMARK 3 T33 0. 0994 T12 0. 0130
REMARK 3 T13 -0. 0239 T23 -0. 0035
REMARK 3. L TENSOR
REMARK 3 Lll 0. 2711 L22 0. 5853
REMARK 3 L33 0. 0692 L12 0. 2310
REMARK 3 L13 0. 0139 L23 0. 0695
REMARK 3 S TENSOR
REMARK 3 Sll -0. 0061 S12 0. 1169 S13 -0. 0413
REMARK 3 S2.1 0. 1642 S22 0. 0576 S23 -0. 1687
REMARK 3 S31 0. 0622 S32 0. 0415 S33 -0. 0217
REMARK 3 TLS GROUP : 7
REMARK 3 SELECTIO : chain Ά and (resseq 210 :237)
REMARK 3 ORIGIN FOR THE GROUP (A) : -0. 7261 11. 4023 -0. 2137
REMARK 3 T TENSOR
REMARK 3 Til 0. 0887 T22: 0. 0832
REMARK 3 T33 0. 0727 T12: -0. 0149
REMARK .3 T13 0. 0049 T23 -0. 0171
REMARK 3 L TENSOR
REMARK 3 Lll 0. 1065 L22 0. 1478
REMARK 3 L33 0. 4157 L12 -0. 0431
REMARK 3 L13 -0. 0342 L23 ' 0. 1201
REMARK 3 S TENSOR
REMARK 3 Sll 0. 0299 S12 0. 0076 S13: -0. 0041
REMARK 3 S21 -0. 0103 S22 0. 0232 S23: -0. 0123
REMARK 3 S31 0. 0905 S32 0. 0 13 S33: 0. 0036
REMARK 3 TLS GROUP : 8
REMARK 3 SELECTION: chain ' B and (resseq 111 :133)
REMARK 3 ORIGIN FOR THE GROUP (A) : -23. 2812 -6. 3110 -24. 3102
REMARK 3 T TENSOR
REMARK 3 Til -0. 0059 T22 0. 1956
REMARK 3 T33 0. 0876 T12 0. 1170
REMARK 3 T13 -0. 0988 T23 -0. 0540
REMARK 3 L TENSOR
REMARK 3 Lll 0. 4161 L22 0. 3354
REMARK 3 L33 0. 5073 L12 0. 3164
REMARK 3 L13 -0. 1076 L23 0. 1119
REMARK 3 S TENSOR
REMARK 3 Sll -0. 2450 S12 -0. 0380 S13 0. 0488
REMARK 3 S21 -0. 2068 S22 -0. 0148 S23- -0. 0886
REMARK 3 S31 -0. 1678 S32 -0. 4302 S33: -0. 1409
REMARK 3 TLS GROUP : 9
REMARK 3 SELECTION: chain 'B and (resseq 134 :144)
REMARK 3 ORIGIN FOR THE GROUP (A) : -14. 8236 -3. 8919 -34. 1322
REMARK 3 T TENSOR
REMARK 3 Til 0. 1917 T22 0. 0953 REMARK 3 T33 : G .0928 T12 : 0.0120
REMARK 3 T13 : -0 .0214 T23 : 0. 0082
REMARK 3 L TENSOR
REMARK 3 Lll : 0 .2461 L22 : 0. 7213
REMARK 3 L33 : 0 .1391 L12 : -0. 0271
REMARK 3 L13 : 0 .1373 L23 : 0. 1865
REMARK 3 S TENSOR
REMARK ■ 3 Sll : -0 .1046 S12 : 0. 1014 S13 0. 0591
REMARK 3 S21 : -0 .4560 S22 : 0. 0891 S23 -0. 0175
REMARK 3 S31 : -0 .3161 S32 : -0. 0384 S33 0. 0250
REMARK 3 TLS GROUP : 10
REMARK 3 SELECTION: chain 'B • and (resseq 145 :153)
REMARK 3 ORIGIN FOR THE GROUP (A) : -13. 564i 3 -2. 8612 -22.0534
REMARK 3 T TENSOR
REMARK 3 Til 0 .0750 T22 : 0. 0820
REMARK 3 T33 0 .1036 T12 0. 0126
REMARK 3 T13 0 .0000 T23 -0. 0053
REMARK 3 L TENSOR
REMARK 3 Lll 0 .1573 L22 0. 2343
REMARK 3 L33 0 .5058 L12 0. 1684
REMARK 3 L13 0 .0192 L23 0. 1594
REMARK 3 S TENSOR
REMARK 3 Sll 0 .0072 S12 -0. 0609 S13 0. 1119
REMARK 3 S21 0 .0523 S22 -0. 0945 S23 0. 0734
REMARK 3 S31 -0 .1822 S32 0. 0812 S33 -0. 0036
REMARK 3 TLS GROUP : 11 '
REMARK 3 SELECTION: chain 'B and (resseq 154 :166)
REMARK 3 ORIGIN "FOR THE GROUP (A) : -17. 7842 - 11. 8945 -14.2189
REMARK 3 T TENSOR
REMARK 3 Til 0 .1263 T22 0. 1438
REMARK 3 T33 0 .1064 T12 -0. 0161
REMARK 3 T13 0 .0420 T23 0. 0083
REMARK 3 L TENSOR
REMARK 3 Lll 0 .2792 L22 0. 6094
REMARK 3 L33 0.2470 L12 -0. 0095
REMARK 3 L13 0 .2318 L23 -0. 1574
REMARK 3 S TENSOR
REMARK 3 Sll -0 .1688 S12 -0. 0102 S13 -0. 0625
REMARK 3 S21 0 .0310 S22 -0. 0029 S23 -0. 0773
REMARK 3 S31 0 .0402 S32 -0. 2628 S33 0. 0312
REMARK 3 TLS GROUP : 12
REMARK 3 SELECTION: chain 'B and (resseq 167 :176)
REMARK 3 ORIGIN FOR THE GROUP (A) : -15. 867£i - 16. 8184 -25.4353
REMARK 3 T TENSOR
REMARK 3 Til 0 .0697 T22 0. 0743
REMARK 3 T33 0 .0853 T12 -0. 0252
REMARK 3 T13 -0 .0009 T23 -0. 0062
REMARK 3 L TENSOR
REMARK 3 Lll 0 .2042 L22 1. 1785
REMARK 3 L33 0 .2773 L12 0. 4404
REMARK 3 L13 0 .1138 L23 0. 0226
REMARK 3 S TENSOR
REMARK 3 Sll 0 .0714 S12 -0. 0768 S13 -0. 0849
REMARK 3 S21 -0 .0022 S22 -0. 0373 S23- -0. 1126
REMARK 3 S31 0 .1020 S32 -0. 1373 S33 -0. 0069
REMARK 3 TLS GROUP : 13
REMARK 3 SELECTION: chain ' B and (resseq 177 :18 4)
REMARK 3 ORIGIN FOR THE GROUP (A) : 2. 1615 -4. 6899
REMARK 3 T TENSOR
REMARK 3 Til -0 .0337 T22 0. 1170
REMARK 3 T33 0 .0773 T12 -0. 0751 REMARK 3 T13 : 0.12-57 T23 : 0.0042
REMARK 3 L TENSOR
REMARK 3 Lll : 0.0006 L22 : 0.0541
REMARK 3 L33 : 0.0844 L12 : -0.0040
REMARK 3 L13 : 0.0030 L23 : -0.0716
REMARK 3 S TENSOR
REMARK 3 Sll : -0.0180 S12 : 0.0237 S13 0.0058
REMARK 3 S21 : -0.0345 S22 0.0389 S23 -0.1223
REMARK 3 S31 . -0.0445 S32 . 0.1959 S33 0.0225
REMARK 3 TLS GROUP : 14
REMARK 3 SELECTION: chain 'B ' and (resseq 185 :193)
REMARK 3 ORIGIN FOR THE GROUP (A): -3.5337 -1.7434 -22 .9954
REMARK 3 T TENSOR
REMARK 3 Til 0.0737 T22 0.0673
REMARK 3 T33 : 0.1215 T12 -0.0041
REMARK 3 T13 ■ -0.0015 T23 0.0022
REMARK 3 L TENSOR
REMARK 3 Lll 0.1079 L22 0.3312
REMARK 3 L33 1.3694 L12 0.1358
REMARK 3 L13 0.2559 L23 -0.0011
REMARK 3 S TENSOR
REMARK 3 Sll: -0.0845 S12 0.0820 S13 -0.1166
REMARK 3 S21: 0.0240 S22 -0.0458 S23 0.0470
REMARK 3 S31 -0.3073 S32 0.1285 S33 0.0388
REMARK 3 TLS GROUP : 15
REMARK 3 SELECTION: chain *B and (resseq 194 :209)
REMARK 3 ORIGIN FOR THE GROUP (A): -1.3036 -9.9492 -20 .8104
REMARK 3 , T TENSOR
REMARK 3 Til 0.1708 T22 0.1452
REMARK 3 T33 0.1140 T12 -0.0012
REMARK 3 T13 -0.0030 T23 -0.0300
REMARK 3 L TENSOR
REMARK 3 Lll 0.4271 L22 0.1183
REMARK 3 L33 0.2529 L12 0.0141
REMARK 3 L13 -0.1182 L23 -0.0052
REMARK 3 S TENSOR
REMARK 3 Sll 0.0506 S12 0.1351 S13 -0.1724
REMARK 3 S21 -0.2656 S22 0.1078 S23 -0.0556
REMARK 3 S31 -0.0990 S32 0.0295 S33 -0.0569
REMARK 3 TLS GROUP : 16
REMARK 3 SELECTION: chain ' B and (resseq 210 :237)
REMARK 3 ORIGIN FOR THE GROUP (A): -11.9072 - 11.1943 -26 .4285
REMARK 3 T TENSOR
REMARK 3 Til- 0.0636 T22 0.0863 \
REMARK 3 T33- 0.0818 T12 0.0083
REMARK 3 T13 0.0040 T23 -0.0185
REMARK 3 L TENSOR
REMARK 3 Lll. 0.4083 L22 0.2347
REMARK 3 L33. 0.2442 L12 0.1391
REMARK 3 L13 0.2058 L23 0.0120
REMARK 3 S TENSOR
REMARK 3 Sll -0.0366 S12 -0.0467 S13: -0.0749
REMARK 3 S21 0.0107 S22 0.0270 S23: -0.0576
REMARK 3 S31 -0.0554 S32 -0.0230 S33 0.0013
REMARK 3
CRYST1 42 170 53.824 57.446 90.00 109.35 90.00 P 1 21 1
SCALE1 0.023714 0.000000 0 008329 0. 00000
SCALE2 0.000000 0.018579 0 000000 0. 00000
SCALE3 0.000000 0.000000 0 018450 0. 00000
ATOM 1 o SER A 111 -17.288 -3.124 10 .455 1. 00 61.64
ANISOU 1 o SER A 111 8130 8203 7088 -1002 200 ATOM 2 N SER A 111 -19.432 -0.525 11.490 1.00 68.71 N
ANISOU 2 N SER A 111 8889 9249 7968 -94*8 221 103 N
ATOM 3 CA SER A 111 -19.061 -1 .936 11 .544 1 .00 68. 78 C
A ISOU 3 CA SER A 111 8954 9211 7969 -986 213 119 C
ATOM 4 C SER A 111 -18.252 -2 .371 10 .320 1 .00 59. 72 C
ANISOU 4 C SER A 111 7835 8012 6842 -992 207 109 C
ATOM 5 CB SER A 111' -20.301 -2 .819 11 .718 1 .00 76. 12 C
ANISOU 5 CB SER A 111 9880 10162 8881 -1032 211 133 C
ATOM 6 OG SER A 111 -19.939 -4 .155 12 .025 1 .00 78. 16 0
ANISOU 6 OG SER A 111 10193 10375 9129 -1067 200 152 o TOM 7 o PRO A 112 -15.448 -2 .005 7 .665 1 .00 33. 81 o
ANISOU 7 o PRO A 112 4602 4622 3622 -956 210 62 . o
ATOM 8 N PRO A 112 -18.647 -1 .914 9 .118 1 .00 51. 08 N
ANISOU 8 N PRO A 112 6711 6934 5763 -987 209 91 N
ATOM 9 CA PRO A 112 -17.823 -2 .172 7 .932 1 .00 44. 85 C
ANISOU 9 CA PRO A 112 5946 6101 4994 -989 206 76 C
ATOM 10 C PRO A 112 -16.482 -1 .446 8 .029 1 .00 35. 83 c
ANISOU 10 C PRO A 112 4815 4931 3867 -950 212 66 c
ATOM 11 CB PRO A 112 -18.656 -1 .579 6 .789 1 .00 46. 14 c
ANISOU 11 CB PRO A 112 6068 6299 5166 -987 206 61 c
ATOM 12 CG PRO A 112 -20.050 -1 .569 7 .297 1 .00 49. 25 c
ANISOU 12 CG PRO A 112 6428 6742 5542 -1003 206 71 c
ATOM 13 CD PRO A 112 -19.931 -1 .285 8 .763 1 .00 54. 73 c
ANISOU 13 CD PRO A 112 7120 7452 6224 -988 211 84 c
ATOM 14 N CYS A 113 -16.512 -0 .212 8 .521 1 .00 34. 90 N
ANISOU 14 N CYS A 113 4660 4851 3750 -910 218 61 N
ATOM 15 CA CYS A 113 -15.302 0 .588 8 .687 1 .00 38. 20 C
ANISOU 15 CA CYS A 113 5084 5250 4181 -869 223 51 C
ATOM 16 C CYS A 113 -15.063 0 .932 10 .153 1 .00 40. 83 C
ANISOU 16 c CYS A 113 5420 5596 4496 -846 224 63 C
ATOM 17 o CYS A 113 -15.998 0 .950 10 .950 1 .00 41. 15 o
ANISOU 17 o CYS A 113 5438 5676 4519 -854 224 74 o
ATOM 18 CB CYS A 113 -15.394 1 .876 7 .865 1 .00 39. 53 C
ANISOU 18 CB CYS A 113 5205 5447 4366 -835 228 31 c
ATOM 19 SG CYS A 113 -15.396 1 .625 6 .072 1 .00 42. 14 s
ANISOU 19 SG CYS A 113 5537 5757 4715 -855 226 15 s
ATOM 20 N PRO A 114 -13.801 1 .204 10 .513 1 .00 38. 90 N
ANISOU 20 N PRO A 114 5205 5319 4257 -819 225 59 N
ATOM 21 CA PRO A 114 -13.459 1 .642 11 .870 1 .00 36. 60 c
ANISOU 21 CA PRO A 114 4918 5040 3948 -792 224 67 C
ATOM 22 C PRO A 114 -14.060 3 .005 12 .193 1 .00 41. 47 C
ANISOU 22 C PRO A 114 5473 5719 4566 -752 230 56 C
ATOM 23 o PRO A 114 -14.465 3 .730 11 .285 1 .00 36. 92 o
ANISOU 23 o PRO A 114 4855 5167 4008 -740 234 40 o
ATOM 24 CB PRO A 114 -11.932 1 .768 11 .821 1 .00 28. 77 C
ANISOU 24 CB PRO A 114 3957 3983 2993 -747 210 50 C
ATOM 25 CG PRO A 114 -11.506 0 .924 10 .682 1 .00 30. 80 c
ANISOU 25 CG PRO A 114 4242 4186 3275 -770 206 42 c
ATOM 26 CD PRO A 114 -12.604 1 .026 9 .674 1 .00 36. 78 c
ANISOU 26 CD PRO A 114 4967 4992 4016 -809 224 43 c
ATOM 27 N ASN A 115 -14.110 3 .349 13 .476 1 .00 39. 29 N
ANISOU 27 N ASN A 115 5192 5466 4269 -733 229 63 N
ATOM 28 CA ASN A 115 -14.482 4 .696 13 .884 1 .00 40. 69 C
ANISOU . 28 CA ASN A 115 5314 5698 4449 -690 234 48 C
ATOM 29 C ASN A 115 -13.490 5 .713 13 .340 1 .00 36. 96 C
ANISOU 29 c . ASN A 115 4830 5214 3998 -644 235 26 C
ATOM 30 o ASN A 115 -12.284 5 .469 13 .344 1 .00 34. 78 o
ANISOU 30 o ASN' A 115 4590 4875 3749 -617 221 19 o
ATOM 31 CB ASN A 115 -14.540 4 .798 15 .413 1 .00 50. 66 C
ANISOU 31 CB ASN A 115 6582 6981 5684 -678 233 58 c
ATOM 32 CG ASN A 115 -15.960 4 .778 15 .950 1 .00 59. 51 c ANISOU 32 CG ASN A 115 7667 8154 6790 -700 239 65 C
ATOM 33 ODl ASN A 115 -16.917 4. 544 15.208 1 .00 63 .21 o
ANISOU 33 ODl ASN A 115 8111 8639 7266 -728 242 65 o
ATOM 34 ND2 ASN A 115. -16.103 5. 019 17. 248 1 .00 62 .55 N
ANISOU 34 ND2 ASN A 115 8049 8566 7152. -687 241 69 N
ATO 35 N ASN A 116 -14.001 6. 851 12. 875 1 .00 35 .33 N
ANISOU 35 N ASN A 116 4566 5049 380? -618 238 10 N
ATOM 36 CA ASN A 116 -13.165 7. 922 12. 345 1 .00 33 .49 C
ANISOU 36 CA ASN A 116 4312 4803 3609 -567 233 -13 C
ATOM 37 C ASN A 116 -12.830 7. 706 10. 869 1 .00 30 .07 C
ANISOU 37 C ASN A 116 3887 4334 3204 -579 232 -20 C
ATOM 38 0 ASN A 116 -12.246 8. 579 10. 224 1 .00 31 .62 o
ANISOU 38 o ASN A 116 4062 4517 3435 -539 227 -39 o
ATOM 39 CB ASN A 116 -11.884 8. 070 13. 178 1 .00 39 .49 C
ANISOU 39 CB ASN A 116 5100 5514 4391 -515 217 -21 C
ATOM 40 CG ASN A 116 -11.147 9. 369 12. 904 1 .00 51 .57 C
ANISOU 40 CG ASN A 116 6596 7036 5963 -451 208 -46 C
ATOM 41 ODl ASN A 116 -11.139 9. 873 11. 781 1 .00 54 .77 o
ANISOU 41 ODl ASN A 116 6978 7441 6393 -446 210 -57 o
ATOM 42 ND2 ASN A 116 -10.517 9. 919 13. 941 1 .00 53 .29 H
ANISOU 42 ND2 ASN A 116 6813 7246 6189 -402 197 -55 N
ATOM 43 N TRP A 117 -13.212 6. 547 10. 335 1 .00 26 .80 N
ANISOU 43 N TRP A 117 3504 3906 2774 -635 238 -6 N
ATOM 44 CA TRP A 117 -12.942 6. 227 8. 933 1 .00 23 .63 C
ANISOU 44 CA TRP A 117 3113 3471 2393 -653 239 -14 C
ATOM 45 C TRP A 117 -14.207 6. 426 8. 110 1 .00 23 .64 c
ANISOU 45 C TRP A 117 3074 3513 2394 -677 238 -12 c
ATOM 46 o TRP A 117 -15.314 6. 327 8. 632 1 .00 26.81 o
ANISOU 46 o TRP A 117 3454 3952 2782 -692 235 -2 o
ATOM ' 47 CB TRP A 117 -12.462 4. 779 8. 790 1 .00 19 .21 c
ANISOU 47 CB TRP A 117 2613 2854 1834 -688 236 -6 c
ATOM 48 CG TRP A 117 -11.107 4. 523 9. 367 1 .00 20 .69 • c
ANISOU 48 CG TRP A 117 2838 2976 2048 -650 222 -14 c
ATOM 49 CD1 TRP A 117 · -10.711 4. 746 10. 656 1 .00 23 .29 c
ANISOU 49 CD1 TRP A 117 3174 3301 2374 -618 212 -10 c
ATOM 50 CD2 TRP A 117 -9.967 3. 974 8. 685 1 .00 15 .11 c
ANISOU 50 CD2 TRP A 117 2167 2197 1379 . -642 214 -30 c
ATOM 51 NE1 TRP A 117 -9.389 4. 385 10. 81.7 1 .00 21 .27 N
ANISOU 51 NE1 TRP A 117 2956 ' 2974 2152 -588 197 -22 N
ATOM 52 CE2 TRP A 117 -8.913 3. 902 9. 625 1 .00 17 .68 c
ANISOU 52 CE2 TRP A 117 2517 2475 1723 -602 199 -35 c
ATOM 53 CE3 TRP A 117 -9.734 3. 545 7. 374 1 .00 16 .74 C
ANISOU 53 CE3 TRP A 117 2384 2374 1602 -664 220 -43 C TOM 54 CZ2 TRP A 117 -7.647 3. 421 9. 295 1 .00 18 .68 C
ANISOU 54 CZ2 TRP A 117 2679 2528 1892 -583 188 -53 c
ATOM 55 CZ3 TRP A 117 -8.472 3. 067 7. 044 1 .00 19 .29 c
ANISOU 55 CZ3 TRP A 117 2740 2624 1963 -645 212 -63 c
ATOM 56 CH2 TRP A 117 -7.445 3. 005 8. 007 1 .00 17 .86 c
ANISOU • 56 CH2 TRP A 117 2583 2398 1806 -605 196 -68 c
ATOM 57 N ILE A 118 -14.043 6. 703 6. 824 1 .00 18 .49 N
ANISOU 57 N ILE A 118 2414 2850 1760 -679 238 -24 ■ N
ATOM 58 CA ILE A 118 -15.191 6. 895 5. 942 1 .00 19 .57 C
ANISOU 58 CA ILE A 118 2517 3017 1900 -699 230 -23 C
ATOM 59 C ILE A 118 -15.277 5. 763 4. 913 1 .00 23 .96 C
ANISOU 59 C ILE A 118 3107 3541 2455 -743 228 -22 ■ C
ATOM 60 o ILE A 118 -14.259 5. 342 4. 363 1 .00 20 .09 o
ANISOU 60 o ILE A 118 2653 3005 1975 -747 234 -31 o
ATOM 61 CB ILE A 118 -15.122 8. 265 5. 234 1 .00 23 .51 C
ANISOU 61 CB ILE A 118 2975 3540 2418 -667 226 -36 C
ATOM 62 CGI ILE A 118 -13.878 8. 360 4. 355 1 .00 26 .48 c
ANISOU 62 CGI ILE A 118 3376 3872 2812 -656 232 -49 c ATOM 63 CG2 ILE•A 118 -15.093 9.386 6.252 1.00 29.28 C
ANISOU 63 CG2 ILE A 118 3670 4306 3151 -624 226 -40 C
ATOM 64 CD1 ILE A 118 -13.818 9. 642 3. 547 1 .00 36. 82 C
A ISOU 64 CD1 ILE A 118 4647 5202 4140 -631 227 -60 C
ATOM 65 N GLN A 119 -16.483 5. 263 4. 655 1 .00 23. 47 N
ANISOU 65 N GLN A 119 3034 3502 2381 -776 221 -15 N
ATOM 66 CA GLN A 119 -16.641 4. 182 3. 679 1 .00 19. 30 C
ANISOU , 66 CA GLN A 119 2537 2947 1851 -817 218 -15 C
ATOM 67 C GLN A 119 -17.063 4. 651 2. 285 1 .00 27. 17 C
ANISOU 67 C GLN A 119 3513 3955 2854 -824 210 -26 C
ATOM 68 o GLN A 119 -17.817 5. 615 2. 121 1 .00 21. 74 o
ANISOU 68 o GLN A 119 2781 3310 2167 -811 203 -26 o
ATOM 69 CB GLN A 119 -17.640 3. 12-4 4. 174 1 .00 22. 05 C
ANISOU 69 CB GLN A 119 2894 3305 2178 -856 214 -1 C
ATOM 70 CG GLN A 119 -17.846 1. 966 3. 190 1 .00 26. 74 C
ANISOU 70 CG GLN A 119 3519 3872 2767 -898 209 , -3 C
ATOM 71 CD GLN A 119 -18.907 0. 972 3. 644 1 .00 34. 07 C
ANISOU 71 CD GLN A 119 4454 4815 3677 -936 204 •12 C
ATOM 72 OEl GLN A 119 -19.603 1. 193 4. 635 1 .00 36. 63 o
ANISOU 72 OEl GLN A 119 4755 5173 3990 -933 205 23 o
ATOM 73 NE2 GLN A 119 -19.031 -0. 134 2. 915 1 .00 37. 84 N
ANISOU 73 NE2 GLN A 119 4962 5267 4149 -973 200 11 N
ATOM 74 N ASN A 120 -16.556 3. 951 .1. 280 1 .00 24. 13 N
,ANISOU 74 N ASN A 120 3162 3534 2474 -845 211 -35 N
ATOM 75 CA ASN- A 120 -17.098 4. 036 -0. 063 1 .00 24. 87 C
ANISOU 75 CA ASN A 120 3247 3637 2566 -864 203 -42 C
ATOM 76 C ASN A 120 -17.034 2. 655 -0. 692 1 .00 26. 36 C
ANISOU 76 C ASN A 120 3479 3792 2746 -904 203 -46 C
ATOM 77 o ASN A 120 -15.956 2. 152 -1. 009 1 .00 24. 29 o
ANISOU 77 o ASN A 120 3251 3484 2492 -906 213 -57 o
ATOM 78 CB ASN A 120 -16.366 5. 083 -0. 914 1 .00 27. 46 c
ANISOU 78 CB ASN A 120 3564 3960 2911 -837 204 -55 c
ATOM 79 CG ASN A 120 -17.036 5. 304 -2. 267 1 .00 38. 36 c
ANISOU 79 CG ASN A 120 4933 5358 4285 -857 192 -60 c
ATOM 80 ODl ASN A 120 -18.059 5. 984 -2. 370- 1 .00 42. 56 o
ANISOU 80 ODl ASN A 120 5427 5932 4810 -857 180 -53 Ό
ATOM 81 ND2 ASN A 120 -16.464 . 4. 721 -3. 306 1 .00 35. 28 N
ANISOU 81 ND2 ASN A 120 4575 4934 3894 -876 196 -73 N
ATOM 82 N ARG A 121 -18.202 2. 035 -0. 833 1 .00 31. 08 N
ANISOU 82 N ARG A 121 4071 4412 3327 -936 194 -38 N
ATOM 83 CA ARG A 121 -18.309 0. 684 -1. 373 1 .00 30. 19 C
ANISOU 83 CA ARG A 121 3997 4272 3203 -976 193' -41 C
ATOM 84 C ARG A 121 -17.433 -0. 307 -0. 597 1 .00 29. 82 C
ANISOU 84 C ARG A 121 3992 4180 3159 -983 202 -38 C
ATOM 85 o ARG A 121 -17.659 -0. 536 0. 591 1 .00 37. 94 o
ANISOU 85 o ARG A 121 5019 5215 4182 -982 202 -22 o
ATOM 86 CB ARG A 121 -17.988 0. 675 -2. 870 1 .00 29. 78 C
ANISOU 86 CB ARG A 121 3958 4205 3153 -986 192 -59 C
ATOM 87 CG ARG A 121. -18.707' 1. 778 -3. 644 1 .00 31. 69 C
ANISOU' 87 CG ARG A 121 4161 4487 3391 -978 181 -60 C
ATOM 88 CD ARG A 121 -18.743 1. 488 -5. 136 1 .00 31. 36 C
ANISOU 88 CD ARG A 121 4137 4438 3341 -1001 177 -73 C
ATOM 89 NE ARG A 121 -19.329 0. 179 -5. 384 1 .00 33. 01 N
ANISOU 89 NE ARG A 121 4371 4638 3532 -1041 174 -72 N
ATOM 90 CZ ARG A 121 -18.898 -0. 666 -6. 311 1 .00 35. 36 C
ANISOU 90 CZ ARG A 121 4705- 4907 3823 -1064 178 -87 C
ATOM 91 NH1 ARG A 121 -17.881 -0. 328 -7. 091 1 .00 41. 19 N
ANISOU 91 NH1 ARG A 121 5459 5622 4570 -1052 187 •105 N
ATOM 92 NH2 ARG A 121 -19.488 -1. 843 -6. 458 1 .00 41. 25 N
ANISOU 92 NH2 ARG A 121 5472 5647 4554 -1098 175 -84 N TOM 93 N GLU A 122 -16.430 -0. 882 -1. 252 1 .00 25. 87 N ANISOU 93 N GLU A 122 3529 3634 2668 -990 209 -53 N
ATOM 94 CA GLU A 122 -15.628 -1. 923 -0. , 607 1. ,00 31. 35 C
ANISOU 94 CA GLU A 122 4266 4279 3365 -1003 215 -51 C
ATOM 95 C GLU A 122 -14.455 -1. 374 0. ,207 1. ,00 26. 29 C
ANISOU 95 C GLU A 122 3633 3615 2742 -970 • 225 -52 C
ATOM 96 o GLU A 122 -13.727 -2. 141 0. ,836 1. 00 28. 86 o
ANISOU 96 o GLU A 122 3995 3896 3073 -979 228 -48 o
ATOM " 97 CB GLU A 122 -15.112 -2. 939 -1. ,638 1. 00 34. 59 C
ANISOU 97 CB GLU A 122 4716 4647 3780 -1029 218 -69 C
ATOM 98 CG GLU A 122 -16.174 -3. 864 -2. 206 1. 00 54. 47 C
ANISOU 98 CG GLU A 122 7239 7178 6278 -1066 208 -64 c
ATOM 99 CD GLU A 122 -15.665 -4. 676 -3. 384 1. 00 73. 23 c
ANISOU 99 CD GLU A 122 9648 9518 8657 -1086 212 -86 c
ATOM 100 OEl GLU A 122 -14.543 -4. 399 -3. 861 1. 00 77. 66 o
ANISOU 100 OEl GLU A 122 10221 10048 9236 -1069 224 ■107 o
ATOM 101 OE2 GLU A 122 -16.385 -5. 592 -3. .835 1. 00 80. 44 o
ANISOU 101 OE2 GLU A 122 10572 10434 9556 -1117 205 -83 o
ATOM 102 N SER A 123 -14.274 -0. 056 0. 196 1. 00 22. 21 N
ANISOU 102 N SER A 123 3083 3124 2234 -934 229 -56 N
ATOM 103 CA SER A 123 -13.106 0. 551 0. 830 1. 00 19. 16 c
ANISOU 103 CA SER A 123 2702 2715 186 -'901 241 -60 c
ATOM 104 C SER A 123 -13.455 1. 404 2. 052 1. 00 22. 12 c
ANISOU 104 C SER A 123 3045 3127 2231 -871 238 -44 c
ATOM 105 .0 SER A 123 -14.549 1. 969 2. 136 1. 00 23. 53 o
ANISOU 105 o SER A 123 3184 3355 2400 -866 230 -35 o
ATOM 106 CB SER A 123 -12.341 1. 412 -0. 177. 1. 00 22. 28 . c
ANISOU 106 CB SER A 123 3087 3102 2278 -878 250 -83 c
ATOM 107 OG SER A 123 -11.797 0. 628 -1. 221 1. 00 27. 64 o
ANISOU 107 OG SER A 123 3798 3740 2965 -903 256 ■103 o
ATO 108 N CYS A 124 -12.509 1. 492 2. 990 1. 00 18. 93 N
ANISOU 108 N CYS A 124 2661 2698 1832 -851 ■246 -41 N
ATOM 109 CA CYS A 124 -12.588 2. 427 4. 117 1. 00 18. 34 C
ANISOU 109 CA CYS A 124 2559 2657 1751 -815 246 -31 c
ATOM. 110 C CYS A 124 -11.385 3. 349 4. 041 1. 00 '19. 83 c
ANISOU 110 C CYS A 124 2742 2821 1972 -760 247 -52 c
ATOM 111 o CYS A 124 -10.277 2. 894 3. 754 1. 00 20. 58 o
ANISOU 111' o CYS A 124 2869 2855 2097 -747 244 -69 o
ATOM 112 CB CYS A 124 -12.509 1; 702. 5. 459 1. 00 19. 40 c
ANISOU 112 CB CYS A 124 2723 2778 1869 -825 243 -12 c
ATOM 113 SG CYS A 124 -13.772 0. 456 5. 712 1. 00 30. 28 s
ANISOU 113 SG CYS A 124 4110 4168 3225 -874 231 8 s
ATOM 114 N TYR A 125 -11.595 4. 629 4. 326 1. 00 15. 49 N
ANISOU 114 N TYR A 125 2150 2313 1424 -723 247 -52 N
ATOM 115 CA TYR A 125 -10.515 5. 607 4. 232 1. 00 15. 24 c
ANISOU 115 CA TYR A 125 2105 2257 1427 -665 243 -73 c
ATOM 116 C TYR A 125 -10.311 6. 386 5. 523 1. 00 13. 75 c
ANISOU 116 C TYR A 125 1898 2084 1244 -617 235 -70 c
ATOM 117 o TYR A 125 -11.258 6. 681 6. 251 1. 00 15. 04 o
ANISOU 117 o TYR A 125 2036 2300 1380 -624 236 -54 o
ATOM 118 CB TYR A 125 -10.791 6. 604 3. 114 1. 00 11. 86 c
ANISOU 118 CB TYR A 125 1641 1859 1005 -660 247 -81 c
ATOM 119 CG TYR A 125 -11.098 5. 966 1. 781 1. 00 13. 11 c
ANISOU 119 CG TYR A 125 1815 2013 1155 -709 254 -85 c
ATOM 120 CDl TYR A 125 -12.410 5. 751 1. 383 1. 00 20. 20 c
ANISOU 120 CDl TYR A 125 _2694 2949 2031 -744 247 -72 c
ATOM 121 CD2 TYR A 125 -10.082 5. 582 0. 922 1. 00 14. 53 c
ANISOU 121 CD2 TYR A 125 2023 2140 1359 -708 259 •106 c
ATOM 122 CE1 TYR A 125 -12.702 5. 171 0. 163 1. 00 18. 26 c
ANISOU 122 CE1 TYR A 125 2462 2693 1785 -777 244 -78~ c
ATOM 123 CE2 TYR A 125 -10.370 4. 992 -0. 306 1. 00 14. 14 c
ANISOU 123 CE2 TYR A 125 1988 2086 1298 -754 266 111 c ATOM 124 CZ TYR A 125· -11.683 4.801 -0.677 1.00 17.00 C
ANISOU 124 CZ TYR A 125 2334 2489 1638 -785 256 -97 C
ATOM 125 OH TYR A 125 -11.997 4. 221 -1. 886 1 .00 17. 10 o
ANISOU 125 OH TYR A 125 2359 2493 1644 -818 252 106 o
ATOM 126 N TYR A 126 -9.065 6. 748 5. 784 1 .00 11. 80 N
ANISOU 126 N TYR A 126 1660 1791 1032. -567 228 -87 N
ATOM 127 CA TYR A 126 -8.770 7. 614 6. 912 1 .00 11. 21 C
ANISOU 127 CA TYR A 126 1566 1729 966 -516 218 -88 C
ATOM 128 C TYR A 126 -7.983 8. 806 6. 435 1 .00 13. 21 C
ANISOU 128 C TYR A 126 1792 1971 1257 -465 215 108 C
ATOM 129 o TYR A 126 -6.939 8. 654 5. 795 1 .00 13. 54 o
ANISOU 129 o TYR A 126 1853 1960 1331 -452 216 126 o
ATOM 130 CB TYR A 126 -7.973 6. 862 7. 975 1 .00 10. 72 C
ANISOU 130 CB TYR A 126 1545. 1618 911 -501 208 -86 C
ATOM 131 CG TYR A 126 -7.533 7. 737 9. 117 1 .00 12. 99 C
ANISOU 131 CG TYR A 126 1815 1911 1209 -446 196 -90 c
ATOM 132 CD1 TYR A 126 -8.397 8. 016 10. 166 1 .00 18. 84 c
ANISOU 132 CD1 TYR •A 126 2537 2705 1916 -448 196 -74 c
ATOM 133 CD2 TYR A 126 -6.257 8. 278 9 . 151 1 .00 15. 43 c
ANISOU 133 CD2 TYR A 126 2127 2174 1563 -392 187 111 c
ATOM 134 CE1 TYR A 126 -8.000 8. 806 11. 224 1 .00 22. 28 c
ANISOU 134 CE1 TYR A 126 2958 3147 2359 -397 185 -80 c
ATOM 135 CE2 TYR A 126 -5.847 9. 078 10. 219 1 .00 14. 55 c
ANISOU 135 CE2 TYR A 126 2000 2066 1462 -340 174 116 c
ATOM 136 CZ TYR A 126 -6.726 -9. 331 11. 248 1 .00 21. 14 c
ANISOU 136 CZ TYR A 126 2817 2954 2259 -343 173 100 c
ATOM 137 OH TYR A 126 -6.342 10. 121 12. 316 1 .00 25. 27 o
ANISOU 137 OH TYR A 126 3326 3485 2791 -292 161 107 o
ATOM 138 N VAL A 127 -8.472 10. 001 6. 750 1 .00 16. 98 N
ANISOU 138 N VAL A 127 2222 2497 1732 -437 212 107 N
ATOM 139 CA VAL A 127 -7.765 11. 209 6. 365 1 .00 14. 27 C
ANISOU 139 CA VAL A 127 1850 2145 1425 -388 206 124 C
ATOM 140 C VAL A 127 -7.037 11. 788 7. 568 1 .00 12. 23 C
ANISOU 140 C VAL A 127 1587 1873 1188 -330 194 132 c
ATOM 141 o VAL A 127 -7.669 12. 148 8. 566 1 .00 15. 90 o
ANISOU 141 o VAL A 127 2030 2379 1632 -319 189 124 ό
ATOM 142 CB VAL A 127 -8.734 12. 269 5. 797 1 .00 13. 41 c
ANISOU 142 CB VAL A 127 1691 2097 1308 -391 206 119 c
ATOM 143 CGI VAL A 127 -7.950 13. 507 5. 340 1 .00 16. 39 c
ANISOU 143 CGI VAL A 127 2042 2461 1726 -342 198 135 c
ATOM 144 CG2 VAL A 127 -9.556 11. 685 4. 645 1 .00 15. 00 c
ANISOU 144 CG2 VAL A 127 1898 2317 1486 -450 215 - 110 c
ATOM 145 N SER A 128 -5.711 11. 887 7. 482 1 .00 8. 57 N
ANISOU 145 ■ N SER A 128 1140 1351 764 -293 . 188 - 150 N
ATOM 146 CA SER A 128 -4.916 12. 309 8. 634 1 .00 8. 10 C
ANISOU 146 CA SER A 128 1081 1269 727 -239 174 159 C
ATOM 147 C SER A 128 -5.067 13. 777 8. 934 1 .00 17. 50 C
ANISOU 147 C SER A 128 2222 2497 1931 -193 165 165 C
ATOM 148 o SER A 128 -5.435 14. 580 8. 068 1 .00 14. 78 o
ANISOU 148 o SER A 128 1842 2179 1594 -194 168 166 o
ATOM 149 CB SER A 128 -3.438 11. 981 8. 419 1 .00 11. 31 C
ANISOU 149 CB SER A 128 1519 1599 1178 -213 169 178 c
ATOM 150 OG SER A 128 -2.789 13. 028 7. 707 1 .00 10. 20 o
ANISOU 150 OG SER A 128 1351 1449 1077 -178 170 196 0
ATOM 151 N GLU A 129 -4.767 14. 109 10. 184 1 .00 15. 40 N
ANISOU 151 N GLU A 129 1952 2230 1669 -153 152 ■168 N
ATOM 152 CA GLU A 129 -4.697 15'. 480 10. 650 1 .00 14. 71 c
ANISOU 152 CA GLU A 129 1820 2168 1602 -100 140 ■178 c
ATOM 153 C GLU A 129 -3.286 15. 775 11. 138 1 .00 12. 06 c
ANISOU 153 C GLU A 129 1497 1775 1311 -45 126 •197 c
ATOM 154 o GLU A 129 -3.044 16. 814 11. 743 1 .00 16. 79 o ANISOU 154 o GLU A 129 2065 2384 1929 5 113 •207 0
ATOM 155 CB GLU A 129 -5.696 15. 703 11. 787 1. .00 18. .15 c
ANISOU 155 CB GLU A 129 2234 2662 2000 -101 138 ■169 C
ATOM 156 CG GLU A 129 -7.142 15. 442 11. 390 1. .00 33. 28 C
ANISOU 156 CG GLU A 129 4133 4638 3874 -154 152 •153 C TOM 157 CD GLU A 129 -8.081 15. 394 12. 585 1. 00 51. 34 C
ANISOU 157 CD GLU A 129 6407 6979 6120 -163 154 ■145 C
ATOM 158 OEl GLU A 129 -7.967 14. 451 13. 397 1. 00 56. 81 o
ANISOU 158 OEl GLU A 129 7140 7657 6788 -180 155 •135 o
ATOM 159 OE2 . GLU A 129 -8.933 16. 300 12. 713 1. 00 52. 40 o
ANISOU 159 OE2 GLU A 129 6492 7171 6248 -154 153 ■149 o
ATOM 160 N ILE A 130 -2.356 14. 866 10. 871 1. 00 12. 56 N
ANISOU 160 N ILE A 130 1604 1775 1392 -53 127 ■202 N
ATOM 161 CA ILE A 130 -0.943 15. 092 11. 155 1. 00 11. 32 C
ANISOU 161 CA ILE A 130 1460 1557 1285 -3 114 ■222 C
ATOM 162 C ILE A 130 -0.126 14. 957 9; 878 1. 00 11. 25 C
ANISOU 162 C ILE A 130 1459 1502 1313 -10 125 •237 C
ATOM 163 0 ILE A 130 -0.574 14. 345 8. 892 1. 00 16. 50 o
ANISOU 163 0 ILE A 130 2136 2174 1960 -59 141 230 o
ATOM 164 CB ILE A 130 -0.388 14. 070 12. 172 1. 00 12. 06 C
ANISOU 164 CB ILE A 130 1601 1607 1375 ' -1 102 220 c
ATOM 165 CGI ILE A 130 -0..592 12. 643 11. 646 1. 00 17. 95 c
ANISOU 165 CGI' ILE A 130 2389 2331 2100 -59 113 210 c
ATOM 166 CG2 ILE A 130 -1.051 14. 251 13. 535 1. 00 16. 29 c
ANISOU 166 CG2 ILE A 130 2130 2185 1874 9 91 208 c
ATOM 167 CD1 ILE A 130 0.136 11. 596 12. 447 1. 00 22. 76 c
ANISOU 167 CD1 ILE A 130 3049 2884 2717 -57 97 210 c
ATOM 168 N TRP A 131 1.079 15. 515 9. 899 1. 00 9. 89 N
ANISOU 168 N TRP A 131 1282 1284' 1192 39 116 258 N
ATOM 169 CA TRP A 131 1.997 15. 411 8. 775 1. 00 9. 58 c
ANISOU 169 CA TRP A 131 1250 1197 1191 36 127 276 C
ATOM 170 C TRP A 131 2.988 14. 323 9. 050 1. 00 14. 30 C
ANISOU 170 C TRP A 131 1893 1728 1813 38 123 290 C
ATOM 171 0 TRP A 131 f 3.326 14. 050 10. 203 1. 00 15. 66 o
ANISOU 171 0 TRP A 131 2084 1879 1990 63 103 290 o
ATOM 172 CB TRP A 131 2.832 16. 682 8. 603 1. 00 13. 78 C
ANISOU 172 CB TRP A 131 1748 1715 1773 89 120 294 C
ATOM 173 CG TRP A 131 2.125 17. 937 8. 207 1. 00 9. 71 C
ANISOU 173 CG TRP A 131 1184 1254 1250 97 120 285 C
ATOM 174 CD1 TRP A 131 0.893 18. 075 7. 636 1. 00 12. 40 c
ANISOU 174 CD1 TRP A 131 1507 1652 1551 57 129 266 c
ATOM 175 CD2 TRP A 131 2.635 19. 253 8. 393 1. 00 9. 68 c
ANISOU 175 CD2 TRP A 131 1143 1249 1286 150 106 - 296 c
ATOM 176 NE1 TRP A 131 0.602 19. 408 7. 449 1. 00 13. 58 N
ANISOU 176 NE1 TRP A 131 1611 1836 1714 83 120 - 265 N
ATOM 177 CE2 TRP A 131 1.662 20. 154 7. 903 1. 00 11. 67 C
ANISOU 177 CE2 TRP A 131 1356 1559 1520 140 106 - 282 C
ATOM 178 CE3 TRP A 131 3.827 19. 759 8. 926 1. 00 13. 62 C
ANISOU 178 CE3 TRP A 131 1637 1701 1835 206 92 - 316 C
ATOM 179 CZ2 TRP A 131 1.843 21. .535 7. 935 1. 00 16. 41 C
ANISOU 179 CZ2 TRP A 131 1912 2170 2152 183 92 - 288 C
ATOM 180 CZ3 TRP A 131 4.012 21. 130 8. 950 1. 00 13. 53 C
ANISOU 180 CZ3 TRP A 131 1583 1703 1855 249 79 321 C
ATOM 181 CH2 TRP A 131 3.024 22. 005 8. 449 i: 00 13. 26 C
ANISOU 181 CH2 TRP A 131 1510 1726 1802 237 79 307 c
ATOM 182 N SER A 132 3.485 13. 728 7. 976 1. 00 10. 68 N
ANISOU 182 N . SER A 132 1452 1234 1372 11 139 - 303 N
ATOM 183 CA SER A 132 4.650 12. 859 8. 038 1. 00 9. 25 C
ANISOU 183 CA SER A 132 1306 979 1229 20 135 325 C
ATOM 184 C SER A 132 5.250 12. 768 6. 641 1. 00 14. 99 c
ANISOU 184 c SER A 132 2032 1679 1983 2 158 347 c ATOM 185 o SER A 132 4.641 13.223 5.665 1.00 10.93 o
ANISOU 185 o SER A 132 1498 1207 1449 -25 176 ■340 o
ATOM 186 CB SER A 132 4.309 11. 480 8. 601 1. 00 16. 24 C
ANISOU 186 CB SER A 132 2235 1848 2087 -14 127 •313 C
ATOM 187 OG SER A 132 3.034 11. 030 8. 187 1. 00 20. 49 O
A ISOU 187 OG SER A 132 2776 2438 2570 ' -70 141 •290 o
ATOM 188 N ILE A 133 6.461 12. 240 6. 548 1. 00 9. 85' N
ANISOU 188 N ILE A 133 1401 960 1380 18 157 376 N
ATOM 189 CA ILE A 133 7.042 11. 976 5. 243 1. 00 7. 25 C
ANISOU 189 CA ILE A 133 1075 604 1074 -4 182 400 C
ATOM 190 C ILE A 133 6.349 10. 757 4. 651 1. 00 9. 80 C
ANISOU 190 C ILE A 133 ■ 1429 935 1360 -66 195 393 C
ATOM 191 0 ILE A 133 5.549 10. 087 5. 330 1. 00 12. 55 o
ANISOU 191 0 ILE A 133 1797 1301 1670 -88 183 369 o
ATOM 192 CB ILE A 133 8.565 11. 750 5. 344 1. 00 12. 07 C
ANISOU 192 CB ILE A 133 1687 1177 1721 34 152 390 C
ATOM 193 CGI ILE A 133 8.841 10. 495 6. 178 1. 00 13. 82 C
ANISOU 193 CGI ILE A 133 1948 1358 1945 ' 33 127 389 C
ATOM 194 CG2 ILE A 133 9.244 12. 991 5. 946 1. 00 18. 49 C
ANISOU 194 CG2 ILE A 133 2474 2003 2549 88 129 371 C
ATOM 195 CD1 ILE A 133 10.298 10. 098 6. 228 1. 00 18. 73 C
ANISOU 195 CD1 ILE A 133 2580 1952 2586 61 100 376 C
ATOM 196 N TRP A 134 6.645 10. 454 3. 397 1. 00 11. 00 N
ANISOU 196 N TRP A 134 1394 1343 1443 308 -239 260 N
ATOM 197 CA TRP A 134 5.852 9. 455 2. 676 1. OQ 10. 42 C
ANISOU 197 CA TRP A 134 1338 1247 1373 293 -243 260 c
ATOM 198 C TRP A 134 6.012 8. 079 3. 314 1. 00 12. 15 c
ANISOU 198 C TRP A 134 1646 1374 1595 328 -319 - 275 c
ATOM 199 o TRP A 134 5.024 7. 399 3. 606 1. 00 15. 00 o
ANISOU 199 o TRP A 134 2076 1677 1946 296 -321 233 o
ATOM 200 CB TRP A 134 6.250 9. 425 1. 207 1. 00 10. 48 c
ANISOU 200 CB TRP A 134 1253 1330 1399 306 -231 - 315 c
ATOM 201 CG TRP A 134 5.414 8. 517 0. 356 1. 00 9. 32 c
ANISOU 201 CG TRP A 134 1115 1170 1255 286 -228 318 c
ATOM 202 CD1 TRP A 134 4.261 8. 835 -0. 274 1. 00 9. 91 c
ANISOU 202 CD1 TRP A 134 1170 1275 1322 227 -167 274 c
ATOM 203 CD2 TRP A 134 5.719 7. 178 -0. 009 1. 00 13. 13 c
ANISOU 203 CD2 TRP A 134 1625 1611 1752 325 -290 371 c
ATOM 204 NE1 TRP A 134 3.801 7. 765 -0. 999 1. 00 10. 99 N
ANISOU 204 NE1 TRP A 134 1319 1392 1465 224 -186 295 N
ATOM 205 CE2 TRP A 134 4.678 6. 728 -0. 843 1. 00 13. 66 C
ANISOU 205 CE2 TRP A 134 1690 1684 1818 284 -262 355 C
ATOM 206 CE3 TRP A 134 6.750 6. 302 0. 316 1. 00 11. 30 C
ANISOU 206 CE3 TRP A 134 1423 1337 1535 393 -369 430 C
ATOM 207 CZ2 TRP A 134 4.652 5. 450 -1. 381 1. 00 14. 85 C
ANISOU 207 CZ2 TRP A 134 1866 1797 1981 307 -310 - 399 c
ATOM 208 CZ3 TRP A 134 6.727 5, 033 -0. 213 1. 00 22. 69 c
ANISOU 208 CZ3 TRP A 134 2891 2739 2992 419 -417 - 473 c
ATOM 209 CH2 TRP A 134 5.685 4. 615 -1. 055 1. 00 18. 62 c
ANISOU 209 CH2 TRP A 134 2372 2228 2474 375 -388 - 459 c
ATOM 210 N HIS A 135 7.252 7. 669 3. 515 1. 00 14. 45 N
ANISOU 210 N HIS A 135 1935 1655 1900 392 -383 334 N
ATOM 211 CA HIS A 135 7.529 6. 372 4. 111 1. 00 17. 69 C
ANISOU 211 CA HIS A 135 2428 1977 2317 433 -464 353 C
ATOM 212 C HIS A 135 6.902 6. 204 5. 491 1. 00 12. 38 C
ANISOU 212 C HIS A 135 1858 1226 1621 407 -475 285 C
ATOM 213 o HIS A 135 6.361 5. 174 5. 797 1. 00 16. 40 o
ANISOU 213 o HIS A 135 2444 1659 2127 399 -511 265 o
ATOM 214 CB HIS A 135 9.036 6. 106 4. 165 1. 00 19. 59 c
ANISOU 214 CB HIS A 135 2642 2228 2576 511 -529 427 c
ATOM 215 CG HIS A 135 9.389 4. 698 4. 532 1. 00 35. 63 c A ISOU 215 CG HIS A 135 4751 4170 4619 561 -617 ■454 C
ATOM 216 ND1 HIS A 135 9.198 4. 184 5. 797 1 .00 36. 25 N
A ISOU 216 ND1 HIS A 135 . 4804 4255 4713 589 -654 ■509 N
ATOM 217 CD2 HIS A 135 9.925 3. 696 3. 798 1 .00 43. 23 C
ANISOU 217 CD2 HIS A 135 5803 5058 5566 562 -663 •417 C
ATOM 218 CE1 HIS A 135 9.598 2. 927 5. 827 1 .00 33. 41 C
ANISOU 218 CE1 HIS A 135 4516 3830 4350 605 -719 •502 C
ATOM 219 NE2 HIS A 135 10.047 2. 608 4. 627 1 .00 38. 09 N
ANISOU 219 NE2 HIS- A 135 5177 4374 4922 587 -725 ■446 N
ATOM 220 N · THR A 136 6.965 7. 237 6. 318 1 .00 10. 88 N
ANISOU 220 N THR A 136 1668 1054 1412 389 -444 ■249 N
ATOM 221 CA THR A 136 6.447 7. 132 7. 678 ί .00 14. 15 C
ANISOU 221 CA THR A 136 2175 1400 1799 367 -455 ■188 C
ATOM 222 C THR A 136 4.924 7. 125 -7. 640 1 .00 13. 44 C
ANISOU 222 C THR A 136 2119 1297 1691 296 -400 120 C
ATOM 223 o THR A 136 4.279 6. 487 8. 469 1 .00 11. 47 o
ANISOU 223 o THR A 136 1957 979 1423 273 -420 -74 o
ATOM 224 CB THR A 136 6.948 8. 305 8. 533 1 .00 12. 18 C
ANISOU 224 CB THR A 136 1913 1182 1535 368 -435 174 C
ATOM 225 OGl THR A 136 8.377 8. 218 8. 668 1 .00 15. 49 o
ANISOU 225 OGl THR A 136 2307 1609 1970 434 -494 236 o
ATOM 226 CG2 THR A 136 6.294 8. 311 9. 915 1 .00 15. 98 c
ANISOU 226 CG2 THR A 136 2485 1604 1982 337 -436 108 c
ATOM 227 N SER A 137 4.354 7. 841 6. 668 1 .00 11. 52 N
ANISOU 227 N SER A 137 1803 1122 1452 259 -330 ■113 N
ATOM 228 CA SER A 137 2.910 7. 833 6. 468 1 .00 11. 40 C
ANISOU 228 CA SER A 137 1806 1104 1422 194 -276 -53 C
ATOM 229 C SER A 137 2.444 6. 416 6. 143 1 .00 14. 79 C
ANISOU 229 C SER A 137 2289 1473 1859 190 -319 -58 c
ATOM 230 o SER A 137 1.438 5. 939 6. 672 1 .00 12. 41 o
ANISOU 230 o SER A 137 2055 1121 1537 147 -313 -2 o
ATOM 231 CB SER A 137 2.498 8. 799 5. 337 1 .00 10. 43 c
ANISOU 231 CB SER A 137 1588 1067 1306 163 -200 -53 c
ATOM 232 OG SER A 137 2.855 10. 138 5. 691 1 .00 12. 55 o
ANISOU 232 OG SER A 137 1814 1384 1569 , 161 -161 -43 o
ATOM 233 N GLN A 138 3.193 5. 736 5. 282 1 .00 15. 89 N
ANISOU 233 N GLN A 138 . 2395 1616 2025 236 -363 125 N
ATOM 234 CA GLN A 138 2.880 4. 348 4. 965 1 .00 14. 81 c
ANISOU 234 CA GLN A 138 2311 1416 1900 240 -413 139 C
ATOM 235 C GLN A 138. 2.955 3. 490 6. 236 1 .00 14. 13 C
ANISOU 235 C GLN A 138 2334 1232 1803 252 -479 112 C
ATOM 236 o GLN A 138 2.081 2. 659 6. 476 1 .00 18. 36 o
ANISOU 236 o GLN A 138 2940 1706 2329 216 -494 -73 o
ATOM 237 CB GLN A 138 3.820 3. 812 3. 885 1 .00 15. 61 C
ANISOU 237 CB GLN- A 138 2357 1541 2033 297 -453 226 C
ATOM 238 CG GLN A 138 3.643 2. 319 3. 582 1 .00 18. 43 C
ANISOU 238 CG GLN A 138 2771 1825 2406 311 -516 251 C
ATOM 239 CD GLN A 138 2.251 1. 952 3. 098 1 .00 22. 43 C
ANISOU 239 CD GLN A 138 3298 2320 2903 243· -478 205 C
ATOM 240 OEl GLN A 138 1.444 2. 816 2. 762 1 .00 14. 96 o
A ISOU 240 OEl GLN A 138 2308 1435 1943 189 -401 164 o
ATOM 241 NE2 GLN A 138 1.965 0. 649 3. 060 1 .00 23. 31 N
ANISOU 241 NE2 GLN A 138 3479 2353 3025 245 -534 213 N
ATOM 242. N GLU A 139 3.982 3. 708 7. 060 1 .00 13. 26 N
ANISOU 242 N GLU A 139 2238 1108 1691 301 -520 132 N
ATOM 243 CA GLU A 139 4.103 2. 943 8. 309 1 .00 1'5. 10 C
ANISOU 243 CA GLU A 139 2575 1250 1911 314 -585 104 c
ATOM 244 C GLU A 139 2.936 3. 234 9. 251 1 .00 18. 86 c
A ISOU 244 C GLU A 139 3109 1709 2348 243 -541 -15 c
ATOM 245 o GLU A 139 2.456 2. 342 9. 953 1 .00 21. 52 o
ANISOU 245 o GLU A 139 3502 2009 2664 211 -561 18 o ATOM 246 CB GLU A 139 5.435 3.219 9.,003. 1.00 22.60 C
ANISOU 246 CB GLU A 139 3510 2214 2861 370 -626 -140 C
ATOM 247 CG GLU A 139 ' 6.616 2. 529 8. 354 1. 00-35. 86 C
ANISOU 247 CG GLU A 139 5147 3908 4569 434 -683 -220 C
ATOM 248 CD GLU A 139 6.495 1. 013 8. .380 · 1. 00 55. 46 c
ANISOU 248 CD GLU A 139 7683 6336 7055 435 -740 -224 c
ATOM 249 OEl GLU A 139 5.871 0. 475 9. 323 1. 00 62. 60 o
ANISOU 249 OEl GLU A 139 8662 7191 7933 395 -751 -168 o
ATOM 250 OE2 GLU A 139 7.023 0. 358 7. 453 1. 00 57. 28 o
ANISOU 250 OE2 GLU A 139 7876 6576 7312 474 · -772 -284 o
ATOM 251 N ASN A 140 2.476 4. 482 9. 256 1. 00 16. 40 N
ANISOU 251 N ASN A 140 2751 1461 2021 207 -464 16 N
ATOM 252 CA ASN A 140 1.298 4. 844 10. 029 1. 00 15. 26 C
ANISOU 252 CA ASN A 140 2651 1307 1838 141 -414 95 C
ATOM 253 C ASN A 140 0.067 4. 069 ' 9. 547 1. 00 17. 95 C
ANISOU 253 C ASN A 140 3016 1629 2175 85 -396 130 C
ATOM 254 o ASN A 140 -0.707 3. 558 10. 348 1. 00 18. 70 o
ANISOU 254 o ASN A 140 3144 1717 2243 40 -386 171 o
ATOM 255 CB ASN A 140 1.042 6. 360 9. 982 1. 00 18. 19 C
ANISOU 255 CB' ASN A 140 2954 1759 2197 116 -331 114 C
ATOM 256 CG ASN A 140 2.094 7. 165 10. 749 1. 00 20. 68 C
ANISOU 256 CG ASN A 140 3259 2092 2508 157 -345 93 C
ATOM 257 ODl ASN A 140 2.783 6. 63.8 11. 632 1. 00 20. 08 o
ANISOU 257 ODl ASN A 140 3242 1963 2426 192 -410 84 o
ATOM 258 ND2 ASN A 140 2.224 8. 439 10. 406 1. 00 20. 34 N
ANISOU 258 ND2 ASN A 140 3139 2122 2469 152 -286 85 N
ATOM 259 N CYS A 141 -0.106 3. 966 8. 233 1. 00 15. 41 N
ANISOU 259 N CYS A 141 - 2637 1339 1879 83 ' ' -377 98 N
ATOM 260 CA CYS A 141 -1.212 3. 180 7. 699 1. 00 15. 45 C
ANISOU 260 CA CYS A 141 2668 1320 1883 33 -367 127 C
ATOM 261 C CYS A 141 -1.065 1. 708 8. 090 1. 00 15. 27 C
ANISOU 261 C CYS A 141 2716 1220 1864 45 -444 117 C
ATOM 262 o CYS A 141 -2.033 1. 059 8. 485 1. 00 18. 16 o
ANISOU 262 o CYS A 141 3113 1577 2210 -7 -433 159 o
ATOM 263 CB CYS A 141 -1.303 3. 342 6. 181 1. 00 15. 92 C
ANISOU 263 CB CYS A 141 2638 1442 1967 32 -333 84 C
ATOM 264 SG CYS A 141 -1.692 5. 045 5. 656 1. 00 15. 42 S
ANISOU 264 SG CYS A 141 2472 1492 1896 3 -229 102 S
ATOM 265 N LEU A 142 0.152 1. 187 7. 980 1. 00 13. 99 N
ANISOU 265 N LEU A 142 2557 1030 1728 115 -514 54 N
ATOM 266 CA LEU A 142 0.385 -0. 222 8. 280 1. 00 19. 50 C
ANISOU 266 CA LEU A 142 3303 1677 2430 129 -582 38 C
ATOM 267 C LEU A 142 0.027 -0. 563 9. 726 1. 00 22. 44 C
ANISOU 267 C LEU A 142 3729 2034 2765 95 -587. 90 C
ATOM 268 o LEU A 142 -0.412 -1. 681 10. 014 1. 00 24. 89 o
ANISOU 268 o LEU A 142 4085 2304 3068 72 -619 106 o
ATOM 269 CB LEU A 142 1.838 -0. 603 7. 986 1. 00 17. 10 c
ANISOU 269 CB LEU A 142 2977 1364 2157^ 212 -649 -40 c
ATOM 270 CG LEU A 142 2.217 -0. 616 6. 499 1. 00 17. 68 c
ANISOU 270 CG LEU A 142 2991 1458 2271 251 -654 111 c
ATOM 271 CD1 LEU A 142 3.712 -0. 816 6. 342 1. 00 26. 03 c
ANISOU 271 CD1 LEU A 142 4008 2532 3350 333 -708 187 c
ATOM 272 CD2 LEU A 142 1.448 -1. 713 5. 750 1. 00 23. 45 c
ANISOU 272 CD2 LEU A 142 3745 2152 3012 222 -669 113 c
ATOM 273 N LYS A 143 0.209 0. 397 10. 629 1. 00 18.72 N
ANISOU 273 N LYS A 143 3249 1593 2270 93 -556 113 N
ATOM 274 *CA LYS A 143^ -0.078 0. 160 12. 042 1. 00 22. 66 C
ANISOU 274 CA LYS A 143 3793 2083 2735 64 -559 154 C
ATOM 275 C LYS A 143 -1.574 -0. 024 12. 246 1. 00 30. 61 C
ANISOU 275 C LYS A 143 4807 3104 3719 -10 -506 210 C
ATOM 276 o LYS A 143 -2.021 -0. 610 13. 235 1. 00 27. 31 o ANISOU 276 o LYS A 143 4429 2670 3279 -38 -517 240 0
ATOM 277 CB LYS A 143 0.437 1. 314 12. 912 1 .00 21. 16 C
ANISOU 277 CB LYS A 143 3586 1927 2527 78 -534 160 C
ATOM 278 CG LYS A 143 1.949 1. 422 12. 973 1 .00 31. 52 C
ANISOU 278 CG LYS A 143 4890 3229 3856 151 * -591 105 C
ATOM 279 CD LYS A 143 2.367 2. 638 13. 786 1 .00 34. 07 C
ANISOU 279 CD LYS A 143 5197 3588 4160 159 -561 114 C
ATOM 280 CE LYS A 143 3.871 2. 674 14. 003 1 .00 42. 74 C
ANISOU 280 CE LYS A 143 6287 4680 5272 228 -621 61 C TOM F 281 NZ LYS A 143 4.264 3. 833 14. 866 1 .00 50. 90 N
ANISOU 281 NZ LYS A 143 7310 5744 6284 232 -596 71 N
ATOM 282 N GLU A 144 -2.346 0. 481 11. 295 1 .00 25. 92 N
ANISOU 282 N GLU A 144 4171 2544 3132 -40 -450 221 N
ATOM 283 CA GLU A 144 -3.795 0. 393 11. 364 1 .00 24. 25 C
ANISOU 283 CA GLU A 144 3950 2361 2904 -106 -394 268 C
ATOM 284 C GLU A 144 -4.325 -0. 701 10. 442 1 .00 21. 06 C
ANISOU 284 C GLU A 144 3563 1924 2515 -126 -419 262 C
ATOM .285 o GLU A 144 -5.515 -0. 736 10. 141 1 .00 27. 02 o
ANISOU 285 o GLU A 144 4296 2708 3262 -177 -371 293 o
ATOM 286 CB GLU A 144 -4.426 1. 737 11. 003 1 .00 29. 42 C
ANISOU 286 CB GLU A 144 4537 3088 3554 -130 -309 288 C
ATOM 287 CG GLU A 144 -5.379 2. 275 12. 057 1 .00 49. 43 C
ANISOU 287 CG GLU A 144 7049. 5670 6062 -166 -251 328 C
ATOM 288 CD GLU A 144 -4.720 2. 431 13. 417 1 .00 60. 66 c
ANISOU 288 CD GLU .A 144 8503 7078 7465 -145 -276 330 c
ATOM 289 OEl GLU A 144 -3.625 3. 029 13. 485 1 .00 67. 77 o
ANISOU 289 OEl GLU A 144 9405 7975 8372 -104 -295 304 o
ATOM 290 OE2 GLU A 144 -5.294 1. 948 14. 417 1 .00 62. 93 o
ANISOU 290 OE2 GLU A 144 8816 7360 7734 -170 -278 356 o
ATOM 291 N GLY A 145 -3.445 -1. 591 9. 995 1 .00 18. 20 N
ANISOU 291 N GLY A 145 3233 1504 2177 -8'2 -494 219 N
ATOM 292 CA GLY A 145 -3.845 -2. 639 9. 071 1 .00 19. 96 c
ANISOU 292 CA GLY A 145 3473 1692 2419 -95 -523 206 c
ATOM 293 C GLY A 145 -4.324 -2. 099 7. 733 1 .00 15. 92 c
ANISOU 293 C GLY A 145 2912 1212 1925 -113 -477 198 c
ATOM 294 o GLY A 145 -5.209 -2. 667 7. 091 1 .00 21. 72 o
ANISOU 294 o GLY A 145 3648 1941 2662 -155 -467 210 o
ATOM 295 N SER A 146 -3.734 -0. 986 7. 316 1 .00 15. 84 N
ANISOU 295 N SER A 146 2857 1234 1925 -84 -451 177 N
ATOM 296 CA SER A 146 -4.110 -0. 346 6. 068 1 .00 16. 51 C
ANISOU 296 CA SER A 146 2895' 1351 2027 -102 -405 169 C
ATOM 2 7 C SER A 146 -2.865 0. 036 5. 278 1 .00 15. 03 C
ANISOU 297 C SER A 146 2665 1171 1875 -31 -432 95 C
ATOM 298 o SER A 146 -1.741 -0. 288 5. 668 1 .00 16. 42 o
ANISOU 298 o SER A 146 2864 1309 2067 33 -496 52 o
ATOM 299 CB SER A 146 -4.993 0. 885 6. 316 1 .00 16. 41 C
ANISOU 299 CB SER A 146 2835 1416 1986 -152 -312 224 C
ATOM 300 OG SER A 146 -4.270 1. 922 6. 973 1 .00 17. 74 o
ANISOU 300 OG SER A 146 2986 1608 2146 -119 -297 223 o
ATOM 301 N THR A 147 -3.077 0. 729 4. 166 1 .00 14. 06 N
ANISOU 301 N THR A 147 2448 1133 1760 -42 -372 73 N
ATOM 302 CA THR A 147 -1.981 1. 153 3. 312 1 .00 14. 64 C
ANISOU 302 CA THR A 147 2439 1265 1860 19 ; -376 -5 C
ATOM 303 C THR A 147 -2.305 2. 541 2. 765 1 .00 13. 94 C
ANISOU 303 C THR A 147 2256 1280 1762 -7 -287 11 C
ATOM 304 0 THR A 147 -3.445 2. 998 2. 849 1 .00 12. 01 o
ANISOU 304 o THR A 147 2009 · 1060 1495 -70 -226 74 o
ATOM 305 CB THR A 147 -1.806 0. 156 2. 143 1 .00 23. 13 C
ANISOU 305 CB THR A 147 3497 2327 2963 40 -415 -69 C
ATOM 306 OGl THR A 147 -0.548 0.377 1. 495 1 .00 25. 00 o
ANISOU 306 OGl THR A 147 3666 2608 3225 111 -436 -150 o ATOM 307 CG2 THR A 147 -2.958 0.282 1.141 1.00 20.01 C
ANISOU 307 CG2 THR A 147 3058 1984 2562 -23 -355 -46 C
ATOM ¾D8 N LEU A 148 -1.314 3. 2.44 2 .236 1 .00 11 .00 N
ANISOU 308 N LEU A 148 1804 969 1404 41 -278 -43 N
ATOM 309 CA LEU A 148 -1.638 4. 551 1 .677 1 .00 11 .85 C
ANISOU 309 CA LEU A 148 1825 1172 1505 13 -196 -25 C
ATOM 310 C LEU A 148 -2.698 4. 404 0 .575 1 .00 11 .34 C
ANISOU 310 C LEU A 148 1723 1146 1439 -38 -152 -11 C
ATOM 311 o LEU A 148 -2.719 3. 410 -0 .164 1 .00 10 .46 o
ANISOU 311 o LEU Α· 148 1618 1014 1343 -32 -188 -50 o
ATOM 312 CB LEU A 148 -0.385 5. 267 1 .166 1 .00 8 .37 C
ANISOU 312 CB LEU A 148 1303 795 1082 68 -197 -87 C
ATOM 313 CG LEU A 148 0.501 5. 923 2 .227 1 .00 11 .73 C
ANISOU 313 CG LEU A 148 1742 1212 1503 104 -212 -87 c
ATOM 314 CDl LEU A 148 1.784 6. 413 1 .598 1 .00 12 .57 c
ANISOU 314 CDl LEU A 148 1768 1379 1629 159 -223 ■156 c
ATOM 315 CD2 LEU A 148 -0.234 7. 076 2 .894 1 .00 13 .49 c
ANISOU 315 CD2 LEU A 148 1964 1461 1702 58 -145 -17 c
ATOM 316 N LEU A 149 -3.595 5. 380 0 .492 1 .00 9 .35 N
ANISOU 316 LEU A 149 1434 949 1170 -89 -76 44 N
ATOM 317· CA LEU A 149 -4.632 5. 415 -0 .544 1 .00 10, .45 c
ANISOU 317 CA LEU A 149 1527 1136 1305 -140 -28 63 C
ATOM 318 C LEU A 149 -4.107 5. 033 -1 .923 1 .00 10, .59 C
ANISOU 318 C LEU A 149 1481 1198 1346 -114 -44 -7 C
ATOM 319 o LEU A 149 -3.093 5. 575 -2 .379 1 .00 11, .11 o
ANISOU 319 o LEU A 149 1483 1313 1424 -70 -45 -57 o
ATOM 320 CB LEU A 149 -5.195 6. 841 -0 .622 1 .00 9 .74 C
ANISOU 320 CB LEU A 149 1377 1123 1203 -174 55 111 C
ATOM 321 CG LEU A 149 -6.231 7. 136 -1 .713 1 .00 12 .18 C
ANISOU 321 CG LEU A 149 1625 1497 1507 -225 114 135 C
ATOM 322 CDl LEU A 149 -7.47.1 6. 261 -1 .583 1 .00 15 .79 C
ANISOU 322 CDl LEU A 149 2138 1911 1949 -280 113 179 C
ATOM 323 CD2 LEU A 149 -6.602 8. 608 -1 .660 1 .00 14 .05 C
ANISOU 323 CD2 LEU A 149 1804' 1800 1734 -246 187 179 C
ATOM 324 N GLN A 150 -4.796 4. 105 -2 .585 1 .00 7 .87 N
ANISOU 324 N GLN A 150 1152 835 1002 -143 -56 -13 N
ATOM 325 CA GLN A 150 -4.542 3. 841 -3 .991 1 .00 10 .45 C
ANISOU 325 CA GLN A 150 1410 1215 1344 -131 -58 -73 C
ATOM 326 C GLN A 150 -5.817 4. 163 -4 .744 1 .00 13 .43 C
ANISOU 326 C GLN A 150 1747 1647 1706 -198 6 -27 C
ATOM 327 o GLN A 150 -6.915 3. 836 -4 .298 1 .00 11 .64 o
ANISOU 327 o GLN A 150 1573 1384 1465 -251 18 31 o
ATOM 328 CB GLN A 150 -4.112 2. 398 -4 .210 1 .00 14.78 c
ANISOU 328 CB GLN A 150 2010 1696 1911 -98 -136 132 c
ATOM 329 CG GLN A 150 -2.879 2. 083 -3 .404 1 .00 14 .94 c
ANISOU 329 CG GLN A 150 2072 1661 1945 -29 -200 •173 c
ATOM 330 CD GLN A 150 -2.392 0. 666 -3 .585 1 .00 16 .35 c
ANISOU 330 CD GLN A 150 2302 1766 2145 11 -283 235 c
ATOM 331 OEl GLN A 150 -1.348· 0. 301 -3 .063 1 .00 21 .47 o
ANISOU 331 OEl GLN A 150 2981 .2370 2808 73 -342 277 o
ATOM 332 NE2 GLN A 150 -3.144 -0. 137 -4 .334 1 .00 15 .90 N
ANISOU 332 NE2 GLN A 150 2255 1695 2090 -23 -288 •243 N
ATOM 333 N ILE A 151 -5.679 4. 860 -5 .858 1 .00 10 .48 N
ANISOU 333 N ILE A 151 1279 1367 1336 -198 47 -50 N
ATOM 334 CA ILE A 151 -6.840 5. 311 -6 .595 1 .00 9 .56 c
ANISOU 334 CA ILE A 151 J 1114 1312 1205 -259 110 -5 c
ATOM 335 C ILE A 151 -6.836 4. 563 -7 .921 1 .00 13 .50 c
ANISOU 335 C ILE A 151 1574 1843 1711 -260 92 -62 c
ATOM 336 0 ILE A 151 -6.019 4. 833 -8 .805 1 .00 13 .85 o
ANISOU 336 0 ILE A 151 1547 1951 1765 -225 92 ■120 o
ATOM 337 CB ILE A 151 -6.782 6. 814. -6 .808 1 .00 14 .28 c ANISOU 337 CB ILE A 151 1633 1995 1798 -265 175 23 C
ATOM 338 CGI ILE A 151 -6.546 7. 514 -5 .454 1 .00 15. 75 C
ANISOU 338 CGI ILE A 151 1861 2145 1979 -253 182 63 c
ATOM 339 CG2 ILE A 151 -8.056 7. 302 -7 .491 1 .00 15. 10 c
ANISOU 339 CG2 ILE A 151 1707 ' 2136 1894 , -300 223 74 c
ATOM 340 CD1 ILE A 151 -6.203 8. 986 -5 .565 1 .00 19. 49 c
ANISOU 340 CD1 ILE A 151 2264 2688 2455 -244 232 78 c
ATOM 341 N GLU A 152 -7.730 3. 595 -8 .041 1 .00 13. 30 N
ANISOU 341 N GLU A 152 1598 1774 1682 -300 76 -48 N
ATOM 342 CA GLU A 152 -7.695 2. 697 -9 .196 1 .00 15. 23 C
ANISOU 342 CA GLU A 152 1820 2032 1933 -297 48 ■109 C
ATOM 343 C GLU A 152 -8.753 3. 004 -10 .245 1 .00 18. 76 C
ANISOU 343. C GLU A 152 2207 f 2557 2366 -356 103 -81 C
ATOM 344 o GLU A 152 -8.873 2. 279 -11 .230 1 .00 21. 08 o
ANISOU 344 o GLU A 152 2481 2866 2662 -363 84 ■127 o
ATOM 345 CB GLU A 152 -7.824 1. 237 -8 .755 1 .00 21. 85 C
ANISOU 345 CB GLU A 152 2756 2763 2781 -295 -23 •128 c
ATOM 346 CG GLU A 152 -6.561 0. 652 -8 .121 1 .00 26. 64 c
ANISOU 346 CG GLU A 152 3413 3300 3409 -222 -95 •185 c
ATOM 347 CD GLU A 152 -6.602 0. 665 -6 .605 1 .00 26. 04 c
ANISOU 347 CD GLU A 152 3421 3144 3329 -222 -113 •130 c
ATOM 348 OEl GLU A 152 -7.515 1. 305 -6 .051 1 .00 19. 07 o
ANISOU 348 OEl GLU A 152 2548 2273 2426 -275 -61 -51 o
ATOM 349 OE2 GLU A 152 -5.737 0. 022 -5 .972 1 .00 27. 13 o
ANISOU 349 OE2 GLU A 152 3616 3209 3482 -170 -178 ■167 o
ATOM 350 N SE A 153 -9.524 4. 067 -10 .040 1 .00 15. 00 N
ANISOU 350 N SER A 153 1698 2127 1873 -398 169 -8 N
ATOM 351 CA SER A 153 -10.635 4. 358 -10 .936 1 .00 13. 96 C
ANISOU 351 CA SER A 153 1537 2034 1732 -412 201 27 C
ATOM 352 C SER A 153 -11.060 5. 801 -10 .827 1 .00 14. 85 C
ANISOU 352 C SER A 153 1655 2124 1861 -330 216 74 C
ATOM 353 o SER A 153 -10.773 6. 468 -9 .830 1 .00 13. 19 o
ANISOU 353 o SER A 153 1464 1886 1660 -306 223 96 o
ATOM i 354 CB SER A 153 -11.842 3. 467 -10 .628 1 .00 13. 59 c
ANISOU 354 CB SER A 153 1551 1937 1676 -466 193 65 c
ATOM 355 OG SER A 153 -12.509 3. 885 -9 .448 1 .00 18. 38 o
ANISOU 355 OG SER A 153 2210 2490 2285 -442 199 125 o
ATOM 356 N LYS A 154 -11.748 6. 283 -11 .857 1 .00 11. 68 N
ANISOU 356 N LYS A 154 1400 1446 1591 -50 -6 367 N TOM 357 CA LYS A 154 -12.279 7. 632 -11 .828 1 .00 12. 44 c
ANISOU 357 CA LYS A 154 1454 1524. 1749 -33 -7 344 C
ATOM 358 C LYS A 154 -13.323 7. 740 -10 .731 1 .00 16. 46 C
ANISOU 358 C LYS A 154 1941 2079 2233 -42 -21 293 C
ATOM 359 o LYS A 154 -13.418 8. 770 -10 .046 1 .00 14. 68 o
ANISOU 359 P LYS A 154 1675 1846 2058 -32 -27 250 o
ATOM 360 CB LYS A 154 -12.884 8. 015 -13 .182 1 .00 10. 26 C
ANISOU 360 CB LYS A 154 1183 1227 1487 -19 3 389 C
ATOM 361 CG LYS A 154 -13.539 9. 3,77 -13 .166 1 .00 14. 31 C
ANISOU 361 CG LYS A 154 1653 1722 2061 -1 2 368 C
ATOM 362 CD LYS A 154 -14.043 9. 757 -14 .548 1 .00 15. 03 c
ANISOU 362 CD LYS A 154 1750 1792 2168 16 12 417 c
ATOM 363 CE LYS A 154 -14.560 11. 183 -14 .569 1 .00 18. 54 c
ANISOU 363 CE LYS A 154 2151 2211 2683 37 12 400 c
ATOM 364 NZ LYS A 154 -15.797 11. 335 -13 .758 1 .00 18. 21 N
ANISOU 364 NZ LYS A 154 2088 2212 2620 31 -1 35 N
ATOM 365 N GLU A 155 -14.094 6. 672 -10 .547 1 .00 13. 30 N
ANISOU 365 N GLU A 155 1566 1727 1759 -62 -26 297 N
ATOM 366 CA GLU A 155 -15.113 6. 671 -9 .502 1 .00 15. 36 c
ANISOU 366 CA GLU A 155 1808 2037 1990 -72 -36 252 C
ATOM 367 C GLU A 155 -14.488 6. 962 -8 .131 1 .00 16. 54 c
ANISOU 367 C GLU A 155 1933 2197 2155 -72 -45 198 c ATOM 368 0 GLU A 155 -14.998 7.778 -7.362 1.00 15.99 o
ANISOU 368. 0 GLU A 155 1826 2140 2109 -65 -52 152 o
ATOM 369 CB GLU A 155 -15.877 5. 346 -9 .480 1 .00 22. 40 C
ANISOU 369 CB GLU A 155 2734 2979 2800 -95 -39 268 C
ATOM 370 CG GLU A 155 -17.009 5. 319 -8 .473 1 .00 34. 59 c
ANISOU 370 CG GLU A 155 4256 4575 4311 -106 -47 227 c
ATOM 371 CD GLU A 155 -17.646 3. 950 -8 .351 1 .00 46. 61 c
ANISOU 371 CD GLU A 155 5811 6144 5755 -132 -49 243 c
ATOM 372 OEl GLU A 155 -18.599 3. 805 -7 .555 1 .00 50. 27 o
ANISOU 372 OEl GLU A 155 6259 6655 6186 -143 -54 215 o
ATOM 373 OE2 GLU A 155 -17.188 3. 019 -9 .049 1 .00 49. 01 o
ANISOU 373 OE2 GLU A 155 6156 6438 6030 -142 -45 284 o
ATOM 374 N GLU A 156 -13.368 6. 309 -7 .842 1 .00 12. 60 N
ANISOU 374 N GLU A 156 1454 1691 1641 -80 -46 205 N
ATOM 375 CA GLU A 156 -12.710 6. 478 -6 .547 1 .00 11. 81 C
ANISOU 375 CA GLU A 156 1335 1605 1549 -80 -56 156 c
ATOM 376 C GLU A 156 -12.068 7. 863 -6 .432 1 .00 15. 15 c
ANISOU 376 C GLU A 156 1717 1981 ' 2060 -59 -58 128 c
ATOM 377 o GLU A 156 -12.112 8. 501 -5 .367 1 .00 12. 45 o
ANISOU 377 o GLU A 156 1341 1652 1737 -54 -69 72 o
ATOM 378 CB GLU A 156 -11.667 5. 383 -6 .336 1 .00 13. 28 c
ANISOU 378 CB GLU A 156 1553 1794 1696 -92 -56 174 c
ATOM 379 CG GLU A 156 -10.970 5. 472 -4 .998 1 .00 13. 76 c
ANISOU 379 CG GLU A 156 1596 1873 1759 -93 -68 125 c
ATOM 380 CD GLU A 156 -10.208 4. 211 -4 .650 1 .00 19. 46 c
ANISOU 380 CD GLU A 156 2353 2614 2427 -107 -70 141 c
ATOM 381 OEl GLU A 156 -10.072 3. 918 -3 .447 1 .00 15. 53 o
ANISOU 381 OEl GLU A 156 1848 2153 1898 -112 -81 105 o
ATOM 382 OE2 GLU A 156 -9.742 3. 504 -5 .571 1 .00 15. 19 o
ANISOU 382 OE2 GLU A 156 1848 2052 1873 -111 -61 191 o
ATOM 383 N MET A 157 -11.481 8. 333 -7 .531 1 .00 13. 02 N
ANISOU 383 N MET A 157 1449 1657 1841 -47 -47 166 N
ATOM 384 CA MET A 157 -10.893 9. 670 -7 .544 1 .00 13. 33 c
ANISOU 384 CA MET A 157 1450 1646 1970 -28 -47 145 c
ATOM 385 C MET A 157 -11.962 10. 710 -7 .233 1 .00 14. 86 C
ANISOU 385 c MET A 157 1605 1845 • 2195 -16 -53 108 c
ATOM 386 o MET A 157 -11.744 11. 610 -6 .421 1 .00 15. 02 o
ANISOU 386 o MET A 157 1588 1855 2264 -7 -63 56 o
ATOM 387 CB MET A 157 -10.223 9. 978 -8 .886 1 .00 13. 68 c
ANISOU 387 CB MET A 157 1504 1633 2062 -16 -31 201 c
ATOM 388 CG MET A 157 -9.495 11. 313 -8 .928 1 .00 16. 36 c
ANISOU 388 CG MET A 157 1803 1915 2499 1 -30 185 c
ATOM 389 SD MET A 157 -8.093. 11. 368 -7 .794 1 .00 16. 66 S
ANISOU 389 SD MET A 157 1823 1944 2564 -2 -42 141 S
ATOM 390 CE MET A 157 -7.415 12. 991 -8 .154 1 .00 17. 91 c
ANISOU 390 CE MET A 157 1934 2026 2844 18 -38 133 c
ATOM 391 N ASP A 158 -13.118 10. 571 -7 .874 1 .00 12. 37 N
ANISOU 391 N ASP A 158 1300 1549 1853 -17 -48 132 N
ATOM 392 CA ASP A 158 -14.244 11. 465 -7 .636 1 .00 14. 56 c
ANISOU 392 CA ASP A 158 1543 1835 2153 -6 -52 100 c
ATOM 393 C ASP A 158 -14.709 11. 407 -6 .184 1 .00 16. 58 c
ANISOU 393 C ASP A 158 1780 2143 2377 -12 -66 36 c
ATOM 394 o ASP A 158 -15.084 12. 429 -5 .601 1 .00 17. 61 o
ANISOU 394 o ASP A 158 1870 2269 2551 2 -74 -11 o
ATOM 395 CB ASP A 158 -15.401 11. 138 -8 .581 1 .00 19. 09 c
ANISOU 395 CB ASP A 158 2135 2427 2692 -7 -45 140 c
ATOM 396 CG ASP A 158 -15.093 11. 503 -10 .025 1 .00 21. 15 c
ANISOU 396 CG ASP A 158 2406 2635 2993 6 -32 197 c
ATOM 397 ODl ASP A 158 -14.147 12. 290 -10 .257 1 .00 22. 77 o
ANISOU 397 ODl ASP A 158 2595 2787 3268 20 -27 201 o
ATOM 398 OD2 ASP A 158 -15.798 11. 012 -10 .933 1 .00 24. 40 o ANISOU 398 OD2 ASP A 158 2842 3060 3369 4 -26 239 o
ATOM 399 N PHE A 159 -14.688 10. 218 -5 .591 1 .00 13. .94 N
ANISOU 399 N PHE A 159 1472 1857 1968 -32 -70 35 N
ATOM 400 CA PHE A 159 -15.054 10. 125 -4 .181 1 .00 15. .61 C
ANISOU 400 CA PHE A 159 1666 2120 2146 -37 -82 -23 C
ATOM 401 C PHE A 159 -14.044 10. 831 -3 .296 1 .00 19. .31 C
ANISOU 401 C PHE A 159 2106 2568 2663 -26 -93 -72 C
ATOM 402 0 PHE A 159 -14.409 11. 570 -2 .379 1 .00 20. .22 o
ANISOU 402 o PHE A 159 2185 2699 2797 -16 -103 •130 o
ATOM 403 ' CB PHE A 159 -15.185 8. 678 -3 .719 1 .00 17. 37 c
ANISOU 403 CB PHE A 159 1924 2397 2279 -61 -82 -10 c
ATOM 404 CG PHE A 159 -15.489 8. 558 -2 .258 1 .00 19. .22 c
ANISOU 404 CG PHE A 159 2140 2686 2476 -64 -93 -66 c
ATOM 405 CD1 PHE A 159 -16.787 8. 677 -1 .801 1 .00 22. 86 c
ANISOU 405 CD1 PHE A 159 2583 3193 2908 -65 -94 -90 c
ATOM 406 CD2 PHE A 159 -14.472 8. 363 -1 .341 1 .00 22. 52 c
ANISOU 406 CD2 PHE A 159 2557 3112 2889 -65 -102 -94 c
ATOM 407 CE1 PHE A 159 -17.073 8. 583 -0 .455 1 .00 25. 63 c
ANISOU 407 CE1 PHE A 159 2917 3598 3223 -66 -102 •141 c
ATOM 408 CE2 PHE A 159 -14.751 8. 272 0 .012 1 .00 22. 06 c
ANISOU. 408 CE2 PHE A 159 2482 3107 2793 -66 -112 ■146 c
ATOM 409, CZ PHE A 159 -16.055 8. 382 0 .450 1 .00 23. 64 c
ANISOU 409 CZ PHE A 159 2666 3354 2963 -66 -Ill •169 c
ATOM 410 N ILE A 160 -12.767 10. 594 -3 .561 1 .00 16. 21 N
ANISOU 410 N ILE A 160 1728 2141 2291 -28 -91 -51 N
ATOM 411 CA ELE A 160 -11.724 11. 183 -2 .740 1 .00 20. 49 C
ANISOU 411 CA ILE A 160 2244 2663 2878 -20 -103 -97 c
ATOM 412 C ILE A 160 -11.744 12. 702 -2 .856 1 .00 24. 31 c
ANISOU 412 C ILE A 160 2683 3099 3454 2 -106 •127 c
ATOM 413 o ILE A 160 -11.687 13. 412 -1 .849 1 .00 23. 36 o
ANISOU 413 o ILE A 160 2528 2986 3361 11 -120 190 0
ATOM 414 CB ILE A 160 -10.332 10. 648 -3 .102 1 .00 13. 38 c
ANISOU 414 CB ILE A 160 1366 1731 1985 -25 -100 -64 c
ATOM 415 CGI ILE A 160 -10.222 9. 171 -2 .716 1 .00 16. 69 c
ANISOU 415 CGI ILE A 160 1826 2200 2315 -45 -100 -45 c
ATOM 416 CG2 ILE A 160 -9.259 11. 496 -2 .410 1 .00 13. 88 c
ANISOU 416 CG2 ILE A 160 1395' 1764 2115 -14 -112 ■111 c
ATOM 417 CD1 ILE A 160 -10.434 8. 876 -1 .218 1 .00 16. 03 c
ANISOU 417 CD1 ILE A 160 1731 2177 2184 -49 -116 ■102 c
ATOM 418 N THR A 161 -11.846 13. 214 -4 .075 1 .00 17. 80 N
ANISOU 418 N THR A 161 1859 2225 2677 10 -93 -83 N
ATOM 419 CA THR A 161 -11.841 14. 665 -4 .226 1 .00 26. 03 C
ANISOU 419 CA THR A 161 2860 3217 1 3812 30 -95 108 C
ATOM 420 C THR A 161 -13.076 15. 277 -3 .555 1 .00 28. 38 C
ANISOU 420 C THR A 161 3129 3548 4107 40 -103 159 c
ATOM 421 o THR A 161 -12.988 16. 322 -2 .909 1 .00 33. 41 o
ANISOU 421 o THR A 161 3726 4166 4802 55 -114 214 o
ATOM 422 CB THR A 161 -11.672 15. 104 -5 .691 1 .00 21. 89 c
ANISOU 422 CB THR A 161 2343 2634 3342 39 -78 -46 c
ATOM 423 OGl THR A 161 -12.713 14. 543 -6 .493 1 .00 24. 51 o
ANISOU 423 OGl THR A 161 2700 2990 3622 35 -67 -2 o
ATOM 424 CG2 THR A 161 -10.328 14. 619 -6 .221 1 .00 25. 70 c
ANISOU 424 CG2 THR A 161 2848 3083 3835 32 -70 -4 c
ATOM 425 N GLY A 162 -14.212 14. 597 -3 .672 1 .00 24. 97 N
ANISOU 425 N GLY A 162 2715 3165 3606 32 -99 142 N
ATOM 426 CA GLY A 162 -15.431 15. 029 -3 .008 1 .00 28. 64 C
ANISOU 426 CA GLY A 162 3155 3671 4057 39 -105, 187 C
ATOM 427 C GLY A 162 -15.301 15. 035 -1 .494 1 .00 31. 19 C
A ISOU 427 C GLY A 162 3458 4036 4357 40 -121 258 C
ATOM 428 o GLY A 162 , -15.708 15. 990 -0 .838 1 .00 38. 20 o
ANISOU 428 o GLY A 162 ' 4308 4926 5282 56 -130 315 o ATOM 429 0 SER A 163 -14.021 15.650 2.021 1.00 35.87 o
ANISOU 429' 0 SER A 163 3990 4691 4947 54 -166 ■439 o
ATOM 430 N SE A 163 -14.733 13. 969 -0. 936 1. 00 16. 42 N
ANISOU 430 N SER A 163 1614 2202 2425 23 -124 •255 N
ATOM 431 CA SER A 163 -14.534 13. 869 0. 505 1. 00 21. 88 C
ANISOU 431 CA SER A 163 2289 2938. 3085 23 -139 318 C
ATOM 432 C SER A 163 -13.663 14. 999 1. 046 1. 00 30. 50 C
ANISOU 432 C SER A 163 3344 3989 4258 41 -154 '373 C
ATOM 433 CB SER A 163 -13.932 12. 508 0. 883 1. 00 26. 94 C
ANISOU 433 CB SER A 163 2968 3618 3652 3 -140 ■297 C
ATOM 434 OG SER A 163 -14.845 11. 449 0. 610 1. 00 30. 82 o
ANISOU 434 OG SER A 163 3489 4154 4065 -15 -129 257 o
ATOM 435 0 LEU A 164 -12.035 18. 477 1. 709 1. 00 46. 76 o
ANISOU 435 0 LEU A 164 5292 5904 6570 90 -186 501 o
ATOM 436 N LEU A 164 -12.521 15. 233 0. 413 1. 00 37. 49 N
ANISOU 436 N LEU A 164 4232 4813 5199 41 -152 347 N
ATOM 437 CA LEU A 164 -11.617 16. 275 0. 882 1. 00 35. 37 C
ANISOU 437 CA LEU A 164 3928 4500 5012 55 -166 396 C
ATOM 438 C LEU A 164 -12.286 17. 637 0. 848 1. 00 38. 66 C
ANISOU 438 C LEU A 164 4302 4886 5500 76 -169 434 C
ATOM 439 CB LEU A 164 -10.332 16. 309 0. 054 1. 00 34. 11 C
ANISOU 439 CB LEU A 164 3778 4277 4906 51 -160 353 C
ATOM 440 CG LEU A 164 -9.470 15. 049 0. 102 1. 00 29. 48 c
ANISOU 440 CG LEU A 164 3229 3713 4261 33 -158 319 c
ATOM 441 CD1 LEU A 164 -8.294 15. 189 -0. 846 1. 00 27. 1.9 ' c
ANISOU 441 CDl' LEU A 164 2945 3355 4030 32 -149 273 c
ATOM 442 CD2 LEU A 164 -9.003 14. 765 1. 523 1. 00 36. 17 c
ANISOU 442 CD2 LEU A 164 4066 4605 5072 32 -179 380 c
ATOM 443 o ARG A 165 -14.913 20. 614 1. 315 1. 00 37. 62 o
ANISOU 443 o ARG A 165 4072 ,4734 5486 133 -179 547 o
ATOM 444 N ARG A 165 -13.136 17. 866 -0. 146 1. 00 32. 12 N
ANISOU 444 N ARG A 165 3480 4040 4684 80 -154 391 N
ATOM 445 CA ARG A 165 -13.803 19. 158 -0. 243 1. 00 35. 85 C
ANISOU 445 CA ARG A 165 3914 4481 5227 101 -157 422 C
ATOM 446 C ARG A 165 -14.620 19. 455 1. 012 1. 00 37. 46 C
ANISOU 446 c ARG A 165 4091 4740 5403 112 -171 498 C
ATOM 447 CB ARG A 165 -14.677 19. 235 -1. 496 1. 00 37. 45 C
ANISOU 447 CB ARG A 165 4129 4666 5436 104 -138 362 C
ATOM 448 CG ARG A 165 -13.920 19. 657 .-2. 742 1. 00 49. 01 C
ANISOU 448 CG ARG A 165 5598 6053 6970 107 -126 304 c
ATOM 449 CD ARG A 165 -14.859 19. 877 -3. 914 1. 00 56. 20 c
ANISOU 449 CD ARG A 165 6516 6947 7889 115 -Ill 251 c
ATOM 450 NE ARG A 165 -14.138 20. 278 -5. 117 1. 00 66. 94 N
ANISOU 450 NE ARG A 165 7882 8238 9314 120 -97 193 N
ATOM 451 CZ ARG A 165 -14.724 20. 611 -6. 263 1. 00 72. 78 C
ANISOU 451 CZ ARG A 165 8626 8951 10076 130 -83 141 c
ATOM 452 NHl ARG A 165 -16,046 20. 592 -6. 365 1. 00 77. 79 N
ANISOU 452 NHl ARG A 165 9259 9622 10675 136 -82 142 N
ATOM 453 NH2 ARG A 165 -13.988 20. 966 -7. 310 1. 00 68. 67 N
ANISOU 453 NH2 ARG A 165 8110 8369 9614 135 -70 -87 N
ATOM 454 o LYS A 166 -15.313 19. 000 5. 284 1. 00 48. 21 o
ANISOU 454 o LYS A 166 5409 6297 6613 126 -216 707 o
ATOM 455 N LYS A 166 " -14.972 18. 402 1. 744 1. 00 31. 64 N
ANISOU ί 455 N LYS A 166 3375 4078 4571 99 -173 507 N
ATOM 456 CA LYS A 166 -15.756 18. 537 2. 967 1. 00 41. 60 C
ANISOU 456 CA LYS A 166 4613 5400 5791 108 -184 574 C
ATOM 457 C LYS A 166 -14.848 18. 818 4. 160 1. 00 44. 94 C
ANISOU 457 C LYS A 166 5016 5832 6228 115 -204 643 C
ATOM 458 CB LYS A 166 -16.563 17. 261 3. 221 1. 00 53. 00 C
ANISOU 458 CB LYS A 166 6088 6923 7126 91 -175 ■549 C
ATOM 459 CG LYS A 166 -17.282 16. 716 1. 992 1. 00 59. 19 C ANISOU 459 CG LYS A 166 6901 7702 7887 79 -156 ■474 C
ATOM 460 CD LYS A 166 -18.309 17. 701 1. 456 1 .00 66. .52 C
ANISOU 460 CD LYS A 166 7802 8608 8864 97 -151 •478 C
ATOM 461 CE LYS A 166 -19.000 17. 167 0. 207 1 .00 68. 96 C
ANISOU 461 CE LYS A 166 8139 8913 9149 86 -134 ■404 c
ATOM 462 NZ LYS A 166 -18.100 17. 152 -0. 980 1 .00 68. 84 N
ANISOU 462 NZ LYS A 166 8145 8832 9179 81 -126 ■344 N
ATOM 463 0 ILE A 167 -11.670 20. 875 3. 588 ' 1 .00 34. 32 o
ANISOU 463' o ILE A 167 4487 4269 4284 245 -128 320 o
ATOM 464 N ILE A 167 -13.546 18. 846 3. 905 1 .00 37. 61 N
ANISOU 464 N ILE A 167 4859 4731 4701 253 -131 ■325 N
ATOM 465 CA ILE A 167 -12.554 19. 101 4. 944 1 .00 41. 22 c
ANISOU 465 CA ILE A 167 5328 5170 5162 237 -130 325 C
ATOM 466 C ILE A 167 -11.941 20. 485 4. 725 1 .00 39. 53 C
ANISOU 466 C ILE A 167 5141 4934 4943 238 -132 325 C
ATOM 467 CB ILE A 167 -11.463 18. 000 4. 956 1 .00 34. 31 C
ANISOU 467 CB ILE A 167 4441 4292 4305 220 -122 317 C
ATOM 468 CGI ILE A 167 -11.992 16. 723 5. 615 1 .00 37. 25 C
ANISOU 468 CGI ILE A 167 4790 4683 4683 215 -123 319 c
ATOM 469 CG2 ILE A 167 -10.232 18. 461 5. 705 1 .00 34. 56 c
ANISOU 469 CG2 ILE A 167 4489 4299 4342 203 -122 315 c.
ATOM 470 CD1 ILE A 167 -13.205 16. 113 4. 941 1 .00 40. 56 c
ANISOU 470 CD1 ILE A 167 5189 5125 5098 229 -125 322 c
ATOM 471 o LYS A 168 -9.040 21. 726 5. 192 1 .00 23. 56 o
ANISOU 471 O' LYS A 168 3156 2857 2939 206 -132 315 o
ATOM . 472 N LYS A 168 -11.749 21. 239 5. 803 1 .00 29. 78 N
ANISOU 472 N LYS A 168 3924 3688 3703 231 -140 330 N
ATOM 473 CA LYS A 168 -11.224 22. 593 5. 672 1 .00 32. 80 C
ANISOU 473 CA LYS A 168 4333 4050 4081 232 -145 - 330 C
ATOM .474 C LYS A 168 -9.876 22. 597 4. 966 1 .00 28. 01 C
ANISOU 474 C LYS A 168 3731 3425 3488 221 -138 321 C
ATOM 475 CB LYS A 168 -11.105 23. 278 7. 033 1 .00 44. 68 C
ANISOU 475 CB LYS A 168 5855 5544 5578 223 -157 338 C
ATOM 476 CG LYS A 168 -10.469 24. 663 6. 959 1 .00 56. 50 C
ANISOU 476 CG LYS A 168 7379 7017 7072 .222 -166 - 337 C
ATOM 477 CD LYS A 168 -10.549 25. 404 8. 290 1 .00 66. 28 C
ANISOU 477 CD LYS A 168 8635 8248 8301 216 -180 346 c
ATOM 478 CE LYS A 168 -9.903 24. 607 9. 413 1 .00 70. 41 c
ANISOU 478 CE LYS A 168 9153 8769 8832 193 -180 345 c
ATOM 479 NZ LYS A 168 -9.902 25. 350 10. 705 1 .00 74. 89 N
ANISOU 479 NZ LYS A 168 9738 9327 9389 186 -195 - 352 N
ATOM 480 o GLY A 169 -9.166 22. 047 1. 888 1 .00 36. 18 o
ANISOU 480 o GLY A 169 4750 4459 4538 235 -113 - 303 o
ATOM 481 N GLY A 169 -9.678 23. 582 4. 101 1 .00 28. 92 N
ANISOU 481 N GLY A 169 3861 3528 3600 230 -138 - 319 N
ATOM 482 CA GLY A 169 -8.415 23. 742 3. 413 1 .00 24. 51 C
ANISOU 482 CA GLY A 169 3308 2949 3055 221 -130 310 C
ATOM 483 C GLY A 169 -8.307 22. 881 2. 175 1 .00 36. 87 C
ANISOU 483 c GLY A 169 4856 4524 4629 227 -115 303 C
ATOM 484 o SER A 170 -4.604 22. 144 -0. 185 1 .00 15. 56 o
ANISOU 484 o SER A 170 2148 1774 1991 205 -70 - 270 o
ATOM 485 N SER A 170 -7.240 23. 100 1. 424 1 .00 35. 23 N
ANISOU 485 N SER A 170 4653 4298 4435 222 -105 295 N
ATOM 486 CA SER A 170 -6.954 22. 280 0. 266 1 .00 14. 37 C
ANISOU 486 CA SER A 170 1995 1662 1802 226 -89 - 286 C
ATOM 487 C SER A 170 -5.563 21. 761 0. 499 1 .00 13. 89 C
ANISOU 487 C SER A 170 1930 1582 1765 208 -80 - 278 C
ATOM . 488 CB SER A 170 -7.009 23. 136 -0. 995 1 .00 29. 91 C
ANISOU 488 CB SER A 170 3975 3623 3765 240 -83 - 283 C
ATOM 489 OG SER A 170 -8.248 23. 822 -1. 067 1 .00 29. 67 o
ANISOU 489 OG SER A 170 3953 3606 3713 256 -95 291 o ATOM 490 o TYR A 171 -4.299 18.802 0.191 1.00 12.38 o
ANISOU 490 o TYR A 171 1691 1402 1611 191 -58 ■261 o
ATOM 491 N TYR A 171 -5.448 20. 899 1. 503 1 .00 10. 65 N
ANISOU 491 N TYR A 171 1508 1177 1360 195 -85 ■280 N
ATOM 492 CA TYR A 171 -4.147 20. 430 1. 948 1 .00 12. 10 • C
ANISOU 492 CA TYR A 171 1688 1342 1566 176 -82 272 C
ATOM 493 C TYR A 171 -3.585 19. 343 1. 043 1 .00 13. 26 C
ANISOU 493 C TYR A 171 1816 1493 1730 178 -65 ■261 C
ATOM 494 CB TYR A 171 -4.244 19. 918 3. 395 1 .00 12. 58 C
ANISOU 494 CB TYR A 171 1746 1409 1626 162 -95 •277 C
ATOM 495 CG TYR A 171 -4.709 20. 984 4. 379 1 .00 12. 08 C
ANISOU 495 CG TYR A 171 1702 1340 1546 . 159 - Ill 287 C
ATOM 496 cpi TYR A 171 -4.470 22. 327 4. 135 1 .00 16. 11 C
ANISOU 496 CD1 TYR A 171 2235 1833 2053 162 - 117 288 C
ATOM 497 CD2 TYR A 171 -5.383 20. 648 5. 548 1 .00 16. 20 C
ANISOU 497 CD2 TYR A 171 2222 1877 2058 155 - 121 294 C
ATOM 498 CE1 TYR A 171 -4.890 23. 314 5. 021 1 .00 19. 09 C
ANISOU 498 CE1 TYR A 171 2631 2206 2416 161 - 134 296 C
ATOM 499 CE2 TYR A 171 -5.796 21. 633 6. 451 1 .00 13. 54 C
ANISOU 499 CE2 TYR A 171 1903 1535 1706 154 - 136 303 C
ATOM 500 CZ TYR A 171 -5.555 22. 962 6. 168 1 .00 13. 81 C
ANISOU 500 CZ TYR A 171 1959 1552 1736 158 - 143 303 C
ATOM 501 OH TYR A 171 -5.975 23. 937 7. 039 1 .00 15. 97 o
ANISOU 501 OH TYR A 171 2251 1821 1995 158 - 159 ■311 o
ATOM 502 N ASP A 172 -2.307 19. 022 1. 253 1 .00 9. 82 N
ANISOU 502 N ASP A 172 1378 1036 1318 163 -61 251 N
ATOM 503 CA ASP A 172 -1.629 17. 974 0. 501 1 .00 8. 60 C
ANISOU 503 CA ASP A 172 1204 882 1182 165 -46 238 C
ATOM 504 C ASP A 172 -1.730 16. 652 1. 238 1 .00 11. 80 C
ANISOU 504 C ASP A 172 1589 1304 1592 156 -53 239 C
ATOM 505 o ASP A 172 -1.438 16. 586 2. 415 1 .00 9. 41 o
ANISOU 505 o ASP A 172 1290 993 1293 140 -67 242 o
ATOM , 506 CB' ASP A 172 -0.140 18. 301 0. 310 1 .00 12. 77 C
ANISOU 506 CB ASP A 172 1739 1377 1737 156 -39 222 C
ATOM 507 CG ASP A 172 0.092 19. 637 -0. 358 1 .00 15 75 C
ANISOU 507 CG ASP A 172 2136 1734 2115 163 -32 219 C
ATOM 508 Dl ASP A 172 -0.833 20. 152 -1. 018 1 .00 14. 66 o
ANISOU 508 ODl AS A 172 2002 1610 1958 178 -28 228 o
ATOM 509 OD2 ASP A 172 1.211 20. 169 -0. 224 1 .00 21. 92 o
ANISOU 509 OD2 ASP A 172 2927 2484 2917 152 -33 205 o
ATOM 510 N TYR A 173 -2.115 15. 595 0. 532 1 .00 7. 86 N
ANISOU 510 N TYR A 173 1069 826 1093 166 -45 237 N
ATOM 511 CA TYR A 173 -2.257 14. 277 1. 147 1 .00 4. 38 C
ANISOU 511 CA TYR A 173 604 401 656 158 -53 239 C
ATOM 512 C TYR A 173 -1.463 13. 250 0. 385 1 .00 9. 93 C
ANISOU 512 C TYR A 173 1288 1106 1380 158 -43 - 226 C
ATOM 513 o TYR A 173 -1.754 12. 984 -0. 784 1 .00 7. 53 o
ANISOU 513 o TYR A 173 978 813 1072 171 -31 223 o
ATOM 514 CB TYR A 173 -3.734 13. 858 1. 131 1 .00 6. 85 c
ANISOU 514 CB TYR A 173 908 745 949 167 -59 250 c
ATOM 515 CG TYR A 173 -4.576 14. 772 1. 971 1 .00 9. 90 c
ANISOU 515 CG TYR A 173 1312 1134 1317 168 -70 261 c
ATOM 516 ,CD1 TYR A 173 -4.763 14. 506 3. 327. 1 .00 10. 83 c
ANISOU 516 CD1 TYR A 173 1427 1255 1433 157 -82 267 c
ATOM 517 CD2 TYR A 173 -5.124 15. 928 1. 442 1 .00 10. 95 c
ANISOU 517 CD2 TYR A 173 1462 1264 1434 180 -68 265 c
ATOM 518 CE1 TYR A 173 -5.503 15. 339 4. 127 1 .00 8. 72 c
ANISOU 518 CE1 TYR A 173 1176 989 1148 158 -91 276 c
ATOM 519 CE2 TYR A 173 -5.873 16. 786 2. 241 1 .00 13. 27 c
ANISOU 519 CE2 TYR A 173 1770 1559 1711 182 -80 275 c
ATOM 520 CZ TYR A 173 -6.060 16. 478 3. 583 1 .00 10. 36 c ANISOU 520 CZ TYR A 173 1400 1195 1341 171 -91 ■280 C
ATOM 521 OH TYR A 173 -6.788 17. 302 4 .411 1 .00 13. 80 o
ANISOU 521 OH TYR A 173 1851 1633 1761 173 - 101 ■290 o
ATOM 522 N TRP A 174 -0.468 12. 651 1 .032 1 .00 9. 13 N
ANISOU 522 N TRP A 174 1175 993 1301 144 -49 •219 N
ATOM 523 CA TRP A 174 0.251 11. 550 0 .395 1 .00 7. 55 C
ANISOU 523 CA TRP A 174 950 796 1120 144 -42 •207 C
ATOM 524 C TRP A 174 -0.700 10. 448 -0 .045 1 .00 12. 47 c
ANISOU 524 C TRP A 174 1554 1449 1733 149 -41 214 c
ATOM 525 .0 TRP A 174 -1.647 10. 111 0 .664 1 .00 12. 72 o
ANISOU 525 o TRP A 174 1581 1498 1753 145 -53 ■226 o
ATOM 526 CB TRP A 174 1.249 10. 908 1 .362 1 .00 8. 33 c
ANISOU 526 CB TRP A 174 r 1036 884 1247 124 -54 200 c
ATOM 527 CG TRP A 174 2.459 11. 705 1 .687 1 .00 '9. 67 c
ANISOU 527 CG TRP A 174 1211 1019 1446 114 -67 193 c
ATOM 528 CD1 TRP A 174 2.886 12. 040 2 .932 1 .00 10. 97 c
ANISOU 528 CD1 TRP A 174 1379 1163 1628 94 -92 199 c
ATOM 529 CD2 TRP A 174 3.430 12. 242 0 .771 1 .00 12. 48 c
ANISOU 529 CD2 TRP A 174 1567 1352 1821 121 -58 178 c
ATOM 530 NE1 TRP A 174 4.043 12. 763 ,2 .858 1 .00 8. 55 N
ANISOU 530 NE1 TRP A 174 1075 822 1352 84 103 185 N
ATOM 531 CE2 TRP A 174 4.403 12. 908 1 .547 1 .00 11. 45 C
ANISOU 531 CE2 TRP A 174 1439 1188 1725 101 -82 173 c
ATOM 532 CE3 TRP A 174 3.555 12. 252 -0 .618 1 .00 14. 70 ' c
ANISOU 532 CE3 TRP A 174 1850 1641 2093 140 -34 165 c
ATOM 533 CZ2 TRP A 174 5.498 13. 560 0 .981 1 .00 12. 34 c
ANISOU 533 CZ2 TRP A 174 . 1551 1272 1866 99 -82 154 c
ATOM 534 CZ3 TRP A 174 • 4.652 12. 897 -1 .185 1 .00 14. 92 c
ANISOU 534 CZ3 TRP A 174 1876 1643 2148 143 -33 150 c
ATOM 535 CH2 TRP A 174 5.604 13. 547 -0 .387 1 .00 14. 74 c
ANISOU 535 CH2. TRP A 174 1853 1584 2164 .121 -56 143 c
ATOM 536 N VAL A 175 -0.438 9. 882 -1 .216 1 .00 83 N
ANISOU 536 N VAL A 175 956 868 1152 157 -29 205 N
ATOM 537 CA VAL A 175 -1.106 8. 642 -1 .633 1 .00 4. 55 C
ANISOU 537 CA VAL A 175 522 476 732 156 -32 - 210 c
ATOM 538 C VAL A 175 -0.066 7. 589 -1 .954 1 .00 7. 36 c
ANISOU 538 C VAL A 175 857 829 1112 149 -28 199 c
ATOM 539 o VAL A 175 1.128 7. 871 -1 .983 1 .00 9. 57 o
ANISOU 539 o VAL A 175 1135 1090 1412 147 -23 185 o
ATOM 540 CB VAL A 175 -2.018 8. 859 -2 .857 1 .00 10. 53 c
ANISOU 540 CB VAL A 175 1287 1245 1468 173 -23 - 213 c
ATOM 541 CGI VAL A 175 -3.078 9. 909 -2 .545 1 .00 9. 84 c
ANISOU 541 CGI VAL A 175 1217 1161 1358 180 -29 225 c
ATOM - 542 CG2 VAL A 175 -1.196 9. 258 -4 .078 1 .00 13. 91 c
ANISOU 542 CG2 VAL A 175 1722 1660 1902 186 -3 198 c
ATOM 543 N GLY A 176 -0.507 6. 362 -2 .216 1 .00 9. 00 N
ANISOU 543 N GLY A 176 1047 1053 1318 144 -32 204 N
ATOM 544 CA GLY A 176 0.429 5. 254 -2 .354 1 .00 12. 78 c
ANISOU 544 CA GLY A 176 1506 1528 1821 133 -31 - 194 C
ATOM 545 C GLY A 176 1.124 5. 135 -3 .707 1 .00 13. 77 C
ANISOU 545 C GLY A 176 1632 1650 1952 146 -14 - 179 C
ATOM 546 o GLY A 176 1.279 4. 028 -4 .224 1 .00 12. 69 o
ANISOU 546 o GLY A 176 1481 1519 1820 143 -13 - 177 o
ATOM 547 N LEU A 177 1.552 6. 262 -4 .269 1 .00 13. 70 N
ANISOU 547 N LEU A 177 1637 1630 1939 161 -1 169 N
ATOM 548 CA LEU A 177 2.213 6. 296 -5 .575 1 .00 10. 19 C
ANISOU 548 CA LEU A 177 1193 1182 1496 177 16 153 C
ATOM 549 .C LEU A 177 3.659 6. 694 -5 .429 1 .00 7. 71 C
ANISOU 549 C LEU A 177 871 850 1207 173 19 136 C
ATOM 550 o LEU A 177 3.969 7. 674 -4 .752 1 .00 11. 44 , o
ANISOU 550 o LEU A 177 1354 1308 1686 170 14 134 o ATOM 551 CB LEU A 177 1.555 7.344 -6.492 1.00 12.51. C
ANISOU 551 CB LEU A 177 1510 14,77 1766 199 30 153 C
ATOM 552 CG LEU A 177 ' 0.493 6 .966 -7 .517 1 .00 21. 14 C
ANISOU 552 CG LEU A 177 2608 2585 2839 210 36 •159 C
ATOM ,553 CDl LEU A 177 0.965 5 .828 -8 .418 1 .00 19. 80 C
ANISOU 553 CD1 LEU A 177 2424 2422 2676 214 42 ■150 C
ATOM 554 CD2 LEU A 177 0.151 8 .199 -8 .341 1 .00 19. 56 C
ANISOU 554 CD2 LEU A 177 2429 2378 2626 228 51 •156 c
ATOM 555 N SE A 178 4.555 5 .960 -6 .074 1 .00 10. 34 N
ANISOU 555 N SER A 178 1188 1185 1556 173 25 ■122 N
ATOM 556 CA SER A 178 5.969 6 .345 -6 .071 1 .00 11. 73 C
ANISOU 556 CA SER A 178 1352 1344 1759 167 27 104 ; C
ATOM 557 e SER A 178 6.586 6 .113 -7 .435 1 .00 17. 46 C
ANISOU 557 C SER A 178 2073 2076 2485 183 41 -90 c
ATOM 558 o SER A 178 6.132 5 .252 -8 .208 1 .00 14. 79 o
ANISOU 558 o SER A 178 1733 1754 2133 193 47 -94 o
ATOM 559 CB SER A 178 6.767 5 .584 -4 .995 1 .00 13. 49 c
ANISOU 559 CB SER A 178 1553 1557 2016 138 13 -98 c
ATOM 560 OG SER A 178 , 6.732 4 .186 -5 .234 1 .00 18. 65 o
ANISOU 560 OG SER A 178 2190· 2221 2673 131 12 ■101 o
ATOM 561 N GLN A 179 7.624 6 .886 -7 .723 1 .00 17; 30 N
ANISOU 561 GLN A 179 2050 2042 2482 186 47 -74 N
ATOM 562 CA GLN A 179 8.241 6 .898 -9 .041 1 .00 23. 18 c
ANISOU 562 CA GLN A 179 2790 2791 3226 202 61 -62 c
ATOM 563 C GLN A 179 9.619 6 .284 -8 .926 1 .00 27. 01 c
ANISOU 563 C GLN A 179 3247 3267 3749 182 61 -42 c
ATOM 564 o GLN A 179 10.425 6 .724 -8 .109 1 .00 25. 55 o
ANISOU 564 o GLN A 179 3053 3063 3594 163 5 -29 o
ATOM 565 CB GLN A 179 8.345. 8 .338 -9 .548 1 .00 32. 38 c
ANISOU 565 CB GLN A 179 3974 3947 4382 220 67 -58 c
ATOM 566 CG GLN A 179 8.813 8 .498 -10 .992 1 .00 39. 96 c
ANISOU 566 CG GLN A 179 4931 4913 5337 240 81 -49 c
ATOM 567 CD GLN A 179 8.912 9 .959 -11 .411 1 .00 42. 43 c
ANISOU 567 CD GLN A 179 5263 5213 5645 254 84 -47 c
ATOM 568 OEl GLN A 179 9.389 10 .805' -10 .653 1 .00 48. 08 o
ANISOU 568 OEl GLN A 179 5982 5906 6378 242 79 -37 o
ATOM 569 NE2 GLN A 179 8.447 10 .264 -12 .616 1 .00 44. 05 N
ANISOU 569 NE2 GLN A 179 5479 5430 5827 279 91 -56 N
ATOM 570 o ASP A 180 12.037 6 .334 -11 .254 1 .00 56. 80 b
ANISOU 570 o ASP A 180 6642 7716 7222 199 312 812 o
ATOM 571 N ASP A 180 9.883 5 .267 -9 .740 1 .00 55. 47 N
ANISOU 571 N ASP A 180 6590 7429 7058 203. 208 - 688 N
ATOM 572 CA ASP A 180 11.200 4 .635 -9 .779 1 .00 66. 41 c
ANISOU 572 CA ASP A 180 7932 8869 8431 239 153 - 780 c
ATOM 573 C ASP A 180 12.264 5 .596 -10 .297 1 .00 59. 10 c
ANISOU 573 C ASP A 180 6946 8000 7509 216 235 838 c
ATOM 574 CB ASP A 180 11.172 3 .372 -10 .644 1 .00 78. 58 c
ANISOU 574 CB ASP A 180 9481 10417 9957 303 70 - 789 c
ATOM 575 CG ASP A 180 11.127 2 .102 -9 .818 1 .00 89. 77 c
ANISOU 575 CG ASP A 180 10925 11813 11369 344 -43 - 800 c
ATOM .576 ODl ASP A 180 11.686 2 .099 -8 .700 1 .00 94. 87 o.
ANISOU 576 ODl ASP A 180 11559 12468 12019 333 -67 835 o
ATOM 577 OD2 ASP A 180 10.534 1 .107 -10 .285 1 .00 91. 61 o
ANISOU 577 OD2 ASP A 180 11192 12023 11594 385 - 108 773 o
ATOM 578 o GLY A 181 15.955 6 .926 -11 .879 1 .00 59. 66 o
ANISOU 578 o GLY A 181 6890 8204 7575 183 307 968 o
ATOM 579 N GLY A 181 13.431 5 .573 -9 .664 1 .00 51. 15 N
ANISOU 579 N GLY A 181 5899 7035 6500 216 217 913 N
ATOM 580 CA GLY A 181 14.513 6 .468 -10 .030 1 .00 55. 76 C
ANISOU 580 CA GLY A 181 6434 7665 7087 185 281 956 C
ATOM 581 C GLY A 181 15.188 6 .126 -11 .3.46 1 .00 57. 32 C ANISOU 581 C GLY A 181 6611 7892 7275 216 262 -970 C
ATOM 582 o HIS A 182 15.045 4. 790 -15. 457 1 .00 63. .57 ' o
ANISOU 582 o HIS A 182 7405 8716 8033 310 229 -942 o
ATOM 583 N HIS A 182 14.905 4. 941 -11. 877 1 .00 58. 47 N
ANISOU 583 N HIS A 182 6763 8042 7410 280 194 -984 N
ATOM 584 CA HIS A 182 15.539 4. 504 -13. 120 1 .00 61, 43 ' C
ANISOU 584 CA HIS A 182 7121 8444 7775 309 174 -1002 c
ATOM 585 C . HIS A 182 14.602 4. 569 -14. 329 1 .00 63. 70 c
ANISOU 585- C HIS A 182 7434 8714 8056 315 206 -946 c
ATOM 586 CB HIS A 182 16.118 3. 093 -12. 961 1 .00 57. 96 c
ANISOU 586 CB HIS A 182 6669 8026 7327 376. 72 -1062 c
ATOM 587 CG HIS A 182 17.208 3. 004 -11. 939 1 .00 60. 53 c
ANISOU 587 CG HIS A 182 6959 8381 7658 373 40 -1119 c
ATOM 588 ND1 HIS A 182 18.472 3. 513 -12. 152 1 .00 61. 28 N
ANISOU 588 ND1 HIS A 182 7013 8516 7756 348 63 -1152 N
ATOM 589 CD2 HIS A 182 17.222 2. 475 -10. 692 1 .00 60. 88 c
ANISOU 589 CD2 HIS A 182 7002 8423 7705 392 -15 -1149 C
ATOM 590 CE1 HIS A 182 19.217 3. 299 -11. 082 1 .00 62. 97 c
ANISOU 590 CE1 HIS A 182 7198 8756 7973 351 26 -1201 c
ATOM 591 NE2 HIS A 182 18.482 ,2. 671 -10. 181 1 .00 61. 21 N
ANISOU 591 NE2 HIS A 182 6998 8509 7750 377 -22 -1200 N
ATOM 592 o SER A 183 11.099 6. 069 -16. 324 1 .00 58. 04 o
ANISOU 592 o SER A 183 6785 7926 7342 258 376 -721 o
ATOM 593 N SER A 183 13.309 4. 384 -14. 088 1 .00 62. 42 N
ANISOU 593 N SER A 183 7303 8518 7894 324 206 -903 N
ATOM 594 CA SER A 183 12.335 4. 362 -15. 172 1 .00 63. 28 C
ANISOU 594 CA SER A 183 7437 8612 7995 329 229 -843' C
ATOM 595 C SER A 183 11.544 5. 662 -15. 249 1 .00 61. 65 C
ANISOU 595 C SER A 183 7238 8383 7803 271 330 -771 C
ATOM 596 CB SER A 183 11.372 3. 187 -14. 997 1 .00 67. 11 C
ANISOU 596 CB SER A 183 7979 9048 8470 361 137 -797 C
ATOM 597 OG SER A 183 10.560 3. 364 -13. 849 1 .00 71. 73 o
ANISOU 597 OG SER A 183 8604 9581 9068 335 130 -743 o
ATOM 598 0 GLY A 184 8.146 7. 853 -14. 006 1 .00 42. 36 o
ANISOU 598 0 GLY A 184 4897 5785 5415 144 494 -509 o
ATOM 599 N GLY A 184 11.375 6. 311 -14. 102 1 .00 62. 92 N
ANISOU 599 N GLY A 184 7409 8516 7982 232 362 -756 N
ATOM 600 CA GLY A 184 10.513 7. 474 -14. 005 1 .00 56. 60 C
ANISOU 600 CA GLY A 184 6628 7677 7200 177 451 -675 C
ATOM 601 C GLY A 1.84. 9.063 7. 030 -14. 005 1 .00 50. 08 C
ANISOU 601 C GLY A 184 5856 6798 6375 182 423 -587 C
ATOM 602 o ARG A 185 7.427 4. 583 -11. 698 1 .00 27. 14 o
ANISOU 602 o ARG A 185 3079 3773 3461 234 193 -521 o
ATOM 603 N ARG A 185 8.861 5. 715 -13. 999 1 .00 48. 04 N
ANISOU 603 N ARG A 185 5627 6528 6099 230 318 -593 N
ATOM 604 CA ARG A 185 7.520 5. 148 -14. 046 1 .00 39. 54 C
ANISOU 604 CA ARG A 185 4607 5399 5019 238 276 -507 C
ATOM 605 C ARG A 185 ' 6.849 5. 057 -12.. 677 1 .00 26. 51 c
ANISOU 605 C ARG A 185 2999 3692 3382 222 248 -469 c
ATOM 606 CB ARG A 185 7.521 3. 782 -14. 733 1 .00 42. 63 c
ANISOU 606 CB ARG A 185 5013 5801 5385 293 179 -526 c
ATOM 607 CG ARG A 185 7.745 3. 857 -16. 235 1 .00 53. 33 c
ANISOU 607 CG ARG A 185 6340 7199 6722 303 210 -535 c
ATOM 608 CD ARG A 185 7.641 2. 486 -16. 885 1 .00 63. 82 c
ANISOU 608 CD ARG A 185 7692 8531 8025 353 116 -550 c
ATOM 609 NE ARG A 185 6.299 1. 919 -16. 778 1 .00 69. 81 N
ANISOU 609 NE ARG A 185 8508 9237 8778 355 70 -466 N
ATOM 610 CZ ARG A 185 5.919 1. 059 -15. 837 1 .00 70. 70 C
ANISOU 610 . cz ARG A 185 8661 9307 8894 376 -11 -458 C
ATOM 611 NHl ARG A 185 6.780 0. 658 -14. 909 1 .00 66. 34 N
ANISOU 611 NHl ARG A 185 8096 8761 8349 398 -55 -527 N ATOM 612 NH2 ARG A 185 4.676 0.,598 -15.,825 1.00 73..35 N
ANISOU 612 NH2 ARG A 185 9049 9597 9225 375 -47 ■378 N
ATOM 613 N TRP A 186 5.606 5. ,507 -12. ,645 1 .00 17. .51 N
ANISOU 613 N TRP A 186 1895 2504 2255 193 288 ■373 N
ATOM 614 CA TRP A 186 4.808 5. 525 -11. ,436 1 .00 13. ,10 C
ANISOU 614 CA TRP A 186 1380 .1886 1710 173 274 ■323 C
ATOM 615 C TRP A 186 4.166 4. 174 -11. 183 1 .00 12. ,84 C
ANISOU 615 C TRP A 186 1395 1821 1663 215 161 ■299 c
ATOM 616 0 TRP A 186 3.640 3. 543 -12. 111 1 .00 14. 52 o
ANISOU 616 o TRP A 186 1623 2033 1861 241 124 •266 o
ATOM 617 CB TRP A 186 3.716 6. 581 -11. 590 1 .00 12. 09 c
ANISOU 617 CB TRP A 186 1268 1718 1605 128 369 ■225 c
ATOM 618 CG TRP A 186 4.239 7. 973 -11. 564 1 .00 11. 92 c
ANISOU 618 CG TRP A 186 ,1208 1716 1605 81 486 ■243 , c
ATOM 619 CD1 TRP A 186 4.477 8. 790 -12. 638 1 .00 16. 22 c
ANISOU 619 CD1 TRP A 186 1713 '2294 2155 65 568 ■236 c
ATOM 620 CD2 TRP A 186 4.583 8. 731 -10. 398 1 .00 11. 12 c
ANISOU 620 CD2 TRP A 186 1103 1599 1522 41 536 ■269 c
ATOM 621 NE1 TRP A 186 4.945 10. 012 -12. 203 1 .00 18. 77 N
ANISOU 621 NE1 TRP A 186 2008 2621 2501 19 668 257 N
ATOM 622 CE2 TRP A 186 5.024 9. 996 -10. 835 1 .00 17. 24 c
ANISOU 622 CE2 TRP A 186 1837 2399 2315 2 650 279 c
ATO 623 CE3 TRP A 186 4.562 8. 461 -9. 029 1 .00 13. 20 c
ANISOU 623 CE3 TRP A 186 1395 1833 1789 31 495 285 c
ATOM 624 CZ2 TRP A 186 5.434 10. 991 -9. 946 1 .00 19. 24 c
ANISOU 624 CZ2 TRP A 186 2084 2642 2585 -45 714 300 c
ATOM 625 CZ3 TRP A 186 4.962 9. 446 -8. 150 1 .00 14. 58 c
ANISOU 625 CZ3 TRP A 186 1557 2002 1981 -18 569 312 c
ATOM 626 CH2 TRP A 186 5.397 10. 694 -8. 609 1 .00 20.85 c
ANISOU 626 CH2 TRP A 186 2312 2819 2791 -57 684 323 c
ATOM 627 o LEU A 187 3.304 3. 460 -7. 310 1 .00 12. 57 o
ANISOU 627 o LEU A 187 1455 1671 1651 191 50 272 o
ATOM 628 N LEU A 187 4.212 3. 734 -9. 928 1 .00 10. 97 N
ANISOU 628 N LEU A 187 1180 1558 ' 1430 219 107 317 N
ATOM 629 CA LEU A 187 3.554 2. 513 -9. 502 1 .00 14. 56 c
ANISOU 629 CA LEU A 187 1682 1975 1875 254- 3 289 C
ATOM 630 C LEU A 187 2.827 2. 729 -8. 183 1 ,00 13. 10 C
ANISOU 630 C LEU A 187 1537 1734 1706 225 3 242 C TOM 631 CB LEU A 187 4.589 1. 413 -9. 298 1 .00 12. 21 C
ANISOU 631 CB LEU A 187 1368 1711 1562 303 -91 378 c
ATOM 632 CG LEU A 187 5.246 0. 860 -10. 551 1 .00 13. 40 c
ANISOU 632 CG LEU A 187 1488 1908 1694 342 -115 - 427 c
ATOM 633 CD1 LEU A 187, 6.291 -0. 186 -10. 170 1 .00 19. 16 c
ANISOU 633 CD1 LEU A 187 2199 2665 2414 390 -203 - 513 c
ATOM 634 CD2 LEU A 187 4.191 0. 271 -11. 486 1 .00 18. 37 c
ANISOU 634 CD2 LEU A 187 2155 2514 2312 360 -144 - 360 c
ATOM 635 N TRP A 188 1.676 2. 091 -8. 034 1 .00 10. 20 N
ANISOU 635 N TRP A 188 1221 1316 1338 238 -47 - 169 N
ATOM 636 CA TRP A 188 1.045 1. 987 -6. 729 1 .00 10. 19 C
ANISOU 636 CA TRP A 188 1261 1263 1348 223 -72 - 132 C
ATOM 637 C TRP A 188 1.860 1. 006 -5. 909 1 .00 13. 49 C
ANISOU 637 C TRP A 188 1675 1697 1753 257 -168 - 206 C
ATOM 638 o. TRP A 188 2.696 0. 265 -6. 455 1 .00 13. 55 o
ANISOU 638 o TRP A 188 1656 1747 1746 299 -223 271 o
ATOM 639 CB TRP A 188 -0.403 1. 502 -6. 846 1 .00 11. 33 C
ANISOU 639 CB TRP A 188 1460 1351 1496 230 -105 -33 c
ATOM 640 CG TRP A 188 -1.274 2. 447 -7. 590 1 .00 11. 38 c
ANISOU 640 CG TRP A 188 1469 1339 1516 196 -13 50 c
ATOM 641 CD1 TRP A 188 -1.792 2. 272 -8. 845 1 .00 15. 05 c
ANISOU 641 CD1 TRP A 188 1935 1814 1972 209 -11 96 c
ATOM 642 CD2 TRP A 188 -1.725 3. 732 -7. 146 1 .00 9. 40 c ANISOU 642 CD2 TRP A 188 1219 1060 1293 142 94 100 C
ATOM 643 NE1 TRP A 188 -2.541 3. 360 -9. 203 1. 00 12. 40 N
ANISOU 643 NE1 TRP A 188 1597 1459 1657 168 88 176 N
ATOM 644 CE2 TRP A 188 -2.515 4. 276 -8. 181 1. 00 9. 64 C
ANISOU 644 CE2 TRP A 188 1248 1083 1332 128 156 180 C
ATOM 645 CE3 TRP A 188 -1.537 4. 478 -5. 977 1. 00 12. 51 C
ANISOU 645 CE3 TRP A 188 1615 1433 1704 103 145 86 c
ATOM 646 CZ2 TRP A 188 -3.117 5. 534 -8. 084 1. 00 10. 31 c
ANISOU 646 CZ2 TRP A 188 1331 1138 1447 79 269 247 c
ATOM 647 CZ3 TRP A 188 -2.141 5. 714 -5. 879 1. 00 13. 50 c
ANISOU 647 CZ3 TRP A 188 1743 1527 1858 54 258 148 c
ATOM 648 CH2 TRP A 188 -2.919 6. 234 -6. 925 1. 00 12. 45 c
ANISOU 648 CH2 TRP A 188 1607 1385 1738 43 320 229 c
ATOM 649 0 GLN A 189 2.699 -2. 133 -3. 083 1. 00 14. 83 0
ANISOU 649 o GLN A 189 2465 1342 1828 255 . -642 ■124 0
ATOM 650 N GLN A 189 1.626 0. 996 -4. 601 1. 00 11. 21 N
ANISOU 650 N GLN A 189 1909 1030 1322 215 -500 -82 N
ATOM 651 C GLN A 189 2.104 -1. 323 -3. 794 1. 00 15. 39 C
ANISOU 651 C GLN A 189 2499 1472 1877 240 -579 •114 C
ATOM 652 CA AGLN A 189 2.408 0. 168 -3. 685 0. 50 10. 19 C
ANISOU 652 CA AGLN A 189 1824 831 1216 233 -575 -95 C
ATOM 653 CB AGLN A 189 2.263 0. 655 -2. 236 0. 50 11. 92 C
ANISOU 653 CB AGLN A 189 2140 991 1397 228 -614 -67 C
ATOM 654 CG AGLN A 189 3.134 1. 853 -1. 910 0. 50 10. 40 C
ANISOU 654 CG AGLN A 189 1933 792 1226 231 -647 -59 c
ATOM 655 CD AGLN A 189 3.099 2. 239 -0. 442 0. 50 15. 35 c
ANISOU 655 CD AGLN A 189 2660 1355 1815 226 -693 -36 c
ATOM 656 OEIAGLN A. 189 2.070 2. 674 0. 078 0. 50 23. 64 0
ANISOU 656 OEIAGLN A 189 3771 2402 2808 206 -655 -12 o
ATOM 657 NE2AGLN A 189 4.231 2. 087 0. 233 0. 50 11. 05 N
ANISOU 657 NE2AGLN A 189 2133 760 1307 240 -775 -44 N
ATOM 658 CA BGLN A 189 2.438 0. 168 -3. 723 0. 50 10. 52 C
ANISOU 658 CA BGLN A 189 1862 874 1259 234 -575 -96 C
ATOM 659 CB BGLN A 189 2.412 0. 724 -2. 300 0. 50 10. 79 C
ANISOU 659 CB BGLN A 189 1988 850 1261 230 -618 -69 C
ATOM 660 CG BGLN A 189 2.912 2. 153 -2. 268 0. 50 12. 60 C
ANISOU 660 CG BGLN A 189 2191 1096 1501 226 -620 -56 C
ATOM 661 CD BGLN A 189 4.217 2. 314 -3. 037 0. 50 16. 52 C
ANISOU 661 CD BGLN A 189 2596 1613 2069 242 -647 -83 C
ATOM 662 OEIBGLN A 189 5.266 1. 868 -2. 582 0. 50 15. 44 o
ANISOU 662 OEIBGLN A 189 2461 1430 1976 260 -716 -99 o
ATOM 663 NE2BGLN A 189 4.154 2. 936 -4. 217 0. 50 10. 14 N
ANISOU 663 NE2BGLN A 189 1707 873 1274 235 -591 -87 N
ATOM 664 0 ASP A 190 1.340 -4. 629 -6. 763 1. 00 16. 00 o
ANISOU 664 0 ASP A 190 2435 1639 2004 242 -480 200 o
ATOM 665 N ASP A 190 1.186 -1. 691 -4. 686 1. 00 13. 16 N
ANISOU 665 N ASP A 190 2187 1238 1574 228 -514 118 N
ATOM 666 CA ASP A 190 0.925 -3. 110 -4. 935 1. 00 15. 34 C
ANISOU 666 CA ASP A 190 ' 2467 1504 1857 233 -512 - 140 · C
ATOM 667 C ASP A 190 1.552 -3. 521 -6. 273 1. 00 17. 31 c
ANISOU 667 C ASP A 190 2617 1800 2159 240 -493 177 c
ATOM 668 CB ASP A 190 -0.576 -3. 421 -4. 898 1. 00 13. 68 c
ANISOU 668 CB ASP A 190 2304 1307 1587 214 -456 122 c
ATOM 669 CG ASP A 190 -1.363 -2. 641 -5. 945 1. 00 17. 07 c
ANISOU 669 CG ASP A 190 2679 1811 1996 195 -382 111 . c
ATOM 670 ODl ASP A 190 -2.609 -2. 606 -5. 861 1. 00 16. 47 o
ANISOU 670 ODl ASP A 190 2639 1746 1874 178 -336 -91 o
ATOM 671 OD2 AS A 190 -0.732 -2. 056· -6. 851 1. 00 13. 45 o
ANISOU 671 OD2 ASP A 190 • 2143 1398 1570 196 -370 123 o
ATOM 672 0 GLY A 191 2.628 -2. 909 -10. 487 1. 00 16. 59 o
ANISOU 672 0 GLY A 191 2243 1906 2154 216 -364 252 o ATOM 673 N GLY A 191 2.324 -2.613 -6.863 1.00 15.88 N
A ISOU 673 N GLY A 191 2369 1653 2010 241 -489 ■184 N
ATOM 674 CA GLY A 191 3.003 -2. 880 -8. 121 1 .00 13. 07 C
ANISOU 674 CA GLY A 191 1919 1342 1705 243 -468 •221 C
ATOM 675 C GLY A 191 2.167 -2. 652 -9. 377 1 .00 12. 71 C
A ISOU 675 C GLY A 191 1825 1374 1630 220 -389 •220 C
ATOM 676 0 SER A 192 0.833 0. 391 -10. 157 1 .00 13. 90 0
A ISOU 676 0 SER A 192 1947 1633 1701 173 -292 ■145 o
ATOM 677 N ,SER A 192 0.936 -2. 182 -9. 223 1 .00S 15. 23 N
A ISOU 677 N SER A 192 2185 1712 1889 202 -350 ■186 N
ATOM 678 CA SER A 192 0.116 -1. 878 -10. 397 1 .00 13. 66 C
ANISOU 678 CA SER A 192 1941 1585 1662 178 -281 •181 C
ATOM 679 C SER A 192 0.392 -0. 469 -10. 913 1 .00 13. 45 C
ANISOU 679 C SER A 192 1868 1603 1638 166 -261 ■164 C
ATOM 680 CB SER A 192 -1.368 -2. 033 -10. 090 1 .00 13. 22 C
ANISOU 680 CB SER A 192 1942 1531 1549 163 -247 152 C
ATOM 681 OG SER A 192 -1.812 -1. 058 -9. 163 1 .00 14. 42 o
ANISOU 681 OG SER A 192 2148 1660 1671 159 -252 ■115 o
ATOM 682 o SER A 193 -1.973 1. 426 -13. 040 1 .00 18. 63 o
ANISOU 682 o SER A 193 2459 2414 2207 98 -119 -99 o
ATOM 683 N SER A 193 0.118 -0. 238 -12. 200 1 .00 13. 96 N
ANISOU 683 N SER A 193 1872 1736 1697 145 -209 169 N
ATOM 684 CA SER A 193 0.378 1. 064 -12. 814 1 .00 15. 39 C
ANISOU 684 CA SER A 193 2005 1963 1879 131 -188 153 C
ATOM 685 C SER A 193 -0.878 1. 924 -12. 797 1 .00 17. 29 C
ANISOU 685 C SER A 193 2271 2229 2070 111 -150 108 C
ATOM 686 CB SER A 1-93 0.823 0. 893 -14. 273 1 .00 21. 16 C
ANISOU 686 CB SER A 193 ' 2655 2754 2629 115 -152 182 C
ATOM 687 OG SER A 193 2.061 0. 204 -14. 363 1 .00 23. 38 o
ANISOU 687 OG SER A 193 2903 3013 2967 133 -182 227 o
ATOM 688 0 PRO A 194 -1.461 4. 178 -14. 997 1 .00 13. 21 o
ANISOU 688 0 PRO A 194 1645 1848 1526 55 -64 -62 o
ATOM 689 N PRO A 194 -0.722 3. 224 -12. 534 1 .00 11. 74 N
ANISOU 689 N PRO A 194 1565 1529 1365 107 -154 -80 N
ATOM 690 CA PRO A 194 -1.895 4. 095 -12. 647 1 .00 12. 20 C
ANISOU 690 CA PRO A 194 1636 1614 138 86 -115 -39 C
ATOM 691 C PRO A 194 -2.291 4. 286 -14. 102 1 .00 13. 77 C
ANISOU 691 C PRO A 194 1773 1885 1573 60 -68 -36 C
ATOM 692 CB PRO A 194 -1.409 5. 429 -12. 070 1 .00 13. 05 C
ANISOU 692 CB PRO A 194 1749 1707 1504 90 -134 -15 C
ATOM 693 CG PRO A 194 0.073 5. 402 -12. 250 1 .00 20. 78 C
ANISOU 693 CG PRO A 194 2685 2681 2529 104 -168 -46 C
ATOM 694 CD PRO A 194 0.479 3. 957 -12. 096 1 .00 15. 03 C
ANISOU 694 CD PRO A 194 1968 1924 1819 121 -193 -84 C
ATOM 695 o SER A 195 -3.131 7. 119 -15. 196 1 .00 12. 17 o
ANISOU 695 o SER A 195 1510 1768 1347 15 -13 42 o
ATOM, 696 N SER A 195 -3.560 4. 585 -14. 323 1 .00 11. 79 N
ANISOU 696 N SER A 195 1535 1658 1288 41 -33 -5 N
ATOM 697 CA SER A 195 -4.029 . 4. 936 -15. 653 1 .00 11. 97 . C
ANISOU 697 CA SER A 195 1504 1747 1297 13 7 6 C
ATOM 698 C SER A 195 -3.561 6. 338 -16. 042 1 .00 13. 41 C
ANISOU 698 C SER A 195 1647 1958 1489 1 11 29 C
ATOM 699 CB SER A 195 -5.553 4. 872 -15. 705 1 .00 13. 99 C
ANISOU 699 CB SER A 195 1785 2012 1518 -3 38 35 C
ATOM 700 OG SER A 195 -6.121 6. 010 -15. 073 1 .00 14. 54 o
ANISOU 700 OG SER A 195 1878 2066 1581 -5 42 76 o
ATOM 701 o PRO A 196 -3.707 10. 264 -17. 314 1 .00 14. 43 o
ANISOU 701 o PRO A 196 1686 2175 1622 -49 48 129 o
ATOM 702 N , PRO A 196 -3.646 6. 661 -17. 337 1 .00 10. 66 N
ANISOU 702 N ' PRO A 196 1244 1672 1133 -26 40 34 N
ATOM ' 703 CA PRO A 196 -3.244 7. 982 -17. 812 1 .00 10. 30 C ANISOU 703 CA PRO A 196 1160 1657 1097 -40 46 58 C
ATOM 704 C PRO A .196 -4.059 9. 108 -17 .160 1 .00 11. 64 C
ANISOU 704 C PRO A 196 1357 1809 1257 -42 49 107 C TOM 705 CB PRO A 196 -3.528 7. 903 -19 .315 1 .00 11. 83 C
ANISOU 705 CB PRO A 196 1305 1919 1271 -75 81 59 C
ATOM 706 CG PRO A 196 -3.316 6. 461 -19 .648 1 .00 14. 80 C
ANISOU 706 CG PRO A 196 1680 2298 1646 -71 84 13 C
ATOM 707 CD PRO A 196 -3.838 5. 705 -18 .444 1 .00 9. 99 C
ANISOU 707 CD PRO A 196 1130 1630 1035 -44 65 9 ■ C
ATOM 708 o GLY A 197 -5.871 11. 014 -13 .683 1 .00 13. 21 o
ANISOU 708 o GLY A 197 1705 1867 1448 -11 33 188 0
ATOM 709 N GLY A 197 -5.152 8. 782 -16 .480 1 .00 9. 62 N
ANISOU 709 N GLY A 197 1149 1523 982 -37 54 122 N
ATOM 710 CA GLY A 197 -5.915 9. 792 -15 .761 1 .00 9. 87 C
ANISOU 710 CA GLY A 197 1208 1530 1010 -37 59 162 C
ATOM 711 C GLY A 197 -5.416 10. 073 -14 .348 1 .00 12. 55 C
ANISOU 711 C GLY A 197 1598 1807 1364 -11 29 159 C
ATOM 712 N LEU A 198 -4.481 9. 253 -13 .886 1 .00 9. 10 N
ANISOU 712 N LEU A 198 1178 1342 939 9 -1 122 N
ATOM 713 CA LEU A 198 -4.025 9. 324 -12 .507 1 .00 12. 52 C
ANISOU 713 CA LEU A 198 1666 1711 1380 32 -36 117 c
ATOM 714 C LEU A 198 -2.508 9. 374 -12 .432 1 .00 13. 12 c
ANISOU 714 c LEU A 198 1718 1777 1489 48 -75 90 c
ATOM 715 o LEU A 198 -1.896 8. 758 -11 .553 1 .00 13. 99 o
ANISOU 715 o LEU A 198 1866 1839 1610 69 - 113 68 o
ATOM 716 CB LEU A 198 -4.5*66 8. 126 -11 .723 1 .00 15. 03 c-
ANISOU 716 CB LEU A 198 2045 1986 1680 44 -43 101 c
ATOM 717 CG LEU A 198 -6.100 8. 136 -11 .676 1 .00 15. 97 c
ANISOU 717 CG LEU A 198 2189 2108 1770 29 -4 128 - c
ATOM 718 CD1 LEU A 198 -6.680 6. 764 -11 .365 1 .00 17. 75 c
ANISOU 718 CD1 LEU A 198 2457 2311 1978 34 -1 109 c
ATOM 719 CD2 LEU A 198 -6.591 9. 188 -10 .682 1 .00 21. 63 c
ANISOU 719 CD2 LEU A 198 2948 2788 2483 28 1 158 c
ATOM 720 N LEU A 199 -1.906 10. 119 -13 .351 1 .00 12. 23 N
ANISOU 720 N LEU A 199 1543 1708 1394 36 -66 93 N
ATOM 721 CA LEU A 199 -0.467 10. 340 -13 .332 1 .00 12. 95 C
ANISOU 721 CA LEU A 199 1604 1792 1523 48 . -98 69 C
ATOM 722 C LEU A 199 -0.181 11. 668 -12 .654 1 .00 15. 29 C
ANISOU 722 C LEU A 199 1911 2066 1831 51 - 113 95 c
ATOM 723 o LEU A 199 -0.780 12. 695 -12 .998 1 .00 17. 91 o
ANISOU 723 o LEU A 199 2228 2425 2153 34 -86 129 o
ATOM 724 CB LEU A 199 0.104 10. 371 -14 .742 1 .00 13168 c
ANISOU 724 CB LEU A 199 1622 1945 1630 . 31 -76 54 c
ATOM 725 CG LEU A 199 -0.057 9. 078 -15 .540 1 .00 20. 98 c
ANISOU 725 CG LEU A 199 ' 2529 2896 2546 25 -58 23 c
ATOM 726 CD1 LEU A 199 0.490 9. 249 -16 .963 1 .00 18. 89 c
ANISOU 726 CD1 LEU A 199 2193 269 2291 1 -30 9 c
ATOM 727 CD2 LEU A 199 0.629 7. 942 -14 .826 1 .00 25; -70 c
ANISOU 727 CD2 LEU A 199 3153 3446 3167 52 -95 -16 c
ATOM 728 N PRO A 200 • 0.731 11. 663 -11 .682 1 .00 13. 06 N
ANISOU 728 N PRO A 200 1656 1734 1572 73 - 159 79 N
ATOM 729 CA PRO A 200 1.031 12. 951 -11 .055 1 .00 15. 98 C
ANISOU 729 CA PRO A 200 2036 2083 1953 75 - 174 102 C
ATOM 730 C PRO A 200 1.562 13. 948 -12 .076 1 .00 17. 03 C
ANISOU 730 C PRO A 200 2098 2266 2108 59 - 155 111 C
ATOM 731 o PRO A 200 2.294 13. 567 -12 .990 1 .00 21. 50 - o
ANISOU 731 o PRO A 200 2609 2865 2694 55 - 151 86 o
ATOM 732 CB PRO A 200 2.119 12. 591 -10 .049 1 .00 14. 40 c
ANISOU 732 CB PRO A 200 1866 1827 1779 99 - 232 76 c
ATOM 733 CG PRO A 200 1.802 11. 164 -9 .661 1 .00 12. 58 c
ANISOU 733 CG PRO A 200 1678 1569 1533 111 - 245 55 c ATOM 734 CD PRO A 200 1.399 10.545 -10.991 1.00 10.22 C
A ISOU 734 CD PRO A 200 1327 1328 1227 96 -202 45 C
ATOM 735 N ALA A 201 1.187 15. 213 -11 .924 1 .00 15 .07 N
A ISOU 735 N ALA A 201 1850 2021 1855 48 -142 146 N
ATOM 736 CA ALA A 201 1.684 16. 266 -12 .808 1 .00 20 .52 C
ANISOU 736 . CA ALA A 201 2477 2754 2566 32 -127 160 C
ATOM 737 C ALA A 201 3.186 16. 449 -12 .615 1 .00 28 .10 C
ANISOU 737 C ALA A 201 3411 3696 3568 46 -164 132 C
ATOM 738 0 ALA A 201 3.708 16. 242 -11 .524 1 .00 32 .55 0
ANISOU 738 o . ALA A 201 4016 4206 4145 68 -207 118 o
ATOM 739 CB ALA A 201 0.945 17. 569 -12 .544 1.00 25 .64 c
ANISOU 739 CB ALA A 201 3137 3401 3206 20 -109 203 c
ATOM 740 o GLU A 202 5.182 19. 253 -12 .910 1 .00 75' .44 o
ANISOU 740 o GLU A 202 9318 9706 9641 35 -184 152 o
ATOM 741 N GLU A 202 3.876 16. 832 -13 .683 1 .00 52 .31 N
ANISOU 7 N GLU A 202 6411 6808 6657 32 -149 126 N
ATOM 742 CA GLU A 202 5.328 16. 971 -13 .647 1 .00 67 .08 C
ANISOU 742 CA GLU A 202 8246 8666 8575 43 -179 97 C
ATOM 743 C GLU A 202 5.767 18. 174 -12 .816 1 .00 71 .59 C
ANISOU 743 C GLU A 202 8831 9203 9166 50 -206 115 C
ATOM 744 CB GLU A 202 5.878 17. 078 -15 .066 1 .00 74 .28 C
ANISOU 744 CB GLU A 202 9084 9637 9502 21 -147 85 C
ATOM 745 CG GLU A 202 5.414 15. 963 -15 .983 1 .00 81 .03 C.
ANISOU 745 CG GLU A 202 9924 10531 10334 9 -116 66 .C
ATOM 746 CD GLU A 202 5.873 16. 157 -17 .415 1 .00 86 .16 C
ANISOU 746 CD GLU A 202 10506 11241 10991 -20 -79 57 c
ATOM 747 OEl GLU A 202 6.410 17. 242 -17 .725 1 .00 86 .52 o
ANISOU 747 OEl GLU A 202 10517 11302 11055 -32 -73 71 o
ATOM 748 OE2 GLU A 202 5.697 15. 226 -18 .230 1 .00 87 .99 o
ANISOU 748 OE2 GLU A 202 10720 11505 11208 -32 -54 34 o
ATOM 749 o GLN A 209 11.573 11. 436 -3 .871 1 .00 41 .46 o
ANISOU 749 o GLN A 209 5379 4836 5539 254 -785 -81 o
ATOM 750 N GLN A 209 14.170 10. 593 -4 .234 1 .00 45 .27 N
ANISOU 750 N GLN A 209 5746 5274 6180 289 -882 148 N
ATOM 751 CA GLN A 209 13.073 9. 768 -4 .729 1 .00 44 .07 C
ANISOU 751 CA GLN A 209 5612 5154 5979 282 -827 146 C
ATOM 752 C GLN A 209 11.771 10. 561 -4 .720 1 .00 39 .40 C
ANISOU 752 C GLN A 209 5064 4598 5307 260 -767 108 C
ATOM 753 CB GLN A 209 12.930 8. 499 -3 .888 1 .00 42 .37 C
ANISOU 753 CB GLN A 209 5464 4881 5755 297 -881 152 C
ATOM 754 CG GLN A 209 11.827 7. 560 -4 .356' 1 .00 46 .80 C
ANISOU 754 CG GLN A 209 6044 5469 6269 290 -828 152 C
ATOM 755 CD GLN A 209 11.589 6. 416 -3 .385 1 .00 51 .76 c
ANISOU 755 CD GLN A 209 6751 6037 6880 302 -882 151 c
ATOM 756 OEl GLN A 209 12.485 6. 029 -2 .635 1 .00 56 .89 o
ANISOU 756 OEl GLN A 209 7418 6625 7573 319 -962 162 o
ATOM 757 NE2 GLN A 209 10.373. 5. 878 -3 .387 1 .00 49 .04 N
ANISOU 757 NE2 GLN A 209 6455 5705 6471 292 -840 ■137 N
ATOM 758 N VAL A 210 10.880 10. 244 -5 .653 1 .00 30 .09 N
ANISOU 758 N VAL A 210 3774 4001 3657 46 88 372 N
ATOM 759 CA VAL A 210 9.663 11. 026 -5 .824 1 .00 24 .23 c
ANISOU 759 CA VAL A 210 3040 3239 2928 12 99 346 C
ATOM 760 C VAL A 210 8.381 10. 225 -5 .550 1 .00 13 .21 C
ANISOU 760 C VAL A 210 1681 1809 1531 8 77 322 C
ATOM 761 o VAL A 210 8.267 9. 047 -5 .910 1 .00 16 .25 o
ANISOU 761 o VAL A 210 2093 2179 1902 27 58 323 o
ATOM 762 CB VAL A 210 9.619 11. 675 -7 .219 1 .00 24 .95 C
ANISOU 762 CB VAL A 210 3126 3335 3019 -3 121 342 C
ATOM 763 CGI VAL A 210 8.363 12. 495 -7 .380 1 .00 18 .54 C
ANISOU 763 CGI VAL A 210 2322 2503 2218 -34 131 ■316 c
ATOM 764 CG2 VAL A 210 10.852 12. 550 -7 .420 1 .00 27 .65 c ANISOU 764 CG2 VAL A 210 3431 3710 3363 -6 146 ■365 C
ATOM ' 765 N CYS A 211 7.435 10. 877 -4 .881 1 .00 12. 40 N
ANISOU 765 N CYS A 211 1577 1694 1440 -17 82 •303 N
ATOM 766 CA CYS A 211 6.180 10. 253 -4 .489 1 .00 12. 44 C
ANISOU 766 CA CYS A 211 1611 1669 1445 -26 64 •282 C
ATOM 767 C CYS A 211 5.002 11. 133 -4 .838 1 .00 10. 67 C
ANISOU 767 C CYS A 211 1387 1436 1233 -56 77 ■259 C
ATOM 768 o CYS A 211 5.147 12. 328 -5 .071 1 .00 14. 49 o
ANISOU 768 o CYS A 211 1847 1932 1726 -71 99 ■259 o
ATOM 769 CB CYS A 211 6.162 10. 003 -2 .981 1 .00 17. 15 C
ANISOU 769 CB CYS A 211 2210 2263 2044 -21 52 •281 c
ATOM 770 SG CYS A 211 7.429 8. 848 -2 .424 1 .00 21. 93 S
ANISOU 770 SG CYS A 211 2822 2878 2633 21 32 ■306 S
ATOM 771 N GLY A 212 3.818 10. 538 -4 .868 1 .00 13. 11 N
ANISOU 771 N GLY A 212 1721 1721 1539 -66 63 241 N
ATOM 772 CA GLY A 212 2.646 11. 275 -5 .297 1 .00 11. 08 c
ANISOU 772 CA GLY A 212 1464 1457 1291 -91 72 •221 c
ATOM 773 C GLY A 212 1.740 11. 691 -4 .146 1 .00 11. 14 c
ANISOU 773 C GLY A 212 1468 1457 1309 -110 71 207 c
ATOM 774 o GLY A 212 1.648 11. 015 -3 .119 1 .00 8. 88 o
ANISOU 774 o GLY A 212 1192 1162 1020 -106 57 206 o
ATOM 775 N TYR A 213 1.039 12. 802 -4 .350 1 .00 9.39 N
ANISOU 775; N TYR A 213 1234 1238 1097 -129 87 194 N
ATOM 776 CA TYR A 213 0.049 · 13. 262 -3 .392 1 .00 8. 06 c
ANISOU 776 CA TYR A 213 1062 1064 939 -147 8 - 179 c
ATOM 777 C TYR A 213 -1.151 13. 872 -4 .089 1 .00 11. 37 c
ANISOU 777 c TYR A 213 1481 1478 1360 -164 93 162 c
ATOM 778 o TYR A 21-3 -1.071 14. 252 -5 .248 1 .00 8. 32 o
ANISOU 778 o TYR A 213 1094 1096 971 -162 102 161 o
ATOM 779 CB TYR A 213 0.655 14. 279 -2 .422 1 .00 6. 45 c
ANISOU 779 CB TYR A 213 833 873 745 -150 103 - 186 c
ATOM 780 CG TYR A 213 1.124 15. 612 -2 .985 1 .00 9. 67 c
ANISOU 780 CG TYR A 213 1220 1294 1161 -156 128 189 c
ATOM 781 CD1 TYR A 213 0.380 16. 773 -2 .782 1 .00 12. 72 c
ANISOU 781 CD1 TYR A 213 1597 1678 1557 -174 143 176 c
ATOM 782 CD2 TYR A 213 2.335 15. 722 -3 .661 1 .00 12. 98 c
A ISOU 782 CD2 TYR A 213 1630 1726 1575 -145 138 207 c
ATOM 783 CE1 TYR A 213 0.826 18. 013 -3 .253 1 .00 16. 09 c
ANISOU 783 CE1 TYR A 213 2010 2115 1990 -180 167 180 c
ATOM 784 CE2 TYR A 213 2.786 16. 957 -4 .148 1 .00 11. 06 c
ANISOU 784 CE2 TYR A 213 1371 1494 1338 -155 164 211 c
ATOM 785 CZ TYR A 213 2.026 18. 098 -3 .940 1 .00 15. 46 c
ANISOU 785 CZ TYR A 213 1922 2046 1905 . -172 178 196 c
ATOM 786 OH TYR A 213 2.486 19. 328 -4 .406 1 .00 15. 04 o
ANISOU 786 OH TYR A 213 1858 2000 1856 -182 205 - 200 ; o
ATOM 787 N VAL A 214 -2.277 13. 931 -3 .383 1 .00 9. 88 N
ANISOU 787 N VAL A 214 1295 1283 1177 -179 88 - 148 N
ATOM- 788 C VAL A 214 -3.628 15. 956 -3 .252 1 .00 11. 28 c
ANISOU 788 C VAL A 214 1449 1468 1370 -202 112 - 127 c
ATOM 789 0 VAL A 214 -3.390 16. 116 -2 .059 1 .00 9. 38 o
■ANISOU 789 o VAL A 214 1200 1229 1136 -204 113 - 129 o
ATOM 790 CA AVAL A 214 -3.463 14. 590 -3 .906 0 .50 12. 78 c
ANISOU 790 CA AVAL A 214 1659 1650 1548 -193 93 - 132 c
ATOM 791 CB AVAL A 214 -4.726 13. 756 -3 .641 0 .50 14. 75 c
ANISOU 791 CB AVAL A 214 1922 1889 1792 -206 76 121 c
ATOM 792 CGIAVAL A 214 -5.949 14. 434 -4 .237 0 .50 9. 83 c
ANISOU 792 CGIAVAL A 214 1293 1271 1171 -218 80 - 107 c
ATOM 793 CG2 VAL A 214 -4.569 12. 361 -4 .212 0 .50 22. 03 c
ANISOU 793 CG2AVAL A 214 2868 2800 2701 -199 57 127 c
ATOM 794 CA BVAL A 214 -3.461 14. 595 -3 .909 0 .50 12. 73 c
ANISOU 794 CA BVAL A .214 1652 1643 1540 -193 93 132 c ATOM T95 CB BVAL A 214 -4.749 13.760 -3.698 0.50 14.82 C
ANISOU 795 CB BVAL A 21 1932 1899 1802 -206 76 121 C
ATOM 796 CG1BVAL A 214 -4.740 12. 527 -4 .575 0 .50 20 .83 C
ANISOU 796 CGIBVAL A 214 2715 2650 2549 -200 59 125 C
ATOM 797 CG2BVAL A 214 -4.924 13. 370 -2 .237 0 .50 6 .30 C
ANISOU 797 CG2BVAL A 214 853 814 725 -213 69 119 C
ATOM 798 N LYS A 215 -4.027 16. 945 -4 .042 1 .00 11 .27 N
ANISOU 798 N LYS A 215 1441 1470 1370 -205 125 119 N
ATOM 799 CA LYS A 215 -4.321 18. 279 -3 .524 1 .00 9 .37 C
ANISOU 799 CA LYS A 215 1186 1234 1140 -212 143 112 C
ATOM 800 C LYS A 215 -5.523 18. 805 -4 .283 1 .00 17 .11 C
ANISOU 800 C LYS A 215 2169 2215 2117 -216 144 -98 C
ATOM 801 o LYS A 215 -5.449 19. 010 -5 .493 1 .00 16 .01 o
ANISOU 801Λ o LYS A 215 2036 2075 1971 -210 149 -96 o
ATOM 802 CB LYS A 215 -3.145 19. 222 -3 .735 1 .00 7 .00 C
ANISOU 802 CB LYS A 215 876 940 844 -208 165 - 123 C
ATOM 803 CG LYS A 215 -3.327 20. 570 -3 .045 1 .00 12 .38 C
ANISOU 803 CG LYS A 215 1545 1623' 1535 -216 184 119 C
ATOM 804 CD LYS A 215 -2.221 21. 555 -3 .459 1 .00 14 .65 c
ANISOU 804 CD LYS A 215 1825 1914 1825 -216 208 129 c
ATOM 805 CE LYS A 215 -2.257 22. 830 -2 .614 1 .00 15 .94 c
ANISOU 805 CE LYS A 215 1979 2078 2000 -226 226 128 c
ATOM 806 NZ LYS A 215 -2.048 22. 540 -1 .157 1 .00 15 .04 N
ANISOU 806 NZ LYS A 215 1854 1968 1893 -229 219 135 N
ATOM 807 N SER A 216 -6.631 19. 004 -3 .576 1 .00 13 .63 N
ANISOU 807 N SER A 216 1724 ■ 1775 1681 -225 140 -87 N
ATOM 808 CA SER A 216 -7.876 19. 378 -4 .216 1 .00 13 .70 C
ANISOU 808 CA SER A 216 1732 1787 1686 -227 138 -74 c
ATOM 809 C SER A 216 -8.253 18. 337 -5 .281 1 .00 15 .54 c
ANISOU 809 C SE A 216 1979 2020 1907 -225 120 -74 c
ATOM .810 o SER A 216 -8.539 17. 189 -4 .957 1 .00 18 .53 o
ANISOU 810 o SER A 216 2364 2395 2282 -232 . 103 -76 o
ATOM 811 CB SER A 216 -7.767 20. 776 -4 .820 1 .00 18 .84 c
ANISOU 811 CB SER A 216 2381 2440 2338 -220 158 -69 c
ATOM 812 OG SER A 216 -7.466 21. 712 -3 .791 1 .00 26 .59 o
ANISOU 812 OG SER A 216 3351 3421 3330 -223 174 -71 o
ATOM 813 N ASN A 217 -8.249 18. 728 -6 .551 1 .00 15 .50 N
ANISOU 813 N AS A 217 1980 2017 1895 -216 : 126 -72 N
ATOM 8Ϊ4 CA ASN A 217 -8.696 17. 814 -7 .601 1 .00 18 .09 c
ANISOU 814 CA ASN A 217 2319 2344 2209 -214 109 -72 C
ATOM 815 C ASN A 217 -7.555 17. 165 -8 .383 1 .00 19 .88 C
ANISOU 815 C ASN A 217 2559 2566 2430 -205 108 1 -83 C
ATOM 816 0 ASN A 217 -7.789 16. 466 -9 .375 1 .00 19 .84 J o
ANISOU 816 0 ASN A 217 2566. 2560 2413 -202 96 -85 o
ATOM 817 CB ASN A 217 -9.613 18. 549 -8 .576 1 .00 23 .23 c
ANISOU 817 CB ASN A 217 2971 3003 2854 -208 111 -62 c
ATOM 818 CG ASN A 217 -10.865 19. 0711 -7 .913 1 .00 32 .11 c
ANISOU 818 CG ASN A 217 4082 4137 3983 -215 109 -52 c
ATOM 819 ODl ASN A 217 -11.549 18. 348 -7 .192 1 .00 26 .94 o
ANISOU 819 ODl ASN A 217 3422 3485 3330 -228 95 -52 o
ATOM 820 ND2 ASN A 217 -11.165 20. 345 -8 .142 1 .00 41 .14 N
ANISOU 820 ND2 ASN A 217 5220 5284 5126 -205 123 -44 N
ATOM 821 N SER A 218 -6.326 17. 398 -7 .936 1 .00 17 .89 N
ANISOU 821 N SER A 218 2303 2311 2183 -201 121 -92 N
ATOM 822 CA SER A 218 -5.153 17. 048 ^8 .729 1 .00 15 .47 C
ANISOU 822 CA SER A 218 2004 2004 1870 -190 125 •104 C
ATOM 823 C SER A 218 -4.210 16. 072 -8 .028 1 .00 19 .12 C
ANISOU 823 C SER A 218 2468 , 2462 2333 -186 116 ■118 C
ATOM 82 0 SER A 218 -4.043 16. 133 -6 .815 1 .00 12 .77 o
ANISOU 82 o SER A 218 1656 1658 1538 -191 117 ■120 o
ATOM 825 CB SER A 218 -4.381 18. 316 -9 .063 1 .00 23 .32 C ANISOU 825 CB SER A 218 2991 3002 2867 -186 151 106 C
ATOM 826 OG SER A 218 -5.202 19. 216 -9 .795 1 .00 24. 89 0
ANISOU 826 OG SER A 218 3194 3202 3062 -185 159 -93 0
ATOM 827 N LEU A 219 -3.591 15. 186 -8 .805 1 .00 17. 17 N
ANISOU 827 N LEU A 219 2234 2214 2076 -176 108 •128 N
ATOM 828 CA LEU A 219 -2.465 14. 388 -8 .325 1 .00 14. 34 C
ANISOU 828 CA LEU A 219 1878 1854 1716 -166 103 144 C
ATOM 829 C LEU A 219 -1.166 15. 085 -8 .708 1 .00 14. 82 c
ANISOU 829 C LEU A 219 1927 1926 1778 -157 123 156 c
ATOM 830 o LEU A 219 -0.964 15. 430 -9 .877 ' 1 .00 15. 09 0
ANISOU 830 o LEU A 219 1965 1964 1805 -152 133 157 0
ATOM 831 CB LEU A 219 -2.490 12. 987 -8 .927 1 .00 15. 42 c
ANISOU 831 CB LEU A 219 2036 1982 1840 -158 82 150 c
ATOM 832 CG LEU A 219 -3.477 12. 009 -8 .306 1 .00 20. 30 c
ANISOU 832 CG LEU A 219 2669 2587 2456 -168 61 143 c
ATOM 833 CD1 LEU A 219 -3.657 10. 775 -9 .190 1 .00 24. 67 c
ANISOU 833 CD1 LEU A 219 3246 3131 2995 -163 42 148 c
ATOM 834 CD2 LEU A 219 -2.958 11. 612 -6 .946 1 .00 19. 47 c
ANISOU 834 CD2 LEU A 219 2563 2479 2357 -166 56 149 c
ATOM 835 N LEU A 220 -0.308 15. 311 -7 .713 1 .00 12. 81 N
ANISOU 835 N LEU A 220 1659 1678 1531 -155 130 167
ATOM 836 CA LEU A 220 0.966 16. 003 -7 .904 1 .00 7. 17 C
ANISOU 836 CA LEU A 220 929 977 819 -150 151 - 181 , C
ATOM 837 C LEU A 220 2.138 15. 168 -7 .427 1 .00.12. 93 C
ANISOU 837 C LEU A 220 1654 1715 1545 -134 143 202 C
ATOM 838 o LEU A 220 1.959 14. 129 -6 .800 1 .00 13. 24 0
ANISOU 838 o LEU A 220 1704 1746 1581 -127 122 - 203 o
ATOM 839 CB LEU A 220 0.953 17. 335 -7 .129 1 .00 10. 43 C
ANISOU 839 CB LEU A 220 1324 1395 1245 -164 171 - 177 c
ATOM 840 CG LEU A 220 -0.228 18. 243 -7 .498 1 .00 17. 80 c
ANISOU 840 CG LEU A 220 2261 2320 2181 -176 179 - 157 c
ATOM 841 CD1 LEU A 220 -0.198 18. 653 -8 .974 1 .00 20. 78 c
ANISOU 841 CD1 LEU A 220 2648 2698 2549 -173 191 - 154 c
ATOM 842 CD2 LEU A 220 -0.258 19. 475 -6 .602 1 .00 16. 37 c
ANISOU 842 CD2 LEU A 220 2066 2141 2013 -188 197 ■ - 154 c
ATOM 843 N SER A 221 3.340 15. 670 -7 .696 1 .00 12. 45 N
ANISOU 843 N SER A 221 1578 1670 1484 -129 161 218 N
ATOM 844 CA SER A 221 4.588 14. 987 -7 .403 1 .00 16. 08 C
ANISOU 844 CA SER A 221 2029 2143 1938 -111 155 241 C
ATOM 845 C SER A 221 5.438 15. 770 -6 .398 1 .00 13. 43 c
ANISOU 845 C SER A 221 1667 1824 1612 -116 169 254 c
ATOM 846 o SER A 221 5.494 17. 001 -6 .446 1 .00 16.45 o
ANISOU 846 o SER A 221 2035 2212 2003 -134 193 252 o
ATOM 847 CB SER A 221 5.378 14. 848 -8 .704 1 .00 17. 83 c
ANISOU 847 CB SER A 221 2251 2376 2149 -101 165 - 253 c
ATOM 848 OG SER A 221 6.620 14. 225 -8 .468 1 .00 33. 51 o
ANISOU 848 OG SER A 221 4225 4378 4128 -81 160 277 o
ATOM 849 N SER A 222 6.117 15. 055 -5 .503 1 .00 11. 49 N
ANISOU 849 N SER A 222 1415 1587 1365 -100 155 269 N
ATOM 850 CA' SER A, .222 6.970 15. 705 -4 .506 1 .00 14. 98 C
ANISOU 850 CA SER A 222 1831 2047 1815 -103 166 285 C
ATOM 851 C SER A 222 8.129 14. 809 -4 .087 1 .00 19. 42 C
ANISOU 851 C SER A 222 2385 2627 2368 -77 152 310 C
ATOM 852 o SER A 222 8.021 13. 583 -4 .109 1 .00 14. 37 o
ANISOU 852 o SER A 222 1765 1977 1717 -56 129 310 o
ATOM 853 CB SER A 222 6.150 16. 067 -3 .264 1 .00 23. 85 C
ANISOU 853 CB SER A 222 2954 3159 2948 -116 161 272 c TOM 854 OG SER A 222 6.977 16. 601 -2 .239 1 .00 23. 68 o
ANISOU 854 OG SER A 222 2908 3156 2934 -117 169 288 o
ATOM 855 N ASN A 223 9.238 15. 422 -3 .690 1 .00 14. ■95 N,
ANISOU 855 N ASN A 223 1789 2088 1805 -77 167 ■332 N ATOM 856 CA ASN A 223 10.254 14.698 -2.942 1.00 15.93 C
ANISOU 856 CA ASN A 223 1900 2230 1921 -51 152 355 C
ATOM 857 C. ASN A 223 9.561 13. 968 -1 .792 1 .00 19. 3 C
ANISOU 857 C ASN A 223 2352 2644 2353 -42 127 343 C
ATOM 858 0 ASN A 223 8.729 14. 555 -1 .092 1 .00 13. 62 o
ANISOU 858 o ASN A 223 1629 1906 1639 -62 130 327 o
ATOM 859 CB ASN A 223 11.286 15. 689 -2 .402 1 .00 16. 98 c
ANISOU 859 CB ASN A 223 1996 '-2395 2062 -61 171 378 c
ATOM 860 CG ASN A 223 12.470 15. 010 -1 .771 1 .00 23. 79 c
ANISOU 860 CG ASN A 223 2841 3284 2915 -32 157 407 c
ATOM 861 ODl ASN A 223 12.326 14. 234 -0 .827 1 .00 22. 81 o
ANISOU 861 ODl ASN A 223 2728 3153 2785 -12 133 406 o
ATOM 862 ND2 ASN A 223 13.659 15. 293 -2 .293 1 .00 34. 02 N
ANISOU 862 ND2 ASN A 223 4109 4612 4207 -28 173 433 N
ATOM 863 N CYS A 224 9.887 12. 690 -1 .608 1 .00 16. 77 N
ANISOU 863 N CYS A 224 2043 2317 2014 -11 102 - 351 N
ATOM 864 CA CYS A 224 9.232 11. 856 -0 .598 1 .00 15. 48 C
ANISOU 864 CA CYS A 224 1904 2131 1845 -2 78 - 339 C
ATOM 865 C CYS A 224 9.537 12. 274 0 .839 1 .00 16. 89 c
ANISOU 865 C CYS A 224 2066 2322 2030 -2 76 - 347 c
ATOM 866 o CYS A 224 8.786 11. 940 1 .774 1 .00 15. 97 o
ANISOU 866 o CYS A 224 1967 2187 1913 -4 63 - 332 o
ATOM 867 CB CYS A 224 9.634 10. 396 -0 .787 1 .00 16. 28 c
ANISOU 867 CB CYS A 224 2031 2226 1928 33 53 - 348 c
ATOM 868 SG CYS A 224 9.253 9. 750 -2 .421 1 .00 24. 65 S
ANISOU 868 SG CYS A 224 3116 3271 2980 36 52 - 340 S
ATOM 869 N ASP A 225 10.648 12. 992 1 .002 1 .00 11. 31 N
ANISOU 869 N ASP A 225 1323 1648 1326 . 0 91 - 371
ATOM 870 CA ASP A 225 . 11.131 13. 432 2 .310 1 .00 13. 57 C
ANISOU 870 CA ASP A 225 1588 1952 1616 1 90 - 384 C
ATOM 871 C ASP A 225 10.643 14. 845 2 .587 1 .00 21. 06 C
ANISOU 871 c ASP A 225 2518 2900 2583 -36 113 - 374 C
ATOM 872 o ASP A 225 11.427 15. 764 2 .844 1 .00 21. 71 o
ANISOU 872 o ASP A 225 2569 3009 2672 -46 131 - 393 o
ATOM 873 CB ASP A 225 12.658 13. 374 2 .356 1 .00 18. 91 C
ANISOU 873 CB ASP A 225 2233 2668 2285 25 90 - 419 C
ATOM 874 CG ASP A 225 13.221 13. 753 3 .724 ' -1 .00 31. 91 C
ANISOU 874 CG ASP A 225 3856 4336 3933 29 87 - 435 C
ATOM 875 ODl ASP A 225 12.584 13. 437 4 .756 1 .00 27. 89 o
ANISOU 875 ODl ASP A 225 3366 3809 3422 33 71 - 421 0
ATOM 876 OD2 ASP A 225 14.309 14. 366 3 .762 1 .00 32. 11 o
ANISOU 876 OD2 ASP A 225 3843 4397 3959 29 99 - 463 o
ATOM 877 N TH A 226 9.329 15. 006 2 .527 1 .00 12. 38 N
ANISOU 877 . N THR A 226 1442 1772 1491 -55 ,115 - 345 N TOM 878 CA THR A 226 8.678 16. 249 2 .894 1 .00 13. 82 C
ANISOU 878 CA THR A 226 1614 1949 1689 -86 134 - 333 C
ATOM 879 C THR A 226 7.459 15. 915 3 .742. 1 .00 10. 32 C
ANISOU 879 C THR A 226 1194 1481 1247 -92 120 309 C
ATOM 880 o THR A 226 6.934 14. 808 3 .662 1 .00 13. 56 o
ANISOU 880 o THR A 226 1632 1872 1647 -79 100 298 o
ATOM 881 CB THR A 226 8.242 17. 041 1 .639 1 .00 19. 45 C
ANISOU 881 CB THR A 226 2327 2654 2409 -109 156 322 C TOM 882 OGl THR A 226 7.404 16. 215 0 .817 1 .00 17. 99 o
ANISOU 882 OGl THR A 226 2172 2447 2218 -103 144 303 o
ATOM 883 CG2 THR A 226 9.458 17. 447 0 .826 1 .00 17. 65 c
ANISOU 883 CG2 THR A 226 2075 2451 2178 -108 174 345 c
ATOM 884 N TRP A 227 6.989 16. 876 4 .530 1 .00 10. 43 N
ANISOU 884 TRP A 227 1198 1493 1273 -112 131 302 N
ATOM 885 CA TRP A 227 5.852 16. 653 5 .433 1 .00 7. 24 C
ANISOU 885 CA TRP A 227 813 1069 870 -119 120 ■281 C
ATOM 886 C TRP A 227 4.527 17. 028 4 .790 1 .00 13. .17 C ANISOU 886 C TRP A 227 1578 1799 1628 -140 128 255 C
ATOM 887 0 TRP A 227 4.332 18. 174 4. 378 1 .00 12. 26 o
ANISOU 887 0 TRP A 227 1449 1685 1523 -159 149 ■252 o
ATOM 888 CB TRP A 227 6.027 17. 489 6. 715 1 .00 10. 28 C
ANISOU 888 CB TRP A 227 1179 1465 1262 -127 127 288 c
ATOM 889 CG TRP A 227 6.881 16. 864 7. 768 1 .00 11. 26 c
ANISOU 889 CG TRP A 227 1297 1604 1376 -104 110 ■307 c
ATOM 890 CD1 TRP A 227 7.361 15. 589 7. 796 1.00 16. 27 c
ANISOU 890 CD1 TRP A 227 1947 ' 2240 1995 -75 88 314 c
ATOM 891 CD2 TRP A 227 7.368 17. 502 8. 957 1· .00 12. 42 c
ANISOU 891 CD2 TRP A 227 1423 1768 1526 -106 114 321 c
ATOM 892 NE1 TRP A 227 8.108 15. 390 8. 939 1 .00 14. 60 N
ANISOU 892 NE1 TRP A 227 1726 2045 1776 -57 77 331 N
ATOM 893 CE2 TRP A 227 8.122 16. 551 9. 665 1 .00 12. 79 c
ANISOU 893 CE2 TRP A 227 1473 1827 1558 -76 92 336 c
ATOM 894 CE3 TRP A 227 7.211 18. 781 9. 503 1 .00 18. 28 C
ANISOU 894 CE3 TRP A 227 2147 2516 2281 -129 132 322 C
ATOM 895 CZ2 TRP A 227 8.735 16. 843 10. 882 1 .00 20. 62 C
ANISOU 895 CZ2 TRP A 227 2448 2838 2548 -69 88 353 C
ATOM 896 CZ3 TRP A 227 7.823 19. 066 10. 709 1 .00 17. 89 C
ANISOU 896 CZ3 TRP A 227 2081 2485 2231 -124 129 - 339 C
ATOM 897 CH2 TRP A 227 8.571 18. 102 11. 384 1 .00 19. 48 C
ANISOU 897 CH2 TRP A 227 2284 2701 2417 -94 107 355 · c
ATOM 898 N LYS A 228 3.622 16. 053 4. 687 1 .00 10. 59 N
ANISOU 898 N LYS A 228 1278 1452 1294 -137 Ill 238 N
ATOM 899 CA LYS A 228 2.284 16. 288 4. 156 1 .00 11. 16 C
ANISOU 899 CA LYS A 228 1363 1507 1372 -155 115 215 C
ATOM 900 C LYS A 228 1.320 15. 476 4. 976 1 .00 10. 20 C
ANISOU 900 C LYS A 228 1262 1369 1245 -158 98 199 c
ATOM 901 0 LYS A 228 1.738 14. 607 5. 735 1 .00 10. 79 o
ANISOU 901 0 LYS A 228 1347 1442 1309 -142 82 206 o
ATOM 902 CB LYS A 228 2.206 15. 856 2. 684 1 .00 8. 62 c
ANISOU 902 CB LYS A 228 1052 1179 1044 -152 114 212 c
ATOM 903 CG LYS A 228 3.166 16. 628 1. 787 1 .00 6. 83 c
ANISOU 903 CG LYS A 228 806 968 820 -151 133 226 c
ATOM 904 CD LYS A 228 3.015 16. 213 0. 330 1 .00 10. 96 c
ANISOU 904 CD LYS A 228 1342 1484 1337 -148 132 222 c
ATOM 905 CE LYS A 228 3.537 17. 310 -0. 595 1 .00 14. 78 c
ANISOU 905 CE LYS A 228 1810 1979 1826 -156 157 228 c
ATOM 906 NZ LYS A 228 4.981 17. 583 -0. 390 1 .00 25. 40 N
ANISOU 906 NZ LYS A 228 3133 3347 3171 -148 167 254 N
ATOM 907 N TYR A 229 0.027 15. 733 4. 842 1 .00 7. 91 N
ANISOU 907 N TYR A 229 979 1067 960 -176 101 180 N
ATOM 908 CA TYR A 229 -0.936 14. 832 5. 466 1 .00 6. 20 C
ANISOU 908 CA TYR A 229 785 ( 836 737 -181 85 ■165 C
ATOM 909 C TYR A 229 -0.931 13. 539 4. 643 1 .00 6. 45 C
ANISOU 909 C TYR A 229 841 854 755 -171 69 ■165 C
ATOM 910 o TYR A 229 -0.186 13. 423 3. 672 1 .00 8. 93 o
ANISOU 910 o TYR A 229 1153 1173 1066 -159 70 •175 o
ATOM 911 CB TYR A 229 -2.324 15. 443 5. 440 1 .00 6. 10 C
ANISOU 911 CB TYR A 229 769 817 731 -203 . 93 ■147 C
ATOM 912 CG TYR A 229 -2.413 16. 755 6. 187 1 .00 8. 17 C
ANISOU 912 CG TYR A 229 1010 1091 1006 -212 110 •146 c
ATOM 913 CD1 TYR A 229 -2.141 17. 972 5. 550 1 .00 8. 69 c
ANISOU 913 CD1 TYR A 229 1057 1165 1081 -216 129 ■150 c
ATOM 914 CD2 TYR A 229 -2.762 16. 777 7. 530 1 .00 10. 49 c
ANISOU 914 CD2 TYR A 229 1302 11383 1299 -217 107 ■143 c
ATOM 915 CEl TYR A 229 -2.216 19. 187 6. 252 1 .00 8. 72 c
ANISOU 915 CEl TYR A 229 1044. 1175 1095 -225 145 •151 c
ATOM 916 CE2 TYR A 229 -2.837 17. 980 8. 234 1 .00 10. 31 c
A ISOU 916 CE2 TYR A 229 1260 1370 1287 -225 123 •144 c ATOM 917 CZ TYR A 229 -2.577 19.175 7.580 1.00 11.40 C
ANISOU ,917 CZ TYR A 229 1382 1515 1435 -229 141 •148 C
ATOM 918 OH TYR A 229 -2.653 20 .368 8 .286 1 .00 10. 79 o
ANISOU . 918 OH TYR A 229 1289 1444 1367 -237 157 •149 o
ATOM 919 N PHE A 230 -1.755 12 .575 5 .036 1 .00 8. 96 N
ANISOU 919 N PHE A 230 1184 1155 1065 -177 54 •153 N
ATOM 920 CA PHE A 230 -1.858 11 .327 4 .283 1 .00 6. 45 C
ANISOU 920 CA PHE A 230 894 823 735 -171 39 152 C
ATOM 921 C PHE A 230 -3.252 10 .717 4 .374 1 .00 7. 56 C
ANISOU 921 C PHE A 230 1055 946 871 -193 31 ■135 C
ATOM 922 o PHE A 230 -4.053 11 .084 5 .226 1 .00 11. 11 o
ANISOU 922 o PHE A 230 1499 1396 1326 -210 36 124 o
ATOM 923 CB PHE A 230 -0.790 10 .328 4 .735 1 .00 6. 92 C
ANISOU 923 CB PHE A 230 971 877 781 -145 24 166 C
ATOM 924 CG PHE A 230 -0.852 9 .980 6 .182 1 .00 10. 36 C
ANISOU 924 CG PHE A 230 1417 1307 1211 -142 17 163 C
ATOM 925 CD1 PHE A 230 -1.655 8 .949 6 .629 1 .00 12. 40 c
ANISOU 925 CD1 PHE A 230 1709 1544 1458 -151 4 151 ' c
ATO 926 CD2 PHE A 230 -0.084 10 .673 7 .095 1 .00 11. 47 c
ANISOU 926 CD2 PHE A 230 1537 1465 1356 -132 23 174 c
ATOM 927 CE1 PHE A 230 -1.695 8 .618 7 .976 1 .00 13. 36 ' c
ANISOU 927 CE1 PHE A 230 1843 1659 1572 -149 -2 148 c
ATOM 928 CE2 PHE A 230 -0.118 10 .356 8 .455 1 .00 13. 16 c
ANISOU 928 CE2 PHE A 230 1762 1675 1564 -128 15 172 c
ATOM 929 CZ PHE A 230 -0.929 9 .336 8 .890 1 .00 12. 18 c
ANISOU 929 CZ PHE A 230 1672 1528 1427 -136 3 - 158 c
ATOM 930 N ILE A 231 -3.562 9 .821 3 .442 1 .00 9. 12 N
ANISOU 930 N ILE A 231 1274 1131 1061 -194 21 133 . N
ATOM 931 CA ILE A 231 -4.835 9 .116 3 .475 1 .00 13. 25 C
ANISOU 931 CA ILE A 231 1818 1639 1578 -217 13 - 119 C
ATOM 932 C ILE A 231 -4.527 7 .630 3 .408 1 .00 9. 05 C
ANISOU 932 C ILE A 231 1324 1085 1029 -206 -5 - 123 C
ATOM 933 o ILE A 231 -3.719 7 .211 2 .576__.l .00 10. 55 o
ANISOU 933 o ILE A 231 1523 1273 1213 -186 -11 - 134 o
ATOM 3*4 CB ILE A 231 -5.739 9 .503 2 .280 1 .00 11. 73 c
ANISOU ( 934 CB ILE A 231 . 1616 1451 1390 -233 18 - 112 c
ATOM 1935 CGI ILE A 231 -6.120 10 .980 2 .377 1 .00 10. 02 c
ANISOU 935 CGI ILE A 231 1366 1254 1189 -241 35 ' - 106 c
ATOM 936 CG2 ILE A 231 -6.996 8 .682 2 .298 1 .00 12. 84 c
ANISOU 936 CG2 ILE A 231 1776 1578 1524 -257 8 - 101 c
ATOM 937 CD1 ILE A 231 -6.646 11 .571 1 .054 1 .00 7. 16 c
ANISOU 937 CD1 ILE A 231 990 899 830 -247 41 - 103 c
ATOM 938 N CYS A 232 -5.139 6 .865 4 .311 1 .00 10. 83 N
ANISOU 938 N CYS A 232 1575 1295 1247 -219 -13 - 115 N
ATOM 939 CA CYS A 232 -4.988 5 .417 4 .346 1 .00 10. 93 C
ANISOU 939 CA CYS A 232 1631 1281 1241 -212 -29 - 116 C
ATOM 940 C CYS A 232 -6.256 4 .782 3 .832 1 .00 10. 32 c
ANISOU 940 c , CYS A 232 1572 1189 1159 -243 -34 - 106 c
ATOM 941 o CYS A 232 -7.337 5 .369 3 .907 1 .00 13. 83 o
ANISOU 941 o CYS A 232 1998 1644 1613 -270 -25 -96 o
ATOM 942 CB CYS A 232 -4.772 4 .903 5 .773 1 .00 15. 44 c
ANISOU 942 CB CYS A 232 2223 1840 1802 -206 -35 - 114 c
ATOM 943 SG CYS A 232 -3.392 5 .618 6 .681 1 .00 17. 10 s
ANISOU 943 SG CYS A 232 2412 2070 2017 -173 -31 - 127 s TOM 944 N GLU A 233 -6.116 3 .553 3 .346 1 .00 11. 33 N
ANISOU 944 N GLU A 233 1739 1294 1272 -238 -48 - 110 N
ATOM 945 CA GLU A 233 -7.233 2 .806 2 .792 1 .00 10. 39 C
ANISOU 945 CA GLU A 233 1641 1160 1146 -267 -53 - 104 C
ATOM 946 C GLU A 233 -7.124 1 .346 3 .196 1 .00 12. 76 C
ANISOU 946 C GLU A 233 1995 1427 1427 -266 -67 - 104 C
ATOM 947 o GLU A 233 -6.047 0 .758 3 .126 1 .00 13. 93 o ANISOU 947 0 GLU A 233 •2166 1563 1565 -233 -76 •114 o
ATOM 948 CB GLU A 233 -7.222 2. 896 1. 265 1 .00 12 .53 C
ANISOU 948 CB GLU A 233 1903 1437 1419 -264 -55 ■110 C
ATOM 949 CG GLU A 233 -8.240 1. 985 0. 585 1 .00 12 .85 C
ANISOU 949 CG GLU A 233 1969. 1461 1451 -292 -63 107 C
ATOM 950 CD GLU A 233 -8.237 2. 089 -0. 926 1 .00 16 .23 C
ANISOU 950 CD GLU A 233 2389 1898 1880 -287 -66 114 C
ATOM 951 OEl. GLU A 233 -9.161 1. 520 -1. 555 1 .00 14 .83 o
ANISOU 95L OEl GLU A 233 2225 1713 1697 -312 -72 113 o
ATOM 952 OE2 GLU A 233 -7.317 2. 722 -1. 493 1 .00 12 .96 o
ANISOU 952 OE2 GLU A 233 1955 1499 1472 -259 -62 122 o
ATOM 953 LYS A 234 -8.241 0. 749 3. 589 1 .00 15 .49 N
ANISOU 953 N LYS A 234 2363 1757 1767 -301 -67 -94 N
ATOM 954 CA LYS A 234 -8.277 -0. 697 3. 753 1 .00 17 .07 C
ANISOU 954 CA LYS A 234 2620 1921 1947 -305 -80 -94 C
ATOM 955 C LYS A 234 -9.625 -1. 251 3. 335 1 .00 19 .92 C
ANISOU 955 C LYS A 234 2994 2272 2304 -350 -80 -88 C
ATOM 956 o LYS A 234 -10.598 -0. 500 3. 206 1 .00 18 .94 o
ANISOU 956 o LYS A 234 2834 2170 2192 -378 -70 -83 o
ATOM 957 CB LYS A 234 -7.961 -1. 098 5. 196 1 .00 20 .47 C
ANISOU 957 CB LYS A 234 3077 2335 2367 -29,7 -81 -88 C
ATOM 958 CG LYS A 234 -8.958 -0. 620 6. 227 1 .00 15 .77 C
ANISOU 958 CG LYS A 234 2466 1749 1777 -331 -68 -75 C
ATOM 959 CD LYS A 234 -8.485 -1. 096 7. 615 1 .00 23 .98 C
ANISOU 959 CD LYS A 234 3538 2771 2803 -317 -71 -71 C
ATOM 960 CE LYS A 234 -9.187 -0. 393 8. 752 1 .00 35 .09 C
ANISOU 960 CE LYS A 234 4922 4192 4217 -341 -57 -59 C
ATOM 961 NZ LYS A 234 -8.770 -0. 952 10. 086 1 .00 28 .41 N
ANISOU 961 NZ LYS A 234 4113 3327 3355 -328 -60 -54 N
ATOM 962 N, TYR A 235 -9.666 -2. 565 3. 126 1 .00 21 .53 N
ANISOU 962 N TYR A 235 3250 2440 2489 -356 -91 -91 N
ATOM 963 CA TYR A 235 -10.891 -3. 246 2. 741 1 .00 23 .84 C
ANISOU 963 CA TYR A 235 . 3562 2721 2776 -401 -91 -88 C
ATOM 964 C TYR A 235 -11.922 -3. 131 3. 852 1 .00 23 .11 C
ANISOU 964 C TYR A 235 3466 2631 2684 -440 -80 -76 C
ATOM 965 o TYR A 235 -11.623 -3. 388 5. 023 1 .00 23 .43 o
ANISOU 965 o TYR A 235 3529 2656 2716 -434 -78 -69 o
ATOM 966 CB TYR A 235 -10.631 -4. 733 2. 478 1 .00 23 .99 C
ANISOU 966 CB TYR A 235 3645 2697 2772 -399 - 105 -93 C
ATOM 967 CG TYR A 235 -9.817 -5. 073 1. 240 1 .00 22 .87 c
ANISOU 967 CG TYR A 235 3514 2549 2626 -367 - 117 107 c
ATOM 968 CDl TYR A 235 -10.298 -4. 804. -0. 036 1 .00 25 .03 c
ANISOU 968 CDl TYR A 235 3763 2839 2907 -380 - 118 - 113 c
ATOM 969 CD2 TYR A 235 -8.582 -5. 708 1. 355 1 .00 25 .61 c
ANISOU 969 CD2 TYR A 235 3897 2875 2960 -323 - 127 114 c
ATOM 970 CE1 TYR A 235 -9.559 -5. 132 -1. 160 1 .00 21 .63 c
ANISOU 970 CE1 TYR A 235 3343 2404 2471 -352 - 128 - 126 c
ATOM 971 CE2 TYR A 235 -7.836 -6. 040 0. 239 1 .00 26 .01 c
ANISOU 971 CE2 TYR A 235 3957 2921 3005 -293 - 137 127 c
ATOM 972 CZ TYR A 235 -8.333 -5. 757 -1. 021 1 .00 27 .88 c
ANISOU 972 CZ TYR A 235 4169 3174 3250 -309 -■ 137 133 c
ATOM 973· OH TYR A 235 -7.596 -6. 095 -2. 146 1 .00 25 .83 o
ANISOU 973 OH TYR A 235 3920 2911 2984 -280 - 147 146 o
ATOM 974 o ALA A 236 -14.421 -5. 137 3. 866 1 .00 25 .08 o
ANISOU 974 o ALA A 236 3782 2839 2909 -549 -78 -70 o
ATOM 975 N ALA A 236 -13.139 -2. 746 3. 485 ·1 .00 25 .41 N
ANISOU 975 N ALA A 236 3728 2943 2984 -479 -73 -73 N
ATOM 976 CA ALA A 236 -14.257 -2. 797 4. 418 1 .00 24 .94 c
ANISOU 976 CA ALA A 236 3667 2885 2923 -522 -63 -63 c
ATOM 977 C ALA A 236 -14.608 -4. 258 4. 714 1 .00 30 .51 c
ANISOU 977 C ALA A 236 4435 3550 3607 -550 -67 -62 c ATOM 978 CB ALA A 236 -15.461 -2.064 3.848 1..00 32.29 C
ANISOU 978 CB ALA A 236 4550 3852 3868 -555 -55 -64 C
ATOM 979 o LEU A 237 -17.380 -5. 636 4 .882 1. 00 39. 84 0
ANISOU 979 o LEU A 237 5647 4718 4771 -673 -55 -59 o
ATOM 980 N LEU A 237 -15.118 -4. 509 5 .912 1. 00 31. 55 N
ANISOU 980 N LEU A 237 4584 3672 3731 -576 -58 -52 N
ATOM 981 CA LEU A 237 -15.477 -5. 863 6 .322 1. 00 40. 28 C
ANISOU 981 CA LEU A 237 5753 4737 4816 -606 -59 -49 C
ATOM 982 C LEU A 237 -16.660 -6. 397 5 .523 1. 00 40. 58 C
ANISOU 982 c LEU A 237 5794 4775 4851 -657 -58 -55 C
ATOM 983 CB LEU A 237 -15.801 -5. 899 7 .815 1. 00 39. 27 C
ANISOU 983 CB LEU A 237 5639 4602 4680 -623 -46 -36 C
ATOM 984 CG LEU A 237 -14.688 -5. 447 8 .756 1. 00 46. 66 C
ANISOU 984 CG LEU A 237 6576 5537 5617 -576 -47 -31 C
ATOM 985 CD1 LEU A 237 -15.155 -5. 525 10 .199 1. 00 50. 26 C
ANISOU 985 CD1 LEU A 237 '7047 5986 6063 -599 -34 -18 C
ATOM 986 CD2 LEU A 237 -13.436 -6. 288 8 .551 1. 00 49. 68 C
ANISOU 986 CD2 LEU A 237 7010 5884 5983 -532 -63 -36 C
TER 987 LEU A 237
ATOM 987 o SER B 111 f -31.297 2. 950 -22 .256 1. 00 79. 83 o
ANISOU 987 o SER B 111 9901 10814 9617 1235 -701 - 1021 o
ATOM 988 N SER B 111 -33.535 0. 478 -21 .356 1. 00 86. 13 N
ANISOU 988 N SER B 111 10546 11832 10346 1179 -584 - 1022 N
ATOM 989 CA SER B 111 -33.315 1. 915 -21 .484 1. 00 86. 95 C
ANISOU 989 CA SER B 111 10700 11878 10458 1243 -648 - 1056 C
ATOM 990 C SER B 111 -31.836 2. 289 -21 -.368 1. 00 77. 30 C
ANISOU 990 C SER B 111 9534 10556 9280 1214 -662 - 1023 C
ATOM 991 CB SER B 111 -34.150 2. 688 -20 .458 1. 00 94. 56 C
ANISOU 991 CB SER B 111 ' - 11650 12876 11401 1293 -67,4 - 1106 C
ATOM 992 OG SER B 111 -33.834 4. 070 -20 .475 1. 00 97. 36 o
ANISOU 992 OG SER B 111 12056 13168 11767 1347 -736 - 1136 o
ATOM 993 o PRO B 112 -27.919 1. 963 -21 .635 1. 00 33. 69 o
ANISOU 993 o PRO B 112 4114 4814 3870 1081 -652 -905 o '
ATOM 994 N PRO B 112 -31.175 1. 872 -20 .272 1. 00 61. 48 ,N
ANISOU 994 N PRO B 112 7526 8538 7296 1164 -634 -997 N
ATOM 995 CA PRO B 112 -29.749 2. 176 -20 .112 1'. 00 53. 24 C
ANISOU 995 CA PRO B 112 6529 7406 6294 1133 -645 -966 C
ATOM 996 C PRO B 112 -28.908 1. 423 -21 .137 1. 00 37. 88 C
ANISOU 996 C PRO B 112 4596 5424 4370 1086 -623 -920 C
ATOM 997 CB PRO B 112 -29.431 1. 656 -18 .704 1. 00 52. 21 C
ANISOU 997 CB PRO B 112 6377 7290 6169 1093 -612 -951 C
ATOM 998 CG PRO B. 112 -30.750 1. 498 -18 .033 1. 00 54. 67 C
ANISOU 998 CG PRO B 112 6642 7686 - 6443 1117 -599 -985 C TOM 999 CD PRO B 112 -31.706 1. 137 -19 .113 "1. 00 59. 21 C
ANISOU 999 CD •PRO B 112 7192 8313 6992 1137 -593 -996 C
ATOM 1000 N CYS B 113 -29.308 0. 192 -21 .442 1. 00 29. 84 N
ANISOU 1000 N CYS B 113 3536 4464 3338 1050 -574 -899 N
ATOM 1001 CA CYS B 113 -28.576 -0. 643 -22 .391 1. 00 33. 55 C
ANISOU 1001 CA CYS B 113 4010 4910 3828 1002 -548 -856 C
ATOM 1002 C CYS B 113 -29.451 -1. 095 -23 .550 1. 00 40. 46 C
ANISOU 1002 C CYS B 113 4857 5841 4676 1015 -537 -863 C
ATOM 1003 o CYS B 113 -30.655 -1. 287 -23 .387 1. 00 40. 62 o
ANISOU 1003 o CYS B 113 4834 5939 4660 1037 -526 -889 ό
ATOM 1004 CB CYS B 113 -28.000 -1. 875 -21 .696 1. 00 31. 25 C
ANISOU 1004 CB CYS B 113 3694 4631 3549 931 -495 -814 < C
ATOM 1005 SG CYS B 113 -26.790 -1. 493 -20 .410 1. 00 40. 49 S
ANISOU 1005 SG CYS B 113 4894 5737 4753 911 -506 -802 S
ATOM 1006 N PRO B 114 -28.834 -1. 276^-24 .726 1. 00 35. 15 N
ANISOU 1006 N PRO B 114 ' 4202 5126 4029 988 -540 -823 N
ATOM 1007 CA PRO B 114 -29.505 -1. 797 -25 .920 1. 00 34. 78 C
ANISOU 1007 CA PRO B 114 4123 5125 3966 980 -529 -806 C ATOM 1008 C PRO B 114 -30.041 -3.209 -25.704 1.00 33.44 . C
ANISOU 1008 C PRO B 114 3896 5044 3766 942 -463 -809 C
ATOM 1009 0 PRO B 114 -29.568 -3. 922 -24 .824 1 .00 27 .75 0
ANISOU 1009 o PRO B 114 3168 4322 3056 895 -427 ■789 o
ATOM 1010 CB PRO B 114 -28,382 -1. 830 -26 .961 1 .00 34 .39 ■ C
ANISOU 1010 CB PRO B 114 4107 4993 3968 926 -540 ■736 C
ATOM 1011 CG PRO B 114 -27.392 -0. 8/37 -26 .492 1 .00 33 .31 C
ANISOU 1011 CG PRO B 114 4026 4764 3866 935 -578 ■731 c
ATOM 1012 CD PRO B 114 -27.436 -0. 898 -24 .998 1 .00 33 .44 c
ANISOU 1012 CD PRO B 114 4034 . 4805 3868 949 -561 ■770 c
ATOM 1013 N ASN B 115 -31.020 -3. 605 -26 .510 1 .00 33 .06 N
ANISOU 1013 N ASN B 115 3807 5068 3688 949 -451 ■818 N
ATOM 1014 CA ASN B 115 -31.542 -4. 962 -26 .465 1 .00 28 .15 C
ANISOU 1014 CA ASN B 115 3129 4525 3043 895 -390 •805 C
ATOM 1015 C ASN B 115 -30.414 -5. 976 -26 .623 1 .00 28 .32 C
ANISOU 1015 C ASN B 115 3158 4505 3097 815 -351 ■750 C
ATOM 1016 o ASN B 115 -29.514 -5. 781 -27 .432 1 .00 24 .91 o
ANISOU 1016 o ASN B 115 2756 4003 2705 791 -370 •707 o
ATO 1017 CB ASN B 115 -32.585 -5. 150 -27 .572 1 .00 39 .20 C
ANISOU 1017 CB ASN B 115 4486 6004 4405 916 -388 825 C
ATOM 1018 CG ASN B 115 -33.236 -6. 520 -27 .544 1 .00 45 .34 C
ANISOU 1018 CG ASN B 115 5206 6856 5165 844 -330 801 C
ATOM 1019 ODl ASN B 115 -33.325 -7. 163 -26 .498 1 .00 39 .60 o
ANISOU 1019 ODl ASN B 115 4470 6134 4444 800 -300 784 o
ATOM 1020 ND2 ASN B 115 -33.706 -6. 970 -28 .702 1 .00 51 .02 N
ANISOU, 1020 ND2 ASN B 115 5889 7634 5863 830 -316 800 N
ATOM 1021 N ASN B 116 -30.466 -7. 051 -25 .84.3 1 .00 27 .55 N
ANISOU 1021 N ASN B 116 3036 4431 3001 752 -306 721 N
ATOM 1022 CA ASN B 116 -29.462 -8. 111 -25 .900 1 .00 31 .79 C
ANISOU 1022 CA ASN B 116 3578 4934 3566 675 -269 671 C
ATOM 1023 C ASN B 116 -28.123 -7. 753 -25 .255 1 .00 29 .93 C
ANISOU 1023 C ASN B 116 3387 4617 3368 673 -286 656 C
ATOM 1024 o ASN B 116 -27.179 -8. 548 -25 .304 1 .00 31 .58 o
ANISOU 1024 o ASN B 116 3602 4795 3602 617 -261 618 o
ATOM 1025 CB ASN B 116 -29.237 -8. 591 -27 .334 1 .00 36 .40 C
ANISOU 1025 CB ASN B 116 4150 5526 4153 644 -254 651 C
ATOM' 1026 CG . ASN B 116 -30.464 -9. 257 -27 .923 1 .00 48 .97 C
ANISOU 1026 CG ASN B 116 5692 7205 5711 622 -228 658 C
ATOM 1027 ODl ASN B 116 -31.026 -8. 782 -28 .910 1 .00 51 .41 o
ANISOU 1027 ODl ASN B 116 5982 7553 5996 661 -246 683 o
ATOM 1028 ND2 ASN B 116 -30.886 -10. 363 -27 .318 1 .00 51 .84 N
ANISOU 1028 ND2 ASN B 116 6033 7596 6068 561 -189 - 637 N
ATOM 1029 N TRP B 117 -28.038 -6. 562 -24 .669 1 .00 24 .41 N
ANISOU 1029 N TRP B 117 2718 3884 2673 731 -328 - 686 N
ATOM 1030 CA TRP B 117 -26.862 -6. 181 -23 .891 1 .00 21 .04 C
ANISOU 1030 CA TRP B 117 2328 3384 2282 725 -346 672 C
ATOM. 1031 C TRP B 117 -27.164 -6. 372 -22 .403 1 .00 22 .43 C
ANISOU 1031 C TRP B 117 2490 3588 2443 722 -334 - 682 C
ATOM 1032 0 TRP B 117 -28.315 -6. 277 -21 .985 1 .00 27 .02 o
ANISOU 1032 0 TRP B 117 3045 4227 2993 744 -331 707 o
ATOM 1033 CB TRP B 117 -26.484 -4. 716 -24 .153 1 .00 22 .40 C
ANISOU 1033 CB TRP B 117 2546 3484 2482 770 -404 - 679 C
ATOM 1034 CG TRP B 117 -25.964 -4. 441 -25 .532 1 .00 21 .30 C
ANISOU 1034 CG TRP B 117 2427 3288 2380 752 -425 - 635 c
ATOM 1035 CD1 TRP B 1-1-7 -26.628 -4. 639 -26 .713 1 .00 24 .06 c
ANISOU 1035 CD1 TRP B 117 . 2757 3672 2712 754 -424 632 c
ATOM 1036 CD2 TRP B 117 -24.678 -3. 904 -25 .880 1 .00 12 .7 c
ANISOU 1036 CD2 TRP B 117 1385 2104 1353 726 -452 588 c
ATO 1037 NE1 TRP B 117 -25.833 -4. 272 -27 .774 1 .00 20 .96 N
ANISOU 1037 NE1 TRP B 117 2395 3207 2361 730 -450 584 N
ATOM 1038 CE2 TRP B 117 -24.635 -3. 814 -27 .292 1 .00 15 .56 c ANISOU 1038 CE2 TRP B 117 1750 2440 1722 714 -466 ■558 C
ATOM 1039 CE3 TRP B 117 -23.559 -3. 505 -25 .142 1. .00 17. ,34 C
ANISOU 1039 CE3 TRP B 117 1995 2616 1975 708 -465 ■567 C
ATOM 1040 CZ2 TRP B 117 -23.522 -3. 341 -27 .976 1. 00 15. 91 C
ANISOU 1040 CZ2 TRP B 117 1833 2397 1815 687 -490 >511 C
ATOM 1041 CZ3 TRP B 117 -22.450 -3. 032 -25 .824 1. 00 21. 41 C
ANISOU 1041 CZ3 TRP B 117 2546 3048 2542 682 -487 ■522 C
ATOM 1042 CH2 TRP B 117 -22.446 -2. 943 -27 .231 1. 00 16. 13 C
ANISOU 1042 CH2 TRP B 117 1888 2359 1881 672 -499 •496 C
ATOM 1043 N ILE B 118 -26.142 -6. 648 -21 .600 1. 00 20. 82 N
ANISOU 1043 N ILE B 118 2301 3345 2265 694 -328 •660 N
ATOM 1044 CA ILE B 118 -26.345 -6. 781 -20 .160 1. 00 20. 92 C
ANISOU 1044 CA ILE B 118 2301 3381 2267 691 -320 •666 C
ATOM 1045 C ILE B 118 -25.668 -5. 620 -19 .432 1. 00 26. 76 C
ANISOU 1045 C ILE B 118 3076 4066 3027 729 -361 ■686 C
ATOM 1046 o ILE B 118 -24.640 -5. 119 -19 .882 1. 00 21. 27 o
ANISOU 1046 o ILE B 118 2411 3295 2375 721 -385 662 o
ATOM 1047 CB ILE B 118 -25.834 -8. 133 -19 .629 1. 00 28. 27 C
ANISOU 1047 CB ILE B 118 3213 4324 3204 630 -280 627 C
ATOM 1048 CGI ILE B 118 -24.354 -8. 321 -19 .953 1. 00 32. 86 C
ANISOU 1048 CGI ILE B 118 3813 4821 3850 589 -284 570 C
ATOM 1049 CG2 ILE B 118 -26.623 -9. 276 -20 .239 1. 00 26. 81 C
ANISOU 1049 CG2 ILE B 118 2996 4192 2998 588 -242 608 C
ATOM 1050 CD1 ILE B 118 -23.823 -9. 681 -19 .540 1. 00 39. 20 C
ANISOU 1050 CD1 ILE B 118 4593 5622 4677 524 -248 ■515 c
ATOM 1051 -N GLN B 119 -26.248 -5. 175 -18 .322 1. 00 28. 21. N
ANISOU 1051 N GLN B 119 3250 4277 3192 755 -370 712 N
ATOM 1052 CA GLN B 119 -25.684 -4. 030 -17 .613 1. 00 26. 15 C
ANISOU 1052 CA GLN B 119 3020 3969 2949 788 -409 734 C
ATOM 1053 C GLN B 119 -24.991 -4. 415 -16 .31.3 1. 00 27. 60 C
ANISOU 1053 C GLN B 119 3198 4146 3142 764 -397 720 C
ATOM 1054 o GLN B 119 -25.405 -5. 341 -15 .615 1. 00 23.80 o
ANISOU 1054 o GLN B 119 2685 3717 2641 738 -363 708 o
ATOM 1055 CB GLN B 119 -26.754 -2. 964 -17 .328 1. 00 28. 76 C
ANISOU 1055 CB GLN B 119 3348 4325 3253 846 -438 781 C
ATOM 1056 CG GLN B 119 -26.200 -1. 682 -16 .695 1. 00 33. 00 C
ANISOU 1056 CG GLN B 119 3920 4810 3808 881 -483 804 C
ATOM 1057 CD GLN B 119 -27.287 -0. 686 -16 .303 1. 00 42. 34 C
ANISOU 1057 CD GLN B 119 5097 6025 4965 939 -512 - 853 c
ATOM 1058 OEl GLN B 119 -28.479 -0. 968 -16 .419 1. 00 43. 01 o
ANISOU 1058 OEl GLN B 119 5149 6176 5018 954 -498 - 872 o
ATOM 1059 NE2 GLN B 119 -26.873 0. 488 -15 .834 1. 00 43. 28 N
ANISOU 1059 NE2 GLN B 119 5248 6099 5098 970 -553 - 875 N
ATOM 1060 N ASN B 120 -23.916 -3. 701 -16 .013 1. 00 25. 65 N
ANISOU 1060 N ASN B 120 2984 3834 2927 771 -426 - 720 N
ATO 1061 CA ASN B 120 -23.340 -3. 708 -14 .682 1. 00 23. 34 c
ANISOU 1061 CA ASN B 120 2686 3533 2649 759 -425 - 709 C
ATOM 1062 C ASN B 120 -22.870 -2. 302 -14 .369 1. 00 25. 29 C
ANISOU 1062 C ASN B 120 2968 3725 2915 793 -472 - 734 C
ATOM 1063 o ASN B 120 -21.933 -1. 795 -14 .990 1. 00 22. 59 o
ANISOU 1063 o ASN B 120 2653 3308 2623 774 -494 - 698 o
ATOM 1064 CB ASN B 120 -22.200 -4. 727 -14 .551 1. 00 '25. 79 C
ANISOU 1064 CB ASN B 120 2981 3801 3015 686 -398 - 623 C
ATOM 1065 CG ASN B 120 -21.779 -4. 944 -13 .100 1. 00 33. 48 C
ANISOU 1065 CG ASN B 120 3940 4783 3998 674 -390 - 609 c
ATOM 1066 ODl ASN B 120 -22.572 -5. 385 -12 .266 1. 00 38. 60 o
ANISOU 1066 ODl ASN B 120 4566 5503 4598 689 -373 - 642 · o
ATOM 1067 ND2 ASN B 120 -20.532 -4. 629 -12 .797 1. 00 31. 34 N
ANISOU 1067 ND2 ASN B 120 3679 4441 ■ 3789 648 -403 - 560 N
ATOM 1068 N ARG B 121 -23.556 -1. 666 -13 .425 1. 00 34. 05 N
ANISOU 1068 N ARG B 121 4071 4870 3997 832 -485 - 777 N ATOM · 1069 CA ARG B 121 -23.258 -0.293 -13.040. 1.00 33.53 C
ANISOU 1069 CA ARG B 121 4035 4760 3944 867 -531 •804 C
ATOM . 1070 C ARG B 121 -23.249 0. 648 -14 .248 1 .00 24. 77 C
ANISOU 1070 C ARG B 121 2962 3602 2847 892 -568 •814 C
ATOM 1071 0 ARG B 121 -24.261 0. 805 -14 .929 1 .00 29. 19 o
ANISOU 1071 0 ARG B 121 3514 4193 3382 919 -571 833 o
ATOM 1072 CB ARG B 121 -21.936 -0. 233 -12 .269 1 .00 34. 62 C
ANISOU 1072 CB ARG B 121 4187 4849 4119· 842 -538 ■784 C
ATOM 1073 CG ARG B 121 -21.805 -1. 335 -11 .223 1 .00 38. 75 C
ANISOU 1073 CG ARG B 121 4672 5413 4640 807 -499 ■752 C
ATOM 1074 CD ARG B 121 -20.733 -1. 024 -10 .190 1 .00 37. 03 c
ANISOU 1074 CD ARG B 121 4454 5152 4462 786 -509 ■721 c
ATOM 1075 NE ARG B 121 -21.142 0. 071 -9 .318 1 .00 43. 01 N
ANISOU 1075 NE ARG B 121 5225 5926 5189 841 -541 ■790 N
ATOM 1076 CZ ARG B 121 -20.377 1. 112 -9 .014 .1 .00 48. 77 c
ANISOU 1076 CZ ARG B 121 5981 6599 5949 849 -576 794 c
ATOM 1077 NH1 ARG B 121 -19.149 1. 196 -9 .506 1 .00 48. 31 N
ANISOU 1077 NH1 ARG B 121 5938 6466 5950 805 -583 732 N
ATOM 1078 NH2 ARG B 121 -20.838 2. 064 -8 .213 1 .00 50. 26 N
ANISOU 1078 NH2 ARG B 121 6177 6805 6112 896 -602 851 N
ATOM 1079 N GLU B 122 -22.104 , 1. 260 -14 .524 1 .00 27. 23 N
ANISOU 1079 N GLU B 122 3313 3837 3198 882 -597 •802 N
ATOM 1080 CA GLU B 122 -22.031 2. 263 -15 .579 1 .00 32. 81 c
ANISOU 1080 CA GLU B 122 4059 4492 3915 904 -638 811 C
ATOM 1081 C GLU B 122 -21.806 1. 650 -16 .963 1 .00 27. 91 C
ANISOU 1081 C GLU B 122 3443 3851 3313 873 -624 771 C
ATOM 1082 o GLU B 122 -21.776 2. 368 -17 .962 1 .00 29. 71 o
ANISOU 1082 o GLU B 122 3703 4038 3547 890 -655 775 o
ATOM 1083 CB GLU B 122 -20.936 3. 294 -15 .274 1 .00 44. 38 C
ANISOU 1083 CB GLU B 122 5563 5882 5416 901 -678 809 C
ATOM 1084 CG GLU B 122 -21.240 4. 196 -14 .082 1 .00 64. 09 C
ANISOU 1084 CG GLU B 122" 8058 8393 7900 935 -699 843 C
ATOM 1085 CD GLU B 122 -20.173 5. 258, -13 .856 1 .00 77. 29 C
ANISOU 1085 CD GLU B 122 9769 9992 9606 930 -740 840 C
ATOM 1086 OEl GLU B 122 -20.502 6. 460 -13 .955 1 .00 81. 70 o
ANISOU 1086 OEl GLU B 122 10353 10533 10158 968 -780 867 o
ATOM 1087 OE2 GLU B 122 -19.011 4. 892 -13 .573 1 .00 78. 40 ' o
ANISOU 1087 OE2 GLU B 122 9912 10095 9782 887 -732 808 o
ATOM 1088 N SER B 123 -21.663 0. 327 -17 .020 1 .00 25. 28 N
ANISOU 1088 N SER B 123 3074 3544 2989 824 -575 724 N
ATOM 1089 CA SER B 123 -21.299 -0. 339 -18 .272 1 .00 23. 39 C
ANISOU 1089 CA SER B 123 2831 3280 2776 781 -556 670 C
ATOM 1090 C SER B 123 -22.411 -1. 215 -18 .861 1 .00 23. 54 C
ANISOU 1090 C SER B 123 2820 3370 2755 789 -525 683 C
ATOM 1091 o SER B 123 -23.204 -1. 801 -18 .122 1 .00 23. 41 o
ANISOU 1091 o SER B 123 2773 3425 2698 802 -500 712 o
ATOM 1092 CB SER B 123 -20.043 -1. 191 -18 .068 1 .00 24. 62 C
ANISOU 1092 CB SER B 123 2970 3397 2989 709 -526 593 C
ATOM 1093 OG SER B 123 -18.911 -0. 391 -17 .773 1 .00 30. 94 O
ANISOU 1093 OG SER B 123 3795 4128 3832 696 -552 573 o
ATOM 1094 N CYS B 124 -22.443 -1. 301 -20 .195 1 .00 19. 37 N
ANISOU 1094 N CYS B 124 2300 2823 2237 780 -527 661 N
ATOM 1095 CA CYS B 124 -23.298 -2. 253 -20 .924 1 .00 18. 10 C
ANISOU 1095 CA CYS B 124 2107 2723 2047 773 -493 660 C
ATOM 1096 C CYS B 124 -22.403 -3. 186 -21 .715 1 .00 15. 63 C
ANISOU 1096 C CYS B 124 1785 2373 1781 703 -464 586 c
ATOM 1097 o CYS B 124 -21.421 -2. 741 -22 .310 1 .00 18. 70 o
ANISOU 1097 o CYS B 124 220.0 2687 2216 681 -483 547 o
ATOM 1098 CB CYS B 124 -24.207 -1. 545 -21 .932 1 .00 19. 62 c
ANISOU 1098 CB CYS B 124 2314 2931 2211 826 -522 •698 c
ATOM 1099 SG CYS B 124 -25.335 -0. 321 -21 .223 1 .00 34. 61 S ANISOU 1099 SG CYS B 124 4224 4868 4060 917 -562 ■787 S
ATOM 1100 N TYR B 125 -22.753 -4. 465 -21. 760 1. 00 12. ,82 N
ANISOU 1100 N TYR B 125 1391 2069 1412 670 -419 568 N
ATOM 1101 CA TYR B 125 -21.906 -5. 442 -22. 444 1. 00 13. ,77 C
ANISOU 1101 CA TYR B 125 1496 2155 •1580 603 -389 ■498 C
ATOM 1102 C TYR B 125 -22.689 -6. 276 -23. 429 1. 00 15. ,61 C
ANISOU 1102 C TYR B 125 1705 2442 1785 590 -361 ■499 C
ATOM 1103 0 TYR B 125 -23.852 -6. 602 -23. 192 1. 00 16. 39 o
ANISOU 1103 0 TYR B 125 1780 2622 1823 616 -346 ■546 o
ATOM 1104 CB TYR B 125 -21.258 -6. 405 -21. 460 1. 00 9. 23 C
ANISOU 1104 CB TYR B 125 897 1576 1034 556 -356 457 C
ATOM 1105 CG TYR B 125 -20.516 -5. 741 -20. 331 1. 00 12. 59 C
ANISOU 1105 CG TYR B 125 1337 1962 1486 563 -376 ■453 C
ATOM 1106 CD1 TYR B 125 -21.133 -5. 525 -19. 116 1. 00 14. 90 C
ANISOU 1106 CD1 TYR B 125 1624 2302 1735 598 -382 500 C
ATOM 1107 CD2 TYR B 125 -19.199 -5. 346 -20. 480 1. 00 14. 38 C
ANISOU 1107 CD2 TYR B 125 1579 2105 1777 535 -388 403 C
ATOM 1108 CE1 TYR B 125 -20.461 -4. 927 -18. 072 1. 00 17. 60 C
ANISOU 1108 CE1 TYR B 125 1977 2610 2099 603 -400 497 C
ATOM 1109 CE2 TYR B 125 -18.513 -4. 744 -19. 438 1. 00 13. 30 C
ANISOU 1109 CE2 TYR B 125 1453 1937 1663 540 -405 400 C
ATOM 1110 CZ TYR B 125 -19.153 -4. 543 -18. 237 1. 00 15. 03 c
ANISOU 1110 CZ TYR B 125 1668 2205 1839 573 -412 447 c
ATOM 1111 OH TYR B 125 -18.492 -3. 950 -17. 193 1. 00 14. 26 o
ANISOU 1111 OH TYR B 125 1578 2078 1762 578 -429 446 o
ATOM 1112 N TYR B 126 -22.026 -6. 631 -24. 519 1. 00 13. 67 N
ANISOU 1112 N TYR B 126 1459 2153 1582 549 -353. 448 N
ATOM 1113 CA TYR B 126 -22.621 -7. 484 -25. 537 1. 00 10. 71 C
ANISOU 1113 CA TYR B 126 1058 1825 1187 526 -325 443 C
ATOM 1114 C TYR B 126 -21.698 -8. 658 -25. 764 1. 00 18. 13 c
ANISOU 1114 C TYR B 126 1975 2732 2183 453 -285 379 c
ATOM 1115 o TYR B 126 -20.501 -8. 478 -26. 015 1. 00 13. 61 o
ANISOU 1115 o TYR B 126 1415 2079 1678 427 -289 327 o
ATOM 1116 CB TYR B 126 -22.811 -6. 728 -26. 845 1. 00 12. 96 c
ANISOU 1116 CB TYR B 126 1364 2091 1471 548 -356 446 c
ATOM 1117 CG TYR B 126 -23.341 -7. 613 -27. 947 1. 00 13. 22 c
ANISOU 1117 CG TYR B 126 1365 2172 1485 516 -327 436 c
ATOM 1118 CD1 TYR B 126 -24.693 -7. 899 -28. 035 1. 00 16. 84 c
ANISOU 1118 CD1 TYR B 126 1795 2726 1879 542 -312 485 c
ATOM 1119 CD2 TYR B 126 -22.491 -8. 159 -28. 890 1. 00 17. 23 c
A ISOU 1119 CD2 TYR B 126 1869 2637 2043 459 -313 381 c
ATOM 1120 CE1 TYR B 126 -25.187 -8. 712 -29. 042 1. 00 24. 39 c
ANISOU 1120 CE1 TYR B 126 2717 3734 2815 506 -284 478 c
ATOM 1121 CE2 TYR B 126 -22.979 -8. 975 -29,907 1. 00 17. 00 c
ANISOU 1121 ' CE2 TYR B 126 1809 2657 1994 422 -287 377 c
ATOM 1122 CZ TYR B 126 -24.327 -9. 242 -29. 974 1. 00 24. 64 c
ANISOU 1122 CZ TYR B 126 2749 3722 2893 444 -273 - 426 c
ATOM 1123 OH TYR B 126 "-24.818 -10. 053 -30. 982 1. 00 27. 20 o
ANISOU 1123 OH TYR B 126 3040 4101 3192 398 -245 425 o
ATOM 1124 N VAL B 127 -22.255 -9. 860 -25. 669 1. 00 16. 32 N
ANISOU 1124 N VAL B 127 1711 - 2563 1926 423 -244 383 N
ATOM 1125 CA VAL B 127 -21.482 -11. 075" -25. 843 1. 00 16. 35 c
ANISOU 1125 CA VAL B 127 1689 2534 1988 356 . -201 326 c
ATOM 1126 C VAL B 127 -21.856 -11. 685 -27. 170. 1. 00 15. 60 c
ANISOU 1126 C VAL B 127 1575 2468 1883 322 -178 327 c
ATOM 1127 o VAL B 127 -22.997 -12. 116 -27. 366 1. 00 14. 54 o
ANISOU 1127 o VAL B 127 1427 2424 1675 324 -166 372 o
ATOM 1128 CB VAL B 127 -21.779 -12. 095 -24. 730 1. 00 10. 26 c
ANISOU 1128 CB VAL B 127 895 1805 1199 335 -171 329 c
ATOM 1129 CGI VAL B 127 -20.922 -13. 348 -24. 927 1. 00 13. 29 c
ANISOU 1129 CGI VAL B 127 1252 2136 1662 270 -121 267 c ATOM 1130 CG2 VAL B 127 -21.528 -11.466 -23.356 1.00 15.64 C
ANISOU 1130 CG2 VAL B 127 1591 2471 1880 368 -195 336 c
ATOM 1131 N SER B 128 -20.900 -11. 726 -28 .090 1 .00 8. 66 N
ANISOU 1131 N SER B 128 694 1519 1076 290 -167 - 278 N
ATOM 1132 CA SER B 128 -21.197 -12. 144 -29 .451 1 .00 8. 55 C
ANISOU 1132 CA SER B 128 663 1531 1056 255 -146 282 C
ATOM 1133 C SER B 128 -21.504 -13. 620 -29 .568 1 .00 16. 73 C
ANISOU 1133 C SER B 128 1665 2604 2089 191 -81 285 C
ATOM 1134 0 SER B 128 -21.104 -14. 428 -28 .721 1 .00 14. 26 o
ANISOU 1134 0 SER B 128 1342 2263 1814 168 -44 261 o
ATOM 1135 CB SER B 128 -20.022 -11. 801 -30 .372 1 .00 9. 18 C
ANISOU 1135 CB SER B 128 746 1522 1219 235 -140 230 C
ATOM 1136 OG SER B 128 -19.052 -12. 846 -30 .393 1 .00 8. 14 o
ANISOU 1136 OG SER B 128 588 1332 1172 180 -57 183 o
ATOM 1137 N GLU B 129 -22.203 -13. 959 -30 .650 1 .00 17. 35 N
ANISOU 1137 N GLU B 129 1729 2744 2119 160 -65 316 N
ATOM 1138 CA GLU B 129 -22.450 -15. 344 -31 .016 1 .00 16. 30 C
ANISOU 1138 CA GLU B 129 1577 2634 1982 82 4 - 323 C
ATOM 1139 C GLU B 129 -21.843 -15. 633 -32 .389 1 .00 14. 82 C
ANISOU 1139 C GLU B 129 1379 2427 1825 14 63 - 329 C
ATOM 1140 o GLU B 129 -22.122 -16. 669 -32 .999 1 .00 16. 83 o
ANISOU 1140 o GLU B 129 1645 2683 2067 -62 125 335 o
ATOM 1141 CB GLU B 129 -23.953 -15. 635 -31 .012 1 .00 22. 93 C.
ANISOU 1141 CB GLU B 129 2412 3601 2702. 83 -17 ■ - 384 C
ATOM 1142 CG GLU B 129 -24.581 -15. 568 -29 .628 1 .00 31. 53 C
ANISOU 1142 CG GLU B 129 3506 4740 3736 134 -45 - 409 C
ATOM 1143 CD GLU B 129 -26.102 -15. 548 -29 .665 1 .00 46. 31 C
ANISOU 1143 CD GLU B 129 5372 6722 5503 156 -52 461 C
ATOM 1144 OEl GLU B 129 -26.672 -15. .298 -30 .749 1 .00 46. o
ANISOU 1144 OEl GLU B 129 5410 6791 5534 149 -47 - 465 o
ATOM 1145 OE2 GLU B 129 -26.727 -15. 777 -28 .606 1 .00 52. 45 o
ANISOU 1145 OE2 GLU B 129 6148 7518 6263 173 -56 464 o
ATOM 1146 N ILE B 130 -21.014 -14. 713 -32 .870 1 .00 12. 63 N
ANISOU 1146 N ILE B 130 1095 2113 1591 38 45 325 N
ATOM ■ 1147 CA ILE B 130 -20.235 -14. 905 -34 .094 1 .00 11. 57 C
ANISOU 1147 CA ILE B 130 966 1931 1500 - 3 123 328 C
ATOM 1148 C ILE B 130 -18.746 -14. 768 -33 .809 1 .00 13. 55 C
ANISOU 1148 C ILE B 130 1238 2041 1869 -40 162 265 C
ATOM 1149 o ILE B 130 -18.330 -14. 136 -32 .824 1 .00 15. 26 o
ANISOU 1149 o ILE B 130 1451 2224 2125 39 100 231 o
ATOM 1150 CB ILE B 130 -20.609 -13. 872 -35 .192 1 .00 9. 59 C
ANISOU 1150 CB ILE B 130 679 1782 1182 -33 63 - 394 C
ATOM 1151 CGI ILE B 130 -20.328 -12. 443 -34 .704 1 .00 16. 46 c
ANISOU 1151 CGI ILE B 130 1570 2618 2067 97 -81 377 c
ATOM 1152 CG2 ILE B 130 -22.076 -14. 033 -35 .594 1 .00 15. 87 c
A ΪSOU 1152 CG2 ILE B 130 1457 2729 1842 -39 24 448 c
ATOM 1153 CDl ILE B 130 -20.478 -11. 395 -35 .797 1 .00 23. 37 c
ANISOU 1153 CDl ILE B 130 2536 3470 2873 109 -168 377 c
ATOM 1154 N TRP B 131 -17.934 -15. 344 -34 .681 1 .00 10. 25 N
ANISOU 1154 N TRP B 131 873 1530 1493 -129 254 242 N
ATOM 1155 CA TRP B 131 -16.495 -15. 221 -34 .553 1 .00 9. 99 C
ANISOU 1155 CA TRP B 131 881 1364 1551 -132 281 193 C
ATOM 1156 C TRP B 131 -15.988 -14. 143 -35 .472 1 .00 10. 38 C
ANISOU 1156 C TRP B 131 933 1356 1654 -160 291 223 C
ATOM 1157 o TRP B 131 -16.541 -13. 914 -36 .548 1 .00 12. 05 o
ANISOU 1157 o TRP B 131 1177 1607 1793 -214 290 258 o
ATOM 1158 CB TRP B 131 - -15.799 -16. 495 -34 .999 1 .00 12. 02 C
ANISOU 1158 CB TRP B 131 1220 1540 1808 -186 338 157 C
ATOM 1159 CG TRP B 131 -16.024 -17. 737 -34 .210 1 .00 7. 66 C
ANISOU 1159 CG TRP B 131 665 992 1253 -182 354 •160 C
ATOM 1160 CDl TRP B 131 ' -16.478 -17. 873 -32 .921 1 .00 13. 07 C ANISOU 1160 CD1 TRP B 131 1304 ' 1732 1929 -136 318 •146 C
ATOM 1161 CD2 TRP B 131 -15.794 -19. 048 -34 .702 1 .00 9. 08 C
ANISOU 1161 CD2 TRP B 131 893 1109 1448 -228 421 •191 C TOM 1162 NE1 TRP B 131 -16.547 -19. 216 -32 .588 1 .00 12. 42 N
ANISOU 1162 NE1 TRP B 131 1238 1624 1858 -163 359 ■161 N
ATOM 1163 CE2 TRP B 131 -16.122 -19. 955 -33 .669 1 .00 14. 63 C
ANISOU 1163 CE2 TRP B 131 1574 1829 2157 -215 422 •191 C
ATOM 1164 CE3 TRP B 131 -15.330 -19. 543 -35 .924 1 .00 9. 46 C
ANISOU 1164 CE3 TRP B 131 1000 1091 1503 -273 481 •225 C
ATOM 1165 CZ2 TRP B 131 -16.008 -21. 335 -33 .830 1 .00 18. 44 C
ANISOU 1165 CZ2 TRP B 131 2088 2259 2658 -247 480 221 C
ATOM 1166 CZ3 TRP B 131 -15.212 -20. 911 -36 .085 1 .00 13. 06 C
ANISOU 1166 CZ3 TRP B 131 1487 1500 1975 -298 541 ■262 C
ATOM 1167 CH2 TRP B 131 '-15.546 -21. 794 -35 .039 1 .00 13. 67 C
ANISOU 1167 CH2 TRP B 131 . 1539 1589 2065 -285 539 259 C
ATOM 1168 N SER B 132 -14.897 -13. 511 -35 .065 1 .00 9. 26 N
ANISOU 1168 N SER B 132 801 1103 1613 -121 274 186 N
ATOM 1169 CA SER B 132 -14.125 -12. 674 -35 .965 1 .00 9. 26 C
ANISOU 1169 CA SER B 132 894 992 1633 -146 258 166 C
ATOM 1170 C SER B 132 -12.705 -12. 617 -35 .427 1 .00 13. 25 C
ANISOU 1170 C SER B 132 . 1416 1372 2248 -123 287 ■114 C
ATOM 1171 o SER B 132 -12.441 -13. 082 -34 .302 1 .00 9. 15 o
ANISOU 1171 o SER B 132 867 859 1750 -64 280 -95 o
ATOM 1172 CB SER B 132 - -14.726 -11. 276 -36 .091 1 .00 16. 32 C
ANISOU 1172 CB SER B 132 1833 1930 2436 -49 94 179 C
ATOM 1173 OG SER B 132 -15.410 -10. 881 -34 .918 1 .00 17. 96 o
ANISOU 1173 • OG SER B 132 1971 2235 2618 72 -10 193 o
ATOM 1174 N ILE B 133 -11.790 -12. 100 -36 .236 1 .00 8. 41 N
ANISOU 1174 N ILE B 133 888 699 1609 -135 281 -87 N
ATOM 1175 CA ILE B 133 -10.435 -11. 829 -35 .776 1 .00 7. 95 C
ANISOU 1175 CA ILE B 133 834 625 1562 -85 265 -45. C
ATOM 1176 C ILE B 133 -10.482 -10. 597 -34 .876 1 .00 8. 08 C
ANISOU 1176 C ILE B 133 830 618 1621 -21 157 -27 C
ATOM 1177 o ILE B 133 -11.515 -9. 910 -34 .791 1 .00 11. 00 o
ANISOU 1177 o ILE B 133 1209 972 1998 33 65 -59 o
ATOM 1178 CB ILE B 133 -9.464 -11. 615 -36 .950 1 .00 12. 00 C
ANISOU 1178 CB ILE B 133 1429 1121 2009 -126 269 -31 C
ATOM 1179 CGI ILE B 133 -9.911 -10. 412 -37 .789 1 .00 12. 69 C
ANISOU 1179 CGI ILE B 133 1600 1157 2063 -155 197 -31 C
ATOM 1180 CG2 ILE B 133 -9.387 -12. 897 -37 .800 1 .00 15. 16 C
ANISOU 1180 CG2 ILE B 133 1832 1531 2397 -162 374 -64 C
ATOM 1181 CD1 ILE B 133 -8.913 -9. 998 -38 .858 1 .00 15. 96 C
ANISOU 1181 CD1 ILE B 133 2084 1544 2438 -190 192 -14 C
ATOM 1182 N TRP B 134 -9.384 -10. 308 -34 .189 1 .00 10. 18 N
ANISOU 1182 N TRP B 134 1508 1168 1192 -210 317 - 133 N
ATOM 1183 CA TRP B 134 -9.421 -9. 278 -33 .144 1 .00 7. 91 C
ANISOU 1183 CA TRP B 134 1198 866 943 -167 286 - 118 C
ATOM 1184 C TRP B 134 -9.783 -7. 899 -33 .702 1 .00 11. 65 C
ANISOU 1184 C TRP B 134. 1812 1276 1337 -161 247 -80 C
ATOM 1185 o TRP B 134 -10.651 -7. 208 -33 .168 1 .00 14. 54 o
ANISOU 1185 o TRP B 134 2181 1641 1703 -93 164 -54 o
ATOM 1186 CB TRP B 134 -8.079 -9. 224 -32 .421 1 .00 9. 31 C
ANISOU 1186 CB TRP B 134 1307 1035 1196 -211 373 152 C
ATOM 1187 CG TRP B 134 -8.061 -8. 334 -31 .210 1 .00 8. 45 C
ANISOU 1187 CG TRP B 134 1153 918 1138 -164 341 144. C
ATOM 1188 CD1 TRP B 134 -8.417 -8. 673 -29 .947 1 .00 6. 65 C
ANISOU 1188 CD1 TRP B 134 806 739 983 -102 299 152 C
ATOM 1189 CD2 TRP B 134 -7.608 -6. 989 -31 .157 1 .00 12. 74 C
ANISOU 1189 CD2 TRP B 134 1775 1401 1666 -179 353 130 C
ATOM 1190 NE1 TRP B 134' -8.218 -7. 608 -29 .089 1 .00 8. 11 N
ANISOU 1190 NE1 TRP B 134 989 897 1196 -75 283 145 N ATOM 1191 CE2 TRP B 134 -7.737 -6.556 -29.817 1.00 10.,92 C
ANISOU 1191 CE2 TRP B 134 1464 1185 1500 -120 313 -131 C
ATOM 1192 CE3 TRP B 134 -7.132 -6. 095 -32. 114 1 .00 12. 10 C
ANISOU 1192 CE3 TRP B 134 1827 1252 1519 -236 393 -116 C
ATOM 1193 CZ2 TRP B 134 -7.387 -5. 280 -29. 410 1 .00 12. 81 C
ANISOU 1193 CZ2 TRP B 134 1747 1375 1744 -116 309 -121 C
ATOM 1194 CZ3 TRP B 134 -6.786 -4. 825 -31. 712 1 .00 20. ,17 c
ANISOU 1194 CZ3 TRP B 134 2893 2223 2547 -234 391 -103 c
ATOM 1195 CH2 TRP B 134 -6.913 -4. 425 -30. 370 1 .00 18. 53 c
ANISOU 1195 CH2 TRP B 134 2599 2032 2408 -173 348 -106 c
ATOM 1196 N HIS B 135 -9.127 -7. 507 -34. 786 1 .00 14. 83 N
ANISOU 1196 N HIS B 135 2334 1625 1674 -233 308 -77 N
ATOM 1197 CA HIS B 135 -9.345 -6. 179 -35. 350 1 .00 16. 24 C
ANISOU 1197 CA HIS B 135 2656 1737 1778 -236 279 -40 C
ATOM 1198 C HIS B 135 -10.774 -5. 993 -35. 854 1 .00 14. 03 C
ANISOU 1198 C HIS B 135 2445 1456 1429 -174 166 -5 C
ATOM 1199 0 HIS B 135 -11.387 -4. 953 -35. 647 1 .00 14. 82 o
ANISOU 1199 o HIS B 135 2599 1525 1507 -128 95 25 o
ATOM 1200 CB HIS B 135 -8.348 -5. 895 -36. 478 1 .00 20. 82 C
ANISOU 1200 CB HIS B 135 3354 2260 2296 -330 375 -44 C
ATOM 1201 CG HIS B 135 -8.511 -4. 539 -37. 093 1 .00 38. 28 C
ANISOU 1201 CG HIS B 135 5719 4397 4426 -340 350 -5 C
ATOM 1202 ND1 HIS B 135 -7.683 -3. 480 -36. 791 1 .00 38. 80 N
ANISOU 1202 ND1 HIS B 135 5820 4412 4511 -373 397 -1 N
ATOM 1203 CD2 HIS B 135 -9.422 -4. 065 -37. 978 1 .00 43. 78 C
ANISOU 1203 CD2 HIS B 135 6547 5062 5027 -319 277 33 C
ATOM 1204 CEl HIS B 135 -8.067 -2. 415 -37. 474 1 .00 36.89 C
ANISOU 1204 CEl HIS B 135 5723 4108 4184 -374 358 39 C
ATOM 1205 NE2 HIS B 135 -9.122 -2. 743 -38. 199 1 .00 38. 84 N
ANISOU 1205 NE2 HIS B 135 6033 4365 4359 -341 283 60 N
ATOM 1206 N THR B 136 -11.294 -7. 021 -36. 515 1 .00 11. 45 N
ANISOU 1206 N THR B 136 2115 1162 1074 -172 147 -11 N
ATOM 1207 CA THR B 136 -12.639 -6. 977 -37. 062 1 .00 15. 94 C
ANISOU 1207 CA THR B 136 2744 1729 1582 -116 39 17 C
ATOM 1208 C THR B 136 -13.667 -6. 981 -35. 935 1 .00 13. 44 C
ANISOU 1208 C THR B 136 2319 1456 1332 -22 -53 24 C
A OM 1209 o THR B 136 -14.722 -6. 352 -36. 052 1 .00 12. 31 o
ANISOU 1209 o THR B 136 2228 1294 1154 36 -151 53 o
ATOM 1210 CB THR B 136 -12.861 -8. 179 -37. 995 1 .00 15. 30 C
ANISOU 1210 CB THR B 136 2673 1675 1465 -140 47 3 C
ATOM 1211 OGl THR B 136 -11.999 -8. 050 -39. 137 1 .00 16. 32 o
ANISOU 1211 OGl THR B 136 2924 1757 1519 -225 129 0 o
ATOM 1212 CG2 THR B 136 -14.312 -8. 268 -38. 448 1 .00 16. 51 C
ANISOU 1212 CG2 THR B 136 2867 1833 1574 -74 -74 27 C
ATOM 1213 N SER B 137 -13.364 -7. 702 -34. 850 1 .00 11. 04 N
ANISOU 1213 N SER B 137 1864 1210 1122 -7 -23 -3 N
ATOM 1214 CA SER B 137 -14.213 -7. 681 -33. 660 1 .00 11. 99 , C
ANISOU 1214 CA SER B 137 1876 1372 1309 78 -95 2 C
ATOM 1215 C SER B 137 -14.299 -6. 261 -33. 117 1 .00 13. 97 c
ANISOU 1215 C SER B 137 2172 1581 1557 110 -130 24 c
ATOM 1216 o SER B 137 -15.382 -5. 775 -32. 787 1 .00 12. 12 o
ANISOU 1216 o SER B 137 1937 1-348 1323 . 182 -222 44 o
ATOM 1217 CB SER B 137 -13.676 -8. 623 -32. 561 1 .00 12. 40 c
ANISOU 1217 CB SER B 137 1770 1484 1458 79 -44 -31 c
ATOM 1218 OG SER B 137 -13.679 -9. 971 -33. 022 1 .00 10. 97 o
ANISOU 1218 OG SER B 137 1541 1343 1284 55 -20 -51 o
ATOM 1219 N GLN B 138 -13.161 -5. 581 -33. 064 1 .00 16. 04 N
ANISOU 1219 N GLN B 138 2474 1801 1819 55 -56 18 N
ATOM 1220 CA GLN B 138 -13.152 -4. 191 -32. 609 1 .00 14. 75 C
ANISOU 1220 CA GLN B 138 2361 1591 1652 79 -85 38 C
ATOM 1221 C GLN B 138 -14.019 -3. 329 -33. 531 1 .00 13. 54 C ANISOU 1221 C GLN B 138 2349 1385 1411 100 -164 7^ C TOM 1222 o GLN B 138 -14.811 -2. 515 -33 .060 1 .00 15. .73 o
ANISOU 1222 o GLN B 138 2635 1650 1693 166 -246 94 o TOM 1223 CB GLN B 138 -11.726 -3. 637 -32 .557 1 .00 16. 01 C
ANISOU 1223 CB GLN B 138 2551 1709 1825 7 12 24 C
ATOM 1224 CG GLN B 138 -11.654 -2. 125 -32 .272 1 .00 18. 01 C
ANISOU 1224 . CG GLN B 138 2876 1902 2065 23 -16 47 c
ATOM 1225 CD GLN B 138 -12.249 -1. 738 -30 .923 1 .00 20. 07 c
ANISOU 1225 CD GLN B 138 3044 2191 2390 107 -81 46 c
ATOM 1226 OEl GLN B 138 -12.526 -2. 590 -30 .086 1 .00 14. 53 o
ANISOU 1226 OEl GLN B 138 2216 1554 1751 147 -90 27 o
ATOM 1227 NE2 GLN B 138 -12.436 -0. 433 -30 .709 1 .00 22. 73 N
ANISOU 1227 NE2 GLN B 138 3446 2479 2712 135 -126 68 N
ATOM 1228 N GLU B 139 -13.877 -3. 522 -34 .844 1 .00 14. 78 N
ANISOU 1228 N GLU B 139 2617 1511 1489 46 -142 83 N
ATOM 1229 CA GLU B 139 -14.688 -2. 773 -35 .814 1 .00 13. 34 c
ANISOU 1229 CA GLU B 139 2579 1275 1215 63 -221 119 c
ATOM 1230 C GLU B 139 -16.172 -3. 068 -35 .595 1 .00 17. 66 C
ANISOU 1230 C GLU B 139 3080 1856 1774 152 -339 128 C
ATOM 1231 0 GLU B 139 -17.013 -2. 175 -35 .691 1 .00 21. 92 o
ANISOU 1231 o GLU B 139 3685 2359 2283 203 -430 154 o
ATOM 1232 CB GLU B 139 -14.298 -3. 137 -37 .247 1 .00 19. 95 C
ANISOU 1232 CB GLU B 139 3536 2081 1964 -10 -174 122 C
ATOM 1233 CG GLU B 139 -12.832 -2. 936 -37 .587 1 .00 36. 78 C
ANISOU 1233 CG GLU B 139 5715 4177 4082 -105 -49 110 C
ATOM 1234 CD GLU B 139 -12.502 -1. 508 -37 .977 1 .00 55. 41 C
ANISOU 1234 CD GLU B 139 8213 6455 6384 -132 -46 141 C
ATOM 1235 / OEl GLU B 139 -12.343 -1. 246 -39 .192 1 .00 64. 30 o
ANISOU 1235 ' OEl GLU B 139 9487 7529 ■7416 -183 -27 159 o
ATOM 1236 OE2- GLU B 139 -12.390 -0. 652 -37 .073 1 .00 54. 83 o
ANISOU 1236 OE2 GLU B 139 8105 6367 6360 -102 -63 146 o
ATOM 1237 N ASN B 140 -16.488 -4. 326 -35 .303 1 .00 14. 72 N
ANISOU 1237 N ASN B 140 2592 1552 1450 171 -338 106 N TOM 1238 CA ASN B 140 -17.859 -4. 702 -34 .976 1 .00 15.11 C
ANISOU 1238 CA ASN B 140 2575 1638 1526 255 -440 110 C
ATOM 1239 C ASN B 140 -18.390 -3. 930 -33 .762 1 .00 18. 04 C
ANISOU 1239 C ASN B 140 2878 2018 1957 329 -495 116 C
ATOM 1240 o ASN B 140 -19.523 -3. 448 -33 .776 1 .00 19. 58 o
ANISOU 1240 o ASN B 140 3059 2213 2167 367 -555 115 o
ATOM 1241 CB ASN B 140 -17.976 -6. 211 -34 .742 1 .00 16. 9 C
ANISOU 1241 CB ASN B 140 2683 1943 1811 257 -417 84 C
ATOM 1242 CG ASN B 140 -17.923 -7. 007 -36 .040 1 .00 19. 44 C
ANISOU 1242 CG ASN B 140 3071 2253 2064 208 -401 80 C
ATOM 1243 ODl ASN B 140 -18.170 -6. 467 -37 .127 1 .00 23. 14 o
ANISOU 1243 ODl ASN B 140 3682 2666 2445 192 -438 101 o
ATOM 1244 ND2 ASN B 140 -17.583 -8. 287 -35 .935 1 .00 20. 98 N
ANISOU 1244 ND2 ASN B 140 3170 2500 2299 . 184 -349 52 N
ATOM 1245 N CYS B 141 -17.575 -3. 809 -32 .714 1 .00 16. 21 N
ANISOU 1245 N CYS B 141 2564 1806 1789 319 -430 100 N
ATOM 1246 CA CYS B 141 -17.985 -3. 038 -31 .548 1 .00 15. 04 C
ANISOU 1246 CA CYS B 141 2357 1665 1693 386 -475 103 C
ATOM 1247 C CYS B 141 -18.177 -1. 569 -31 .923 1 .00 16. 43 C
ANISOU 1247 C CYS B 141 2645 1775 1824 384 -510 122 C
ATOM 1248 o CYS B 141 -19.145 -0. 932 -31 .503 1 .00 16. 45 o
ANISOU 1248 o CYS B 141 2594 1797 1860 408 -537 109 o
ATOM 1249 CB CYS B 141 -16.974 -3. 174 -30 .405 1 .00 13. 24 C
ANISOU 1249 CB CYS B 141 2027 1465 1537 368 -395 79 C
ATOM .1250 SG CYS B 141 -16.802 -4. 866 -29 .768 1 .00 14. 40 S
ANISOU 1250 SG CYS B 141 2014 1700 1758 365 -344 47 S
ATOM 1251 N LEU B 142 -17.250 -1. 032 -32 .709 1 .00 14. 12 N
ANISOU 1251 N LEU B 142 2484 1417 1465 324 -471 140 N ATOM 1252 CA LEU B 142 -17.302 0 388 -33.050 1.00 18.95 C
ANISOU 1252 CA LEU B 142 3216 1955 202 325 -509 164 C TOM 1253 C LEU B 142 -18.595 0 734 -33.787 1.00 27.35 C
ANISOU 1253 C LEU B 142 4300 3020 3071 342 -574 156 C
ATOM 1254 0 LEU B 142 -19.125 1 837 -33.637 1.00 23.79 O
ANISOU 1254 0 LEU B 142 ' 3862 2550 2629 361 -609 153 O
ATOM 1255 CB LEU B 142 -16.076 0 795 -33.877 1.00 20.54 C
ANISOU 1255 CB LEU B 142 3536 2096 2171 232 -424 174 C
ATOM 1256 CG LEU B 142 -14.749 0 848 -33.108 1.00 21.75 C
ANISOU 1256 CG LEU B 142 3623 2256 2383 185 -320 151 C
ATOM 1257 CD1 LEU B 142 -13.578 1 005 -34.066 1.00 30.33 C
ANISOU 1257 CD1 LEU B 142 4815 3293 3418 84 -224 153 C
ATOM 1258 CD2 LEU B 142 -14.761 1 981 -32.086 1.00 19.87 C
ANISOU 1258 CD2 LEU B 142 3363 1996 2189 231 -354 155 C
ATOM > 1259 N LYS B 143 -19.105 -0 213 -34.572 1.00 20.76 N
ANISOU 1259 N LYS B 143 3470 2206 2212 335 -588 153 N
ATOM 1260 C LYS B 143 -21.508 0 238 -34.409 1.00 30.21 C
ANISOU 1260 C LYS B 143 4559 3442 3476 402 -676 122 C
ATOM 1261 o LYS B 143 -22.504 0 865 -34.780 1.00 28.86 O
ANISOU 1261 o LYS B 143 4411 3252 3302 419 -723 117 O
ATOM 1262 CA LYS B 143 -20.327 0 018 -35.341 1.00 22.45 C
ANISOU 1262 CA LYS B 143 3707 2411 2412 351 -647 145 C
ATOM 1263 CB LYS B 143 -20.645 -1 175 -36.249 1.00 29.20 C
ANISOU 1263 CB LYS B 143 4573 3283 3238 337 -654 143 C
ATOM 1264 CG LYS B 143 -19.640 -1 453 -37.355 1.00 37.76 ■ C
ANISOU 1264 CG LYS B 143 5797 4322 4227 272 -614 163 C
ATOM 1265 CD LYS B 143 -20.211 -2 474 -38.337 1.00 44.00 C
ANISOU 1265 CD LYS B 143 6601 5127 4990 264 -635 155 C
ATOM 1266 CE LYS B 143 . -19.183 -2 911 -39.368 1.00 52.40 C
ANISOU 1266 CE LYS B 143 7790 6160 5961 189 -575 168 C
ATOM 1267 NZ LYS B 143 -18.566 -1 760 -40.079 1.00 56.10 N
ANISOU 1267 NZ LYS B 143 8405 6551 6358 · 138 -548 187 N
ATOM 1268 N GLU B 144 -21.408 -0 309 -33.203 1.00 21.73 N
ANISOU 1268 N GLU B 144 3354 2431 2473 420 -638 109 N
ATOM 1269 CA GLU B 144 -22.491 -0 195 -32.239 1.00 22.72 C
ANISOU 1269 CA GLU B 144 3358 2606 2668 449 -639 91 C
ATOM 1270 C GLU B 144 -22.162 0 873 -31.210 1.00 20.80 C
ANISOU 1270 C GLU B 144 3092 2358 2451 459 -624 88 C
ATOM 1271 o GLU B 144 -22.761 0 910 -30.134 1.00 23.34 O
ANISOU 1271 o GLU B 144 - 3311 2727 2829 471 -600 77 O
ATOM 1272 CB GLU B 144 -22.751 -1 538 -31.554 1.00 24.71 C
ANISOU 1272 CB GLU B 144 3479 2931 2977 448 -595 80 C
ATOM 1273 CG GLU B 144 -22.840 -2 714 -32.520 1.00 38.37 C
ANISOU 1273 CG GLU B 144 5228 4669 4683 436 . -605 81 C
ATOM 1274 CD GLU B 144 -23.910 -2 528 -33.581 1.00 51.84 C
ANISOU 1274 CD GLU B 144 6994 6344 6357 444 -661 81 C
ATOM 1275 OEl GLU B 144 -24.888 -1 790 -33.324 1.00 52.56 O
ANISOU 1275 OEl GLU B 144 7069 6431 6469 463 -684 76 O
ATOM 1276 OE2 GLU B 144 -23.769 -3 118 -34.677 1.00 53.87 O
ANISOU 1276 OE2 GLU B 144 7320 6581 6568 430 -681 86 O
ATOM 1277 N GLY B 145 -21.205 1 736 -31.539 1.00 15.35 N
ANISOU 1277 N GLY B 145 1948 1566 2316 407 -138 -88 N
ATOM 1278 CA GLY B 145 -20.788 2 786 -30.628 1.00 19.03 C
ANISOU 1278 CA GLY B 145 2450 1980 2801 402 -160 -113 C
ATOM 1279 C GLY B 145 -20.192 2 258 -29.332 1.00 17.01 C
ANISOU 1279 C GLY B 145 2178 1739 2546 390 -144 -132 C
ATOM 1280 0 GLY B 145 -20.363 2 837 -28.261 1.00 19.90 O
ANISOU 1280 0 GLY B 145 2561 2078 2923 389 -141 -170 O
ATOM 1281 N SER B 146 -19.485 1 143 -29.441 1.00 16.01 N
ANISOU 1281 N SER B 146 2017 1658 2408 380 -133 -105 N
ATOM 1282 CA SER B 146 -18.863 0 521 -28.286 1.00 14.07 C ANISOU 1282 CA SER B 146 1747 1433 2164 370 -117 -119 C
ATOM 1283 C SER B 146 -17.420 0. 172 -28 .638 1 .00 12. 63 C
ANISOU 1283 c SER B 146 1560 1284 1953 329 - 126 -65 C
ATOM 1284 ό SER B 146 -16.918 0. 558 -29 .690 1 .00 17. 17 o
ANISOU 1284 0 SER B 146 2155 1846 2522 318 - 153 -28 o
ATOM 1285 CB SER B 146 -19.642 -0. 734 -27 .870 1 .00 15. 07 C
ANISOU 1285 CB SER B 146 1834 1623 2269 374 -70 -128 C
ATOM 1286 OG SER B 146 -19.570 -1. 735 -28 .874 1 .00 '17. 13 o
ANISOU 1286 OG SER B 146 2073 1930 2505 372 -58 -87 o
ATOM 1287 N THR B 147 -16.763 -0. 567 -27 .755 1 .00 11. 88 N
ANISOU 1287 N THR B 147 1441 1243 1830 299 -99 -58 N
ATOM 1288 CA THR B 147 -15.381 -0. 964 -27 .969 1 .00 12. 53 C
ANISOU 1288 CA THR B 147 1513 1372 1873 253 - 101 -7 C
ATOM 1289 C THR B 147 -15.202 -2. 357 -27 .359 1 .00 11. 89 C
ANISOU 1289 C THR B 147 1386 1355 1778 256 -68 -3 C
ATOM 1290 o THR B 147 -16.051 -2. 823 -26 .607 1 .00 13. 67 o
ANISOU 1290 o THR B 147 1596 1582 2017 280 -44 -41 O
ATOM 1291 CB THR B 147 -14.437 0. 049 -27 .285 1 .00 19. 96 C
ANISOU 1291 CB THR B 147 2493 2304 2786 196 - 112 -1 C
ATOM 1292 OGl THR B 147 -13.084 -0. 159 -27 .712 1 .00 22. 52 " o
ANISOU 1292 OGl THR B 147 2811 2673 3075 N 152 - 120 49 o
ATOM 1293 CG2 TH B 147 -14.540 -0. 063 -25 .764 1 .00 19. 72 c
ANISOU 1293 CG2 THR B 147 2459 2288 2744 181 -87 -35 c
ATOM 1294 N LEU B 148 -14.129 -3. 051 -27 .706 1 .00 9. 88 N
ANISOU 1294 N LEU B 148 1108 1151 1495 232 -66 42 N
ATOM 1295 CA LEU B 148 -13.913 -4. 350 -27 .079 1 .00 8. 86 C
ANISOU 1295 CA LEU 'B 148 939 1077 1349 235 -38 47 C
ATOM 1296 C LEU B 148 -13.857 -4. 208 -25 .551 1 .00 8. 97 C
ANISOU 1296 C LEU B 148 963 1099 1347 . 210 -21 18 C
ATOM. 1297 o LEU B 148 -13.347 -3. 214 -25 .022 1 .00 8. 01 o
ANISOU 1297 o LEU B 148 872 960 1210 172 -34 14 o
ATOM 1298 CB LEU B 148 -12.640 -5. 014 -27 .617 1 .00 10. 25 C
ANISOU ,1298 CB LEU B 148 1092 1307 1495 211 -41 98 C
ATOM 1299 CG LEU B 148 -12.772 -5. 693 -28 .987 1 .00 11. 47 C
ANISOU 1299 CG LEU B 148 1223 1472 1664 241 -44 125 C
ATOM 1300 CD1 LEU B 148 -11.408 -6. 118 -29 .511 1 .00 8. 91 C
ANISOU 1300 CD1 LEU B 148 879 1198 1309 212 -50 173 c
ATOM 1301 CD2 LEU B 148 -13.711 -6. 899 -2-8 .890 1 .00 12. 52 c
ANISOU 1301 CD2 LEU B 148 1337 1617 1804 275 -19 96 c
ATOM 1302 N LEU B 149 -14.395 -5. 196 -24 .849 1 .00 9. 53 N
ANISOU 1302 N LEU B 149 1010 1193 1419 229 7 -3 N
ATOM 1303 CA LEU B 149 -14.375 -5. 224 -23 .381 1 .00 9. 43 C
ANISOU 1303 CA LEU B 149 1004 1192 1387 _ 205 25 -29 C
ATOM 1304 C LEU B 149 -13.028 -4. 813 -22 .795 1 .00 8. 41 C
ANISOU 1304 C LEU B 149 887 1090 1219 151 13 -2 c
ATOM 1305 o LEU B 14'9 -11.985 -5. 336 -23 .192 1 .00 11. 18 o
ANISOU 1305 o LEU B 149 1218 1484 1546 135 6 41· o
ATOM 1306 CB LEU B 149 -14.704 -6. 644 -22 .904 1 .00 10. 15 c
ANISOU 1306 CB LEU B 149 1064 1323 1472 224 55 -34 c
ATOM 1307 CG LEU B 149 -14.564 -6. 896 -21 .402 1 .00 9. 68 c
ANISOU 1307 CG LEU B 149 1009 1283 1385 196 74 -54 c
ATOM 1308 CD1 LEU B 149 -15.470 -5. 955 -20 .614 1 .00 14. 13 c
ANISOU 1308 CD1 LEU B 149 1600 1803 1965 193 81 -110 c
ATOM 1309 CD2 LEU B 149 -14.881 -8. 359 -21 .075 1 .00 11. 56 c
ANISOU 1309 CD2 LEU B 149 1221 1557 1614 216 101 -54 c
ATOM 1310 N GLN B 150 -13.050 -3. 884 -21 .839 1 .00 6. 71 N
ANISOU 1310 N GLN B 150 702 851 996 121 12 -30 N
ATOM 1311 CA GLN B 150 -11.850 -3. 594 -21 .055 1 .00 12. 55 c
ANISOU 1311 CA GLN B 150 1451 1622 1694 66 5 -11 c
ATOM 1312 C GLN B 150 -12.154 -3. 921 -19 .60 1 .00 15. 28 C
ANISOU 1312 C GLN B 150 1799 1981 2025 53 29 -43 c ATOM 1313 0 GLN B 150 -13.218 -3.,582 -19.,103 1..00 10..71 O
ANISOU 1313 0 GLN B 150 1236 1365 1468 69 43 -91 O
ATOM 1314 CB GLN B 150 -11.430 -2. ,134 -21. ,214 1. ,00 13. 85 C
ANISOU 1314 CB GLN B 150 1657 1750 1856 31 -19 -11 C
ATOM 1315 CG GLN B 150 ■ -11.174 -1. ,786 -22. 659 1. ,00 14. ,33 C
ANISOU 1315 CG GLN B 150 1721 1795 1929 40 -43 20 C
ATOM 1316 CD GLN B 150 -10.605 -0. 405 -22. 843 1. ,00 15. 74 C
ANISOU 1316 CD GLN B 150 1942 1940 2097 -2 -68 26 C
ATOM 1317 OEl GLN. B- 150 -10.235 -0. 031 -23. 947 1. ,00 20. 55 0
ANISOU 1317 OEl GLN B 150 2561 2539 2709 -6 -88 54 o
ATOM 1318 NE2 GLN B 150 -10.517 ' 0. 35 -21. 756 1. 00 14. 49 N
ANISOU 1318 NE2 GLN B 150 1815 1767 1925 . -37 -65 -1 N
ATOM 1319 N ILE B 151 -11.250 -4. 634 -18. 953 1. 00 11. 42 N
ANISOU 1319 N ILE B 151 1294 1547 1500 27 33. -18 N
ATOM 1320 CA ILE B 151 -11.500 -5. 078 -17. 592 1. 00 10. 19 C
ANISOU 1320 CA ILE B 151 1141 1407 1325 13 55 -44 C
ATOM 1321 C ILE B 151 -10.560 -4. 300 -16. 670 1. 00 14. 40 C
ANISOU 1321 C ILE B 151 1696 1953 1823 -46' 44 -41 C
ATOM 1322 o ILE B 151 -9.356 -4. 554 -16. 633 1. 00 14. 95 o
ANISOU 1322 o ILE B 151 1752 2069 1860 -74 30 -1 o
ATOM 1323 CB ILE B 151- -11.286 -6. 579 -17. 466 1. 00 11. 63 C
ANISOU 1323 CB ILE B 151 1290 1638 1491 31 69 -21 C
ATOM 1324 CGI ILE B 151 -12.087 -7. 322 -18. 556 1. 00 14. 38 C
ANISOU 1324 CGI ILE B 151 1615 1975 1872 85 78 -19 C
ATOM 1325 CG2 ILE B 151 -11.624 -7. 059 -16. 042 1. 00 12. 96 C
ANISOU 1325 CG2 ILE B 151 1467 1821 1638 15 92 -49 C-
ATOM 1326 CD1 ILE B 151 -11.732 -8. 793 -18. 683 1. 00 16. 61 C
ANISOU 1326 CD1 ILE B 151 1867 2305 2139 104 87 12 C
ATOM 1327 N GLU B 152 -11.108 -3. 331 -15. 950 1. 00 14. 34 N
ANISOU 1327 N GLU B 152 1721 1905 1820 -64 50 -84 N
ATOM 1328 CA GLU ,B 152 -10.265 -2. 420 -15. 168 1. 00 17. 21 C
ANISOU 1328 CA GLU B 152 2113 2275 2153 -123 38 -83 C
ATOM 1329 C GLU B 152 -10.231 -2. 724 -13. 675 1. 00 17. 62 C
ANISOU 1329 C GLU B 152 2171 2350 2174 -154 56 104 C
ATOM ' 1330 o GLU B 152 -9.618 -1. 982 -12. 904 1. 00 19. 48 o
ANISOU 1330 o GLU B 152 2430 2590 2381 -205 49 •109 o
ATOM 1331 CB GLU B 152 -10.692 -0. 970 -15. 387 1. 00 24. 96 C
ANISOU 1331 CB GLU B 152 3135 3193 3157 -132 28 112 C
ATOM 1332 CG GLU B 152 -10.300 -0. 389 -16. 747 1. 00 29. 83 C
ANISOU 1332 CG GLU B 152 3758 3790 3788 -125 2 -82 C
ATOM 1333 CD GLU B 152 -11.437 -0. 424 -17. 747 1. 00 29. 87 C
ANISOU 1333 CD GLU B 152 3759 3749 3840 -65 3 -98 C
ATOM 1334 OEl GLU B 152 -11.316 0. 195 -18. 826 1. 00 27. 23 o
ANISOU 1334 OEl GLU B 152 3438 3387 3521 -58 . -19 -81 o
ATOM 1335 OE2 GLU B 152 -12.456 -1. 065 -17. 441 1. 00 22. 47 o
ANISOU 1335 OE2 GLU B 152 2807 2807 2924 -28 25 129 o
ATOM 1336 N SER B 153 -10.874 -3. 812 -13. 267 1. 00 12. 41 N
ANISOU 1336 N SER B 153 1493 1707 1516 -127 79 117 N
ATOM 1337 CA SER B 153 -10.997 -4. 124 -11. 850 1. 00 14. 83 c
ANISOU 1337 CA SER B 153 1809 2031 1794 -155 98 142 c
ATOM 1338 C SER B 153 -11.381 -5. 574 -11. 645 1. 00 12. 38 c
ANISOU 1338 c SER B 153 1475 1751 1478 -127 117 136 c
ATOM 1339 o SER B 153 -11.935 -6. 218 -12. 541 1. 00 11. 10' o
ANISOU 1339 o SER B 153 1291 1582 1344 -80 123 130 o
ATOM 1340 CB SER B 153 -12.061 -3. 241 -11. 186 1. 00 15. 72 c
ANISOU 1340 CB SER B 153 1954 2093 1926 -162 117 205 c
ATOM 1341 OG SER B 153 -13.366 -3. 576 -11. 645 1. 00 16. 20 o
ANISOU 1341 OG SER B 153 2004 2123 2028 -110 136 238 o
ATOM 1342 N LYS B 154 -11.087 -6. 082 -10. 453 1. 00 13. 61 ' N
ANISOU 1342 N LYS B' 154 1893 1446 1831 236 -103 348 N
ATOM 1343 CA LYS B 154 -11.503 -7. 419 -10. 093 1. 00 13. 17 C ANISOU 1343 CA LYS B 154 1837 1412 1756 188 -12 334 C
ATOM 1344 C LYS B 154 -13.021 -7. 503 -10. 073 1. .00 13. 06 C
ANISOU 1344 C LYS B 154 1802 1512 1649 182 16 275 C
ATOM 1345 o LYS B 154 -13.590 -8. 521 -10. 457 L. 00 12. 83 o
ANISOU 1345 o LYS B 154 1755 1522 1599 123 101 253 o
ATOM 1346 CB LYS B 154 -10.927 -7. 824 -8. 733 1. 00 12. 49 c
ANISOU 1346 CB LYS B 154 1785 1267 1693 210 -29 367 c
ATOM 1347 CG LYS B' 154 • -11.441 -9. 160 -8. 238 1. 00 12. 33 • c
ANISOU 1347 CG LYS B 154 1765 1273 1646 164 58 353 c
ATOM 1348 CD LYS B 154 , -10.694 -9. 616 -6. 983 1. 00 13. 04 c
ANISOU 1348 CD LYS B 154 1893 1291 1772 179 41 394 c
ATOM 1349 CE LYS ,B 154 -11.078 -11. 039 -6. 597 1. 00 17. 70 c
ANISOU 1349 CE LYS B 154 2483 1897 2346 124 131 388 c
ATOM 1350 NZ LYS B 154 -12.526 -11. 180 -6. 215 1. 00 13. 17 N
ANISOU 1350 NZ LYS B 154 1896 1432 1674 123 160 339 N
ATOM 1351 N GLU B 155 -13.675 -6. 425 -9. 651 1. 00 13. 02 N
ANISOU 1351 N GLU B 155 1798 1559 1591 243 -57 251 N
ATOM 1352 CA GLU B 155 -15.133 -6. 409 -9. 601 1. 00 16. 46 c
ANISOU 1352 CA GLU B 155 2209 2104 1942 245 -35 196 C
ATOM 1353 C GLU B 155 -15.708 -6. 676 -10. 990 1. 00 15. 36 C
ANISOU 1353 C GLU B 155 2032 2014 1790 187 17 168 C
ATOM 1354 o GLU B 155 -16.671 -7. 427 -11. 151 1. 00 15. 49 o
ANISOU 1354 o GLU B 155 2026 2098 1763 145 83 135 o
ATOM 1355 CB GLU B 155 ' -15.633 -5. 059 -9. 094 1. 00 21. 32 C
ANISOU 1355 CB GLU B 155 2829 2759 2512 325 -129 175 c
ATOM 1356 CG GLU B 155 -17.140 -4. 930 -9. 075 1. 00 29. 67 c
ANISOU 1356 CG GLU B 155 3856 3931 3487 333 -Ill 119 c
ATOM 1357 CD GLU B 155 -17.600 -3. 555 -8. 628 1. 00 42. 40 c
ANISOU 1357 CD GLU B 155 5473 5578 5058 417 -206 96 c
ATOM 1358 OEl GLU B 155 -16.739 -2. 666 -8. 445 1. 00 44. 89 o
ANISOU 1358 OEl GLU B 155 5817 5828 5410 465 -290 123 . o
ATOM 1359 OE2 GLU B 155 -18.824 -3. 365 -8. 460 1. 00 46. 30 o
ANISOU 1359 OE2 GLU B 155 5942 6164 5486 435 -197 52 . o
ATOM 1360 N GLU B 156 -15.104 -6. ,054 -11. 993 1. 00 10. 59 N
ANISOU 1360 N GLU B 156 1425 1375 1225 185 -15 182 N
ATOM 1361 CA GLU B 156 -15.596 -6. 188 -13. 356 1. 00 13. 00 C
ANISOU 1361 CA GLU B 156 1702 1722 1516 134 24 156 C
ATOM 1362 C GLU B 156 -15.243 -7. 567. -13. 903 1. 00 13. 62 C
ANISOU 1362 C GLU B 156 1780 1769 1626 57 124 166 C
ATOM 1363 o GLU B 156 -16.045 -8. 209 -14. 591 1. 00 11. 80 o
ANISOU 1363 o GLU B 156 1529 1593 1360 4 185 133 o
ATOM 1364 CB GLU B 156 -15.023 -5. 085 -14. 246 1. 00 12. 60 c
ANISOU 1364 CB GLU B 156 1651 1640 1494 156 -41 172 c
ATOM 1365 CG GLU B 156 -15.556 -5. 138 -15. 674 1. 00 14. 56 c
ANISOU 1365 CG GLU B 156 1876 1934 1721 106 -7 145 c
ATOM 1366 CD GLU B 156 -15.234 -3. 9.00 -16. 487 1. 00 15. 48 c
ANISOU 1366 CD GLU B 156 1992 2039 1851 133 -82 155 c
ATOM 1367 OEl GLU B 156 -14.224 -3. 213 -16. 198 1. 00 13. 18 o
ANISOU 1367 OEl GLU B 156 1719 1679 1612 173 -143 197 o
ATOM 1368 OE2 GLU B 156 -15.996 -3. 614 -17. 439 1. 00 13. 29 o
ANISOU 1368 OE2 GLU B 156 1695 1820 1535 Ill -83 123 o
ATOM 1369 N MET B 157 -14.040 -8. 034 -13. 593 1. 00 13. 13 N
ANISOU 1369 N MET B 157 1741, 1616 1632 50 140 212 N
ATOM 1370 CA MET B 157 -13.651 -9. 381 -14. 003 1. 00 12. 90 C
ANISOU 1370 CA MET B 157 1714 1550 1637 -18 234 222 c
ATOM 1371 C MET B 157 -14.618 -10. 419 -13. 436 1. 00 15. 18 c
ANISOU 1371 C MET B 157 1995 1895 1879 -52 296 193 c
ATOM 1372 o MET. B 157 -15.061 -11. 326 -14. 147 1. 00 13. 86 o
ANISOU 1372 o MET B 157 1814 1752 1698 -115 369 170 o
ATOM 1373 CB MET B 157 -12.218 -9. 695 -13. 577 1. 00 12. 88 c
ANISOU 1373 CB MET B 157 1736 1439 1720 -12 234 278 c ATOM 1374 CG MET B 157 -11.717 -11.05.9 -14.023 1.00 12.28 C
ANISOU 1374 CG MET B 157 1662 1317 1687 -79 329 290 C
ATOM 1375 SD MET B 157 -11.578 -11. 163 -15. 821 1 .00 16. 22 S
ANISOU 1375 SD MET B 157 2148 1817 2197 -130 380 276 S
ATOM 1376 CE MET B 157 -10.880 -12. 801 -16. 013 1 .00 16. 50 C
ANISOU 1376 CE MET B 157 2194 1784 2289 -194 486 293 C
ATOM 1377 N ASP B 158 -14.957 -10. 276 -12. 160 1 .00 15. 40 N
ANISOU 1377 N ASP B 158 2031 1943 1878 -11 265 193 N
ATOM 1378 CA ASP B 158 -15.882 -11. 212 -11. 525 1 .00 14. 07 C
ANISOU 1378 CA ASP B 158 1853 1830 1663 -40 322 170 C
ATOM 1379 C ASP B 158 -17.268 -11. 163 -12. 175 1 .00 15. 40 C
ANISOU 1379 C ASP B 158 1986 2102 1764 -64 343 119 c
ATOM 1380 o ASP B 158 -17.924 -12. 197 -12. 321 1 .00- 16. 32 o
ANISOU 1380 o ASP B 158 2087 2254 1860 -121 413 101 o
ATOM 1381 CB ASP B 158 -15.977 -10. 952 -10. 021 1 .00 18. 33 c
ANISOU 1381 CB ASP B 158 2412 2374 2179 15 281 182 c
ATOM 1382 CG ASP B 158 -14.698 -11. 335 -9. 275 1 .00 20. 87 c
ANISOU 1382 CG ASP B 158 2771 2593 2567 24 273 233 c
ATOM 1383 ODl ASP B 158 -13..880 -12. 098 -9. 827 1 .00 21. 15 o
ANISOU 1383 ODl ASP B 158 2810 2561 2664 -23 320 257 o
ATOM 1384 OD2 ASP B 158 -14.514 -10. 880 -8. 134 1 .00 20. 64 o
ANISOU 1384 OD2 ASP B 158 2766 2548 2528 78 220 249 o
ATOM 1385 N PHE B 159 -17.715 -9. 971 -12. 566 1 .00 12. 22 N
ANISOU 1385 N PHE B 159 1568 1744 1331 -22 280 97 N
ATOM 1386 CA PHE B 159 -19.008 -9. 860 -13. 234 1 .00 12. 41 c
ANISOU 1386 CA PHE B 159 1556 1862 1297 -44 292 50 c
ATOM 1387 C PHE B 159 -18.961 -10. 558 -14. 58 1 .00 15. 23 c
ANISOU 1387 C PHE B 159 1905 2209 1671 -118 350 41 c
ATOM 1388 o PHE B 159 -19.885 -11. 291 -14. 956 1 .00 17. 38 o
ANISOU 1388 o PHE B 159 2155 2538 1911 -169 402 12 o
ATOM 1389 CB PHE B 159 -19.411 -8. 399 -13. 447 1 .00 18. 37 c
ANISOU 1389 CB PHE B 159 2299 2659 20.23 17 207 31 c
ATOM 1390 CG PHE B 159 -20.662 -8. 246 -14. 265 1 .00 18. 85 c
ANISOU 1390 CG PHE B 159 2321 2808 2033 -7 214 -15 c
ATOM 1391 CDl PHE B 159 -21.911 -8. 310 -13. 667 1 .00 20. 49 c
ANISOU 1391 CD1 PHE B 159 2498 3102 2183 4 224 -47 c
ATOM 1392 CD2 PHE B 159 -20.588 -8. 071 -15. 637 1 .00 17. 29 c
ANISOU 1392 CD2 PHE B 159 2118 2606 1846 -44 .214 -24 c
ATOM 1393 CE1 PHE B 159 -23.061 -8. 184 -14. 418 1 .00 23. 60 c
ANISOU 1393 CE1 PHE B 159 2853 3575 2537 -20 228 -86 c
ATOM 1394 CE2 PHE B 159 -21.737 -7. 939 -16. 39 1 .00 23. 28 c
ANISOU 1394 CE2 PHE B 159 2843 3443 2559 -69 216 -65 c
ATOM 1395 CZ PHE B 159 -22.972 -7. 999 -15. 781 1 .00 25. 14 c
ANISOU 1395 CZ PHE B 159 3045 3763 2744 -57 221 -95 c
ATOM 1396 N ILE B 160 -17.888 -10. 315 -15. 331 1 .00 12. 47 N
ANISOU 1396 N ILE B 160 1576 1789 1374 -123 339 66 N
ATOM 1397 CA ILE B 160 -17.761 -10. 905 -16. 657 1 .00 14. 29 c
ANISOU 1397 CA ILE B 160 1805 2006 1617 -188 393 58 c
ATOM 1398 C ILE B 160 -17.701 -12. 428 -16. 581 1 .00 18. 99 c
ANISOU 1398 C ILE B 160 2407 2578 2229 -252 483 60 c
ATOM 1399 o ILE B 160 -18.442 -13. 118 -17. 280 1 .00 16. 93 o
ANISOU 1399 o ILE B 160 2135 2359 1941 -309 532 29 o
ATOM 1400 CB · ILE B 160 -16.528 -10. 368 -17. 431 1 .00 9. 62 c
ANISOU 1400 CB ILE B 160 1235 1339 1082 · -178 370 90 c
ATOM 1401 CGI ILE B 160 ' -16.698 -8. 875 -17. 735 1 .00 15. 38 c
ANISOU 1401 CGI ILE B 160 1956 2097 1792 -124 281 85 c
ATOM 1402 CG2 ILE B 160 -16.326 -11. 191 -18. 706 1 .00 11. 30 c
ANISOU 1402 CG2 ILE B 160 1455 1532 1308 -248 442 82 'c
ATOM 1403 CDl ILE B 160 -17.888 -8. 545 -18. 629 1 .00 21. 54 c
ANISOU 1403 CDl ILE B 160 2713 2962 2512 -146 272 38 c
ATOM 1404 N THR B 161 -16.837 -12. 964 -15. 727 1 .00 16. 22 N ANISOU 1404 N THR B 161 2077 2160 1925 -244 501 96 N
ATOM 1405 CA THR B 161 -16.720 -14 .419 -15. 657 1 .00 19. 66 C
ANISOU 1405 CA THR B 161 2522 2567 2382 -305 583 100 C
ATOM 1406 C THR B 161 -18.024 -15 .047 -15. 162 1 .00 21. 31 C
ANISOU 1406 C THR B 161 · 2707 2855 2533 -333 613 69 C
ATOM 1407 0 THR B 161 -18.463 -16 .079 -15. 677 1 .00 22. 96 o
ANISOU 1407 Ό THR B 161 2908 3073 2743 -378 644 52 o
ATOM 1408 CB THR B 161 -15.519 -14 .869 -14. 815 1 .00 16. 61 C
ANISOU 1408 CB THR B 161 2161 2088 2061 -292 592 147 C
ATOM 1409 OGl THR B 161 -15.667 -14 .406 -13. 468. 1 .00 20. 13 o
ANISOU 1409 OGl THR B 161 2610 2548 2489 -238 542 160 o
ATOM 1410 CG2 THR B 161 -14.234 -14. .306 -15. 410 1 .00 18. 26 C
ANISOU 1410 CG2 THR B 161 2387 2218 2334 -271 567 180 C
ATOM 1411 0 GLY B 162 -21.796 -15 .757 -15. 010 1 .00 26. 98 o
ANISOU 1411 o GLY B 162 3338 3803 3111 -376 626 -19 o
ATOM 1412 N GLY B 16.2 -18.662 -14 .400 -14. 193 1 .00 21. 00 N
ANISOU 1412 N GLY B 162 2655 2871 2454 -283 567 63 N
ATOM 1413 CA GLY B 162 -19.989 -14 .812 -13. 757 1 .00 22. 98 C
ANISOU 1413 CA GLY B 162 2876 3208 2646 -302 591 34 ' C
ATOM 1414 C GLY B 162 -21.016 -14 .813 -14. 881 1 .00 25. 89 C
ANISOU 1414 C GLY B 162 3215 3646 2977 -343 605 -7 C
ATOM 1415 o SER B 163 -22.611 -15 .344 -18. 413 1 .00 25. 05 o
ANISOU 1415 o SER B 163 3083 3586 2847 -444 599 -84 o
ATOM 1416 N SER B 163 -21.025 -13 .757 -15. 694 1 .00 18. 82 N
ANISOU 1416 N ' SER B 163 2315 2765 2072 -317 553 -22 N
ATOM 1417 CA SER B 163 - -21.944 -13 .670 -16. 827 1 .00 21. 65 C
ANISOU 1417 CA SER B 163 2648 3183 2395 -354 557 -60 C
ATOM 1418 C SER B 163 -21.679 -14 .755 -17. 868 1 .00 25. 19 c
ANISOU 1418 c · SER B 163 3115 3577 2879 -413 59 -60 c
ATOM 1419 CB SER B 163 -21.876 -12 .287 -17. 484 1 .00 24. 11 c
ANISOU 1419 CB SER B 163 2956 3507 2696 -309 483 -69 c
ATOM 1420 OG SER B 163 -22.278 -11 .274 -16. 574 1 .00 24. !8
ANISOU 1420 OG SER B 163 2949 3558 2678 -236 420 -72 o
ATOM 1421 o LEU ■B 164 -20.779 -18 .337 -19. 392 1 .00 33. 89 o
ANISOU 1421 o LEU B 164 4275 4511 4090 -499 657 -49 o
ATOM 1422 N LEU B 164 -20.410 -15 .024 -18. 149 1 .00 25. 66 N
ANISOU 1422 N LEU B 164 3208 3550 2991 -416 612 -32 N
ATOM 1423 CA LEU B 164 -20.085 -16 .052 -19. 134 - 1 .00 30. 09 C
ANISOU 1423 CA LEU B 164 3789 4054 3588 -448 628 -33 C
ATOM 1424 C LEU B 164 -20.447 -17 .440 -18. 615 1 .00 32. 94 C
ANISOU 1424 C LEU B 164 4148 4401 3965 -466 647 -31 C
ATOM 1425 CB LEU B 164 -18.606 -16 .000 -19. 519 1 .00 29. 71 C
ANISOU 1425 CB LEU B 164 3771 3921 3595 . -438 635 -4 C
ATOM 1426 CG LEU B 164 -18.175 -14 .740 -20. 268 1 .00 27. 54 C
ANISOU 1426 CG LEU B 164 3503 3648 3312 -429 620 -1 - C
ATOM 1427 CD1 LEU B 164 -16.685 -14 .794' -20. 582 1 .00 24. 36 C
ANISOU 1427 CD1 LEU B 164 3127 3155 2974 - 15 627 35 C
ATOM 1428 CD2 LEU B 164 -18.992 -14 .561 -21. 539 1 .00 29. 41 c
ANISOU 1428 CD2 LEU B 164 3735 3933 3507 -459 612 -39 c
ATOM 1429 o ARG B 165 -22.513 -20 .461 -16. 987 1 .00 36. 90 o
ANISOU 1429 o ARG B 165 4616 4946 4458 -518 684 -35 o
ATOM 1430 N ARG B 165 -20.375 -17 .613 -17. 300 1 .00 23. 52 N
ANISOU 1430 N ARG B 165 2949 3210 2778 -446 651 -9 N
ATOM 1431 CA ARG B 165 -20.729 -18 .886 -16. 676 1 .00 34. 08 c
ANISOU 1431 CA ARG B 165 4283 4539 4126 -462 667 -3 c
ATOM 1432 C ARG B 165 -22.166 -19 .281 -17. 007 1 .00 32. 17 C
ANISOU 1432 C ARG B 165 4017 4360 3848 -491 669 -33 C
ATOM 1433 CB ARG B 165 -20.567 -18 .799 -15. 159 1 .00 38. 10 C
ANISOU 1433 CB ARG B 165. 4789 5053 4634 -435 671 24 C
ATOM 1434 CG ARG B 165 -19.420 -19 .606 -14. 587 1 .00 50. .09 C
ANISOU 1434 CG ARG B 165 6333 6494 6205 -434 680 56 C ATOM 1435 CD ARG B 165 -19.469 -19.578 -13.067 1.00 57.,10 C
ANISOU 1435 CD ARG B 165 7220 7395 7082 -414 685 80 C
ATOM 1436 NE ARG B 165 -18.228 -20. 049 -12 .461 1 .00 69. 66 N
ANISOU 1436 NE ARG B 165 8838 8906 8724 -408 685 113 N
ATOM 1437 CZ ARG B 165 -18.034 -20. 172 -11 .150 1 .00 76. 68 C
ANISOU 1437 CZ ARG B 165 9737 9785 9612 -395 690 139 C
ATOM 1438 NH1 ARG B 165 -19.004 -19. 861 -10 .299 1 .00 78. 35. N
ANISOU 1438 NH1 ARG B 165 9933· 10067 9769 -384 698 135 N
ATOM 1439 NH2 ARG B 165 . -16.869 -20. 608 -10 .690 1 .00 76. 40 N
ANISOU 1439 NH2 ARG B 165 9729 9671 9630 -391 685 168 N
ATOM 1440 ■ o LYS B 166 -25.754 -19. 092 -19 .502 1 .00 44. 95 o
ANISOU 1440 o LYS B 166 5573 6146 5361 -588 644 ■140 o
ATOM 1441 N LYS B 166 -22.996 -18. 286 -17 .303 1 .00 30. 60 N
ANISOU 1441 N LYS B 166 3794 4227 3604 -485 651 -58 N
ATOM 1442 CA •LYS B 166 -24.406 -18. 515 -17 .603 1 .00 39. 76 C
ANISOU 1442 CA LYS B 166 4925 5452 4730 -512 648 -87 C
ATOM 1443 C LYS B 166 -24.634 -18. 799 -19 .083 1 .00 41. 06 C
ANISOU 1443 C LYS B 166 5101 5607 4893 -555 646 ■115 C
ATOM 1444 CB LYS B 166 -25.246 -17. 307 -17 .183 1 .00 44. 73 C
ANISOU 1444 CB LYS B 166 5519 6167 5310 -483 624 103 C
ATOM 1445 CG LYS B 166 -25.370 -17. 126 -15 .683 1 .00 51. 72 C
ANISOU 1445 CG LYS B 166 6391 7079 6183 -443 628 -84 C
ATOM 1446 CD LYS B 166 -26.175 -15. 885 -15 .344 1 .00 54. 02 C
ANISOU 1446 CD LYS B 166 6646 7459 6421 -406. 599 107 C
ATOM 1447 CE LYS B 166 -26.450 -15. 811 -13 .853 1 .00 58. 41 C
ANISOU 1447 CE LYS B 166 7190 8046 6956 -366 605 -92 C
ATOM 1448 NZ LYS B 166 -25.194 -15. 833 -13 .055 1 .00 60. 53 N
ANISOU 1448 NZ LYS B 166 7496 8258 7246 -342 617 -60 N
ATOM 1449 o ILE B 167 -22.063 -20. 724 -21 .07 1 .00 27. 15 o
ANISOU 1449 o ILE B 167 3373 3677 3264 -593 -99 31 o
ATOM 1450 N ILE B 167 -23.573 -18. 701 -19 .875 1 .00 31. 18 N
ANISOU 1450 N ILE B 167 3819 4304 3725 -600 -17 -9 N
ATOM 1451 CA ILE B 167 1 -23.665 -18. 960 -21 .305 1 .00 31. 34 C
ANISOU 1451 CA ILE B 167 3828 4304 3778* -563 -30 -13 C
ATOM 1452 C ILE B 167 -23.11 -20. 351 -21 .591 1 .00 28. 23 c
ANISOU 1452. C ILE B 167 3477 3858 3391 -587 -74 14 c
ATOM 1453 CB ILE B 167 -22.897 -17. 897 -22 .122 1 .00 27. 79 c
ANISOU 1453 CB ILE B 167 3369 3829 3361 -490 -18 -32 · c
ATOM 1454 CGI ILE B 167 -23.460 -16. 504 -21 .829 1 .00 34. 75 c
ANISOU 1454 CGI ILE B 167 4209 4759 4237 -465 22 -60 c
ATOM 1455 CG2 ILE B 167 -22.976 -18. 194 -23 .612 1 .00 30. 02 c
ANISOU 1455 CG2 ILE B 167 3655 4083 3667 -461 , -30 -37 c
ATOM 1456 CD1 ILE B 167 -22.948 -15. 423 -22 .763 1 .00 33. 87 c
ANISOU 1456 CD1 ILE B 167 4086 4628 4155 -397 33 -80 c
ATOM 1457 o LYS B 168 -21.568 -21. 521 -23 .964 1 .00 20. 48 o
ANISOU 1457 o LYS B 168 2549 2748 2484 -523 -112 19 o
ATOM 1458 N LYS B 168 -23.840 -21. 126 -22 .390 1 .00 28. 36 N
ANISOU 1458 N LYS B 168 3492 3876 3407 -603 -86 19 N
ATOM 1459 CA LYS B 168 -23.398 -22. 471 -22 .739 1 .00 30. 43 C
ANISOU 1459 CA LYS B 168 3796 4087 3678 -625 -125 42 C
ATOM 1460 C LYS B 168 -21.969 -22. 449 -23 .263 1 .00 23. 29 C
ANISOU 1460 C LYS B 168 2918 3115 2818 -578 -136 41 C
ATOM 1461 CB LYS B 168 -24.330 -23. 101 -23 .775 1 .00 34. 86 C
ANISOU 1461 CB LYS B 168 4355 4655 4235 -636 -134 43 c
ATOM 1462 CG LYS B 168 -23.646 -24. 123 -24 .673 1 .00 51. 11 c
ANISOU 1462 CG LYS B 168 6459 6646 6316 -631 -163 54 c
ATOM 1463 CD LYS B 168 -24.638 -24. 850 -25 .581 1 .00 63. 37 c
ANISOU 1463 CD LYS B 168 8020 8204 7855 -649 -176 59 c
ATOM 1464 CE LYS B 168 -25.474 -23. 881 -26 .409 1 .00 67. 57 c
ANISOU 1464 CE LYS B 168 8516 8771 8386 -630 -161 38 c
ATOM 1465 NZ LYS B 168 -26.286 -24. 588 -27 .440 1 .00 69. 13 N ANISOU 1465 NZ LYS B 168 8731 8960 8575 -652 -186 45 N
ATOM 1466 0 GLY B 169 -19.262 -21. 877 -21. 811 1. ,00 32. 52 0
ANISOU 1466 0 GLY B 169 4113 4196 4047 -542 -163 57 0
ATOM 1467 N GLY B 169 -21.205 -23. .473 -22. 908 1. 00 26. 59 N
ANISOU 1467 N GLY B 169 3371 3480 3251 -600 -171 64 N
ATOM 1468 CA GLY B 169 -19.843 -23. 608 -23. 379 1. 00 24. 17 C
ANISOU 1468 CA GLY B 169 3083 3100 3000 -559 -180 63 C
ATOM 1469 C GLY B 169 -18.875 -22. 732 -22. 610 1. 00 31. 25 C
ANISOU 1469 C GLY B 169 3969 3985 3919 -533 -178 62 C
ATOM 1470 o SER B 170 -14.617 -21. 960 -23. 619 1. 00 18. 44 o
ANISOU 1470 o SER B 170 2335 2155 2515 -390 -170 41 o
ATOM 1471 N SER B 170 -17.596 -22. 950 -22. 855 1. 00 27. 29 N
ANISOU 1471 N SER B 170 3477 3411 3478 -503 -191 64 N
ATOM 1472 CA SER B 170 -16.578 -22. 120 -22. 246 1. 00 14. 70 C
ANISOU 1472 CA SER B 170 1872 1797 1917 -475 -194 64 C
ATOM 1473 C SER B 170 -15.773 -21. 580 -23. 399 1. 00 14. 10 C
ANISOU 1473 C SER B 170 1783 1678 1895 -412 -159 38 C
ATOM 1474 CB SER B 170 -15.721 -22. 957 -21. 300 1. 00 24. .73 c
ANISOU 1474 CB SER B 170 3164 3012 3221 -506 -253 98 C
ATOM 1475 OG SER B 170 -16.551 -23. 634 -20. 370 1. 00 26. 63 o
ANISOU 1475 OG SER B 170 3428 3290 3400 -559 -277 120 o
ATOM 1476 N TYR B 171 -16.414 -20. 705 -24. 164 1. 00 12. 23 N
ANISOU 1476 N TYR B 171 1533 1485 1630 -385 -115 11 N
ATOM 1477 CA TYR B 171 -15,844 -20. 243 -25. 414 1. 00 15. 03 C
ANISOU 1477 CA TYR B 171 . 1887 1801 2021 -334 -77 -16 C
ATOM 1478 C TYR B 171 -14.785 -19. 166 -25. 188 1. 00 11. 77 C
ANISOU 1478 C TYR B 171 1455 1368 1648 -292 -61 -27 c
ATOM 1479 o TYR B 171 -14.653 -18. 631 -24. 080 1. 00 13. 54 o
ANISOU 1479 o TYR B 171 .1665 1617 1862 -301 -80 -14 o
ATOM 1480 CB TYR B 171 -16.960 -19. 742 -26. 345 1. 00 11. 85 c
ANISOU 1480 CB TYR B 171 1485 1446 1571 -328 -49 -37 c
ATOM 1481 CG TYR B 171 -17.986 -20. 818 -26. 687 1. 00 13. 64 c
ANISOU 1481 CG TYR B 171 ■ 1731 1687 1764 -370 -67 -25 c TOM 1482 CD1 TYR B 171 -17.638 -22. 156 -26. 675 1. 00 14. 62 c
ANISOU 1482 CD1 TYR B 171 1882 1763 1911 -396 -92 -8 c
ATOM 1483 CD2 TYR B 171 -19.295 -20. 492 -27. 013 1. 00 16. 22 c
ANISOU 1483 CD2 TYR B 171 ; 2048 2073 2043 -383 -63 -31 c TOM 1484 CE1 TYR B 171 -18.563 -23. 153 -26. 985 1. 00 18. 75 c
ANISOU 1484 CE1 TYR B 171 2426 2297 2401 -437 -Ill 3 c
ATOM 1485 CE2 TYR B 171 -20.229 -21. 480 -27. 323 1. 00 16. 56 c
ANISOU 1485 CE2 TYR B 171 2106 2126 2058 -424 -84 -19 c
ATOM 1486 CZ TYR B 171 -19.854 -22. 807 -27. 299 1. 00 16. 62 c
ANISOU 1486 CZ TYR B 171 2145 2087 2081 -452 -107 -2 c TOM 1487 OH TYR B 171 -20.776 -23. 785 -27. 609 1. 00 17. 80 o
ANISOU 1487 OH TYR B 171 2314 2248 2203 -495 -129 11 o
ATOM 1488 N ASP B 172 -14.041 -18. 858 -26. 248 1. 00 10. 64 N
ANISOU 1488 N ASP B 172 1316 1178 1549 -251 -25 -51 N
ATOM 1489 CA ASP B 172 -13.016 -17. 821 -26. 206 1. 00 8. 42 c
ANISOU 1489 CA ASP B 172 1015 874 1311 -209 -3 -64 c
ATOM . 1490 C ASP B 172 -13.614 -16. 502 -26. 652 1. 00 11. 45 c
ANISOU 1490 C ASP B 172 1392 1309 1648 -185 30 -87 c
ATOM 1491 o ASP B 172 -14.275 -16. 441 -27. 678 1. 00 9. 61 o
ANISOU 1491 o ASP B 172 1177 1088 1385 -180 54 ■104 o
ATOM 1492 CB ASP B 172 -11.861 -18. 153 -27. 154 1. 00 12. 93 c
ANISOU 1492 CB ASP B 172 1593 1361 1958 -180 28 -81 c
ATOM 1493 CG ASP B 172 -11.199 -19. 485 -26. 844 1. 00 18. 83 c
ANISOU 1493 CG ASP B 172 2342 2043 2768 -198 -4 -62 c
ATOM 1494 ODl ASP B 172 -11.356 -19. 995 -25. 717 1. 00 15. 18 o
ANISOU 1494 ODl ASP B 172 1875 1594 2298 -231 -59 -30 o
ATOM 1495 OD2 ASP B 172 -10.514 -20. 015 -27. 742 1. 00 23. 80 o
ANISOU 1495 OD2 ASP B 172 2982 2604 3456 -181 29 -80 o ATOM 1496 N TYR B 173 -13.359 -15.440 -25.893 1.00 9.12 N
ANISOU 1 96 N TYR B 173 1075 1041 1349 -170 29 -87 N
ATOM 1497 CA TYR B 173 -13.929 -14. 137 -26 .217 1 .00 5. 61 C
ANISOU 1497 CA TYR B 173 622 646 865 -145 56 ■108 C
ATOM 1498 C TYR B 173 -12.848 -13. 083 -26 .262 1 .00 9. 56 C
ANISOU 1498 C TYR B 173 1111 1119 1404 -105·' 77 •121 C
ATOM 1499 o TYR B 173 -12.211 -12. 802 -25 .237 1 .00 6. 64 o
ANISOU 1499 o TYR B 173 726 743 1055 -107 56 107 o
ATOM 1500 CB TYR B 173 -14.940 -13. 735 -25 .134 1 .00 5. 62 C
ANISOU 1500 CB TYR B 173 607 719 811 -170 39 -98 C
ATOM 1501 CG TYR B 173 -16.134 -14. 642 -25 .138 1 .00 9. 23 C
ANISOU 1501 CG TYR B 173 1069 1210 1228 -210 24 -88 C
ATOM 1502 CD1 TYR B 173 -17.238 -14. 345 -25 .934 1 .00 9. 00 C
ANISOU 1502 CD1 TYR B 173 1036 1218 1166 -206 39 103 C
ATOM 1503 CD2 TYR B 173 -16.154 -15. 801 -24 .384 1 .00 12. 55 C
ANISOU 1503 CD2 TYR B 173 1499 1621 1647 -254 -9 -62 C
ATOM 1504 CE1 TYR B 173 -18.330 -15. 172 -25 .970 1 .00 9. 24 C
ANISOU 1504 CE1 TYR B 173 1068 1277 1167 -244 23 -93 C
ATOM 1505 CE2 TYR B 173 -17.252 -16. 644 -24 .413 1 .00 12. 04 C
ANISOU 1505 CE2 TYR B 173 1441 1588 1548 -294 -22 -53 C
ATOM 1506 CZ TYR B 173 -18.335 -16. 316 -25 .208 1 .00 10. 54 C
ANISOU 1506 CZ TYR B 173 1242 1436 1329 -288 -4 -69 c
ATOM 1507 OH TYR B 173 -19.444 -17. 127 -25 .270 1 .00 13. 31 o
ANISOU 1507 OH TYR B 173 1594 1816 1650 -328 -18 -59 o
ATOM 1508 N TRP B 174 -12.651 -12. 470 -27 .424 1 .00 8. 80 N
ANISOU 1508 N TRP B 174 1027 1004 1314 -74 117 147 N
ATOM 1509 CA TRP B 174 -11.684 -11, 381 -27 .520 1 .00 6. 26 C
ANISOU 1509 CA TRP B 174 696 659 1025 -38 141 162 C
ATOM 1510 C TRP B 174 -12.028 -10. 279 -26 .536 1 .00 12. 09 C
ANISOU 1510 C TRP B 174 1413 1453 1728 -32 126 158 C
ATOM 1511 0 TRP B 174 -13.206 -9. 965 -26 .301 1 .00 10. 40 o
ANISOU 1511 o TRP B 174 1195 1299 1458 -43 120 - 159 o
ATOM 1512 CB TRP B 174 -11.691 -10. 752 -28 .920 1 .00 8. 10 c
ANISOU 1512 CB TRP B 174 955 876 1248 -13 185 192 c
ATOM 1513 CG TRP B 174 -11.062 -11. 553 -30 .000 1 .00 6. 20 c
ANISOU 1513 CG TRP B 174 743 567 1046 -13 218 205 c
ATOM 1514 CD1 TRP B 174 -11.644 -11. 915 -31 .170 1 .00 9. 91 c
ANISOU 1514 CD1 TRP B 174 1256 1026 1483 -24 237 - 220 c
ATOM 1515 CD2 TRP B 174 -9.721 -12. 079 -30 .040 1 .00 9. 52 c
ANISOU 1515 CD2 TRP B 174 1154 914 1549 -5 237 - 208 c
ATOM 1516 NE1 TRP B 174 -10.772 -12. 635 -31 .937 1 .00 8. 03 N
ANISOU 1516 NE1 TRP B 174 1041 716 1293 -25 274 233 N
ATOM 1517 CE2 TRP B 174 -9.585 -12. 764 -31 .269 1 .00 10. 25 c
ANISOU 1517 CE2 TRP B 174 1283 976 1637 -13 267 215 c
ATOM 1518 CE3 TRP B 174 -8.641 -12. 065 -29 .154 1 .00 10.29 c
ANISOU 1518 CE3 TRP B 174 1213 980 1716 5 221 - 194 c
ATOM 1519 CZ2 TRP B 174 -8.408 -13. 400 -31 .649 1 .00 11. 46 c
ANISOU 1519 CZ2 TRP B 174 1425 1096 1833 -11 279, - 203 c
ATOM 1520 CZ3 TRP B 174 -7.464 -12. 690 -29 .533 1 .00 9. 40 c
ANISOU 1520 CZ3 TRP B 174 1089 836 1648 6 228 179 c
ATOM 1521 CH2 TRP B 174 -7.362 -13. 367 -30 .765 1 .00 11. 80 c
ANISOU 1521 CH2 TRP B 174 1422 1125 1937 -1 261 187 c
ATOM 1522 N VAL B 175 -10.993 -9. 680 -25 .968 1 .00 7. 38 N
ANISOU 1522 N VAL B 175 802 832 1170 -16 123 154 N TOM 1523 CA VAL B 175 -11.156 -8. 457 -25 .173 1 .00 6. 31 C
ANISOU 1523 CA VAL B 175 655 739 1004 -6 118 156 C
ATOM 1524 C VAL B 175 -10.204 -7. 394 -25 .688 1 .00 7. 99 C
ANISOU 1524 C VAL B 175 865 921 1251 33 146 174 C
ATOM 1525 o VAL B 175 -9.363 -7. 672 -26 .536 1 .00 10. 03 o
ANISOU 1525 o VAL B 175 1128 1123 1561 47 170 183 o
ATOM 1526 CB VAL B 175 -10.920 -8. 689 -23 .666 1 .00 10. 01 C ANISOU 1526 CB VAL B 175 1116 1215 1472 -36 75 -127 C
ATOM 1527 CGI VAL B 175 -11.883 -9. 745 -23. 142 1 .00 7. 19 C
ANISOU 1527 CGI VAL B 175 767 889 1077 -80 51 -110 C
ATOM 1528 CG2 VAL B 175 -9.460 -9. 069 -23. 398 1 .00 10. 85 C
ANISOU 1528 CG2 VAL B 175 1214 1251 1658 -33 52 -110 C
ATOM 1529 N GLY B 176 -10.322 -6. 168 -25. 182 1 .00 10. 28 N
ANISOU 1529 N GLY B 176 1148 1245 1513 8 147 -180 N
ATOM 1530 CA GLY B 176 -9.615 -5. 043 -25. 782 1 .00 13. 04 C
ANISOU 1530 CA GLY B 176 1502 1574 1879 82 173 -196 C
ATOM 1531 C GLY B 176 -8.160 -4. 899 -25. 361 1 .00 13. 13 C
ANISOU 1531 C GLY B 176 1504 1536 1950 85 160 -180 C
ATOM 1532 0 GLY B 176 -7.692 -3. 794 -25. 085 1 .00 10. 56 o
ANISOU 1532 o GLY B 176 1184 1219 1607 95 152 -173 o
ATOM 1533 N LEU B 17.7 -7.441 -6. 013 -25.- 338 1 .00 12, 93 N
ANISOU 1533 N LEU B 177 1554 1633 1725 -173 339 -47 N
ATOM 1534 CA LEU B 177 -6.076 -6. 052 -24. 854 1 .00 8. 74 C
ANISOU 1534 CA LEU B 177 996 1132 1193 -184 346 -44 C
ATOM 1535 C LEU B 177 -5.174 -6. 490 -25. 975 1 .00 6. 75 C
ANISOU 1535 C LEU B 177 728 896 939 -192 360 -45 C
ATOM 1536 o LEU B 177 -5.474 -7. 459 -26. 663 1 .00 10. 50 o
ANISOU 1536 o LEU B 177 1208 1365 1418 -182 361 -43 o
ATOM 1537 CB LEU B 177 ' -5.974 -7. 071 -23. 706 1 .00 14. 01 C
ANISOU 1537 CB LEU B 177 1654 1804 1866 -168 336 -37 C
ATOM 1538 CG LEU B 177 -4.871 -6. 955 -22. 663 1 .00 23. 55 C
ANISOU 1538 CG LEU B 177 2838 3038 3073 -174 336 -33 C
ATOM 1539 CD1 LEU B 177 -5.125 -7. 987 -21. 579 1 .00 23. 27 C
ANISOU 1539 CD1 LEU B 177 2800 2999 3042 -155 324 -26 C
ATOM 1540 CD2 LEU B 177 -4.825 -5. 561 -22. 072 1 .00 17. 80 C
ANISOU 1540 CD2 LEU B 177 2113 2310 2340 -187 335 -37 C
ATOM 1541 N SER B 178 -4.068 -5. 784 -26. 176 1 .00 9. 39 N
ANISOU 1541 N SER B 178 1045 1253 1270 -211 371 -47 N
ATOM 1542 CA SER B 178 -3.094 -6. 193 -27. 18 1 .00 10. 61 C
ANISOU 1542 CA SER B 178 1183 1426 1424 -219 386 -48 C
ATOM 1543 C SER B 178 -1.689 -5. 827 -26. 758 1 .00 16. 25 C
ANISOU 1543 C SER B 178 1868 2169 2137 -232 394 -47 C
ATOM 1544 o SER B 178 -1.493 -4'. 933 -25. 927 1 .00 12. 93 o
ANISOU 1544 o SER' B 178 1446 1753 1715 -241 391 -48 o
ATOM · 1545 CB SER B 178 -3.406 -5. 566 -28. 555 1 .00 15. 14 C
ANISOU 1545 CB SER B 178 > 1769 1989 1993 -231 395 -55 C
ATOM 1546 OG SER B 178 -3.235 -4. 158 -28. 521 1 .00 16. 33 o
ANISOU 1546 OG SER B 178 1924 2142 2138 -249 398 -61 o
ATOM 1547 N GL B 179 -0.707 -6. 522 -27. 324 1 .00 16. 82 N
ANISOU 1547 N GLN B 179 1919 2262 2211 -233 406 -44 N
ATOM 1548 CA GLN B 179 0.689 -6. 262 -26. 990 1 .00 18. 56 C
ANISOU 1548 CA GLN B 179 2110 2510 2432 -244 415 -43 C
ATOM 1549 C GLN B 179 1.314 -5. 299 -27. 975 1 .00 28. 24 C
ANISOU 1549 .C GLN B 179 3331 3745 3653 -266 429 -49 C
ATOM 1550 O GLN B 179 1.366 -5. 572 -29. 171 1 .00 34. 28 o
ANISOU 1550 0 GLN B 179 4099 4510 4418 -268 439 -50 o
ATOM 1551 CB GLN B 179 1.491 -7. 557 -26. 993 1 .00 25. 52 C
ANISOU 1551 CB GLN B 179 2968 ' 3411 3318 -232 421 -37 C
ATOM 1552 CG GLN B 179 1.593 -8. 243 -25. 661 1 .00 33. 98 C
ANISOU 1552 CG GLN B 179 4029 4489 4393 -217 410 -30 C
ATOM 1553 CD GLN B 179 2.726 -9. 242 -25. 635 1 .00 38. 50 c
ANISOU 1553 CD GLN B 179 4573 5087 4970 -210 419 -25 c
ATOM 1554 OEl GLN B 179 3.449 -9. 393 -26. 616 1 .00 43, 86 o
ANISOU 1554 OEl GLN B 179 5238 5780 5648 -215 434 -26 o
ATOM 1555 NE2 GLN B 179' 2.892 -9. 928 -24. 511 1 .00 42. 80 N
ANISOU 1555 NE2 GLN B 179 5106 5638 5517 -196 411 -20 N
ATOM 1556 0 ASP B 180 4.656 -4. 206 -28. 060 1 .00 55. 06 o
ANISOU 1556 0 ASP B 180 6662 7205 7052 -303 ■ 460 -49 o ATOM 1557 N ASP B 180 1.789 -4.169 -27.466 1.00 38.59 N
ANISOU 1557 N ASP B 180 4636 5063 4962 -282 431 -51 N
ATOM 1558 CA ASP B 180 2.500 -3. 195 -28 .280 1 .00 50 .74 C
ANISOU 1558 CA ASP B 180 6169 6613 6497 -304 444 -57 C
ATOM 1559 C ASP B 180 3.776 -3. 826 -28 .823 1 .00 54 .69 C
ANISOU 1559 C ASP B 180 6640 7140 7001 -306 458 -54 C
ATOM 1560 CB ASP B 180 2.838 -1. 960 -27 .441 1 .00 57 .97 C
ANISOU 1560 CB ASP B 180 7082 7534 7410 -320 441 -59 C
ATOM 1561 CG ASP B 180 3.538 -0. 881 -28 .245 1 .00 67 .36 c
ANISOU 1561 CG ASP B 180 8266 8734 ■ 8594 -344 453 -65 c
ATOM 1562 ODl ASP B 180 3.154 -0. 666 -29 .414 1 .00 70 .33 o
ANISOU 1562 ODl AS B 180 8656 9101 8966 -350 459 -70 o
ATOM 1563 OD2 ASP B 180 4.474 -0. 250 -27 .708 1 .00 68 .76 o
ANISOU 1563 OD2 ASP B 180 8426 8929 8771 -358 457 -65 o
ATOM 1564 0 GLY B 181 7.405 -4. 499 -30 .497 1 .00 73 .67 o
ANISOU 1564 0 GLY B 181 8965 9614 9411 -325 504 -48 o
ATOM 1565 N GLY B 181 3.872 -3. 940 -30 .143 1 .00 60 .65 N
ANISOU 1565 N GLY B 181 7396 7894 7753 -311 470 -56 N
ATOM 1566 CA GLY B 181 5.026 -4. 562 -30 .767 1 .00 66 .80 C
ANISOU 1566 CA GLY B 181 8149 8697 8535 -312 484 -53 C
ATOM 1567 C GLY B 181 6.341 -3. 881 -30 .429 1 .00 72 .23 C
ANISOU 1567 C . GLY B 181 8811 9410 9225 -328 494 -53 C
ATOM 1568 0 HIS B 182 9.186 -2. 350 -28 .179 1 .00 52 .09 o
ANISOU 1568 0 HIS B 182 6197 6912 6683 -360 500 -50 o
ATOM 1569 N HIS B 182 6.268 -2. 606 -30 .059 1 .00 70 .05 . N
ANISOU 1569 N HIS B 182 8542 9129 8944 -346 490 -58 N
ATOM 1570 CA HI'S B 182 7.461 -1. 817 -29 .767 1 .00 63 .15 C
ANISOU 1570 CA HIS B 182 7646 8278 8072 -364 498 -58 c
ATOM 1571 C HIS B 182 8.001 -2. 069 -28 .359 1 .00 55 .•90 c
ANISOU 1571 C HIS B 182 6708 7371 7159 -358 491 -54 c
ATOM 1572 CB HIS B 182 7.175 -0. 325 -29 .960 1 .00 62 .57 c
ANISOU 1572 CB HIS B 182 7589 8195 7990 -386 497 -66 c
ATOM 1573 o SER B 183 7.746 -4. 010 -24 ,474 1 .00 47 .46 o
ANISOU 1573 o SER B 183 5622 6304 6107 -310 458 -39 o
ATOM 1574 N SER B 183 7.128 -1. 961 -27 .363 1 .00 49 .10 N
ANISOU 1574 N SER B 183 5865 6494 6298 -350 476 -54 N
ATOM 1575 CA SER B 183 7.529 -2. 151 -25 .975 1 .00 46 .57 C
ANISOU 1575 CA SER B 183 5530 6183 5983 -344 468 -50 C
ATOM 1576 C SER B 183 7.240 -3. 571 -25 .506 1 .00 47 .37 C
ANISOU 1576 C SER 183 5628 6282 6090 -318 462 -43 C
ATOM 1577 CB SER B 183 6.793 -1. 162 -25 .073 1 .00 45 .31 C
ANISOU 1577 CB SER B 183 5391 6007 5818 -350 • 455 -53 C
ATO 1578 OG SER B 183 5.399 -1. 411 -25 .098 1 .00 46 .12 o
ANISOU 1578 OG SER B 183 5522 6083 5917 -337 444 -53 o
ATOM 1579 0 GLY B 184 4.636 -6. 696 -24 .264 1 .00 40 .67
ANISOU 1579 o GLY B 184 4814 5392 5246 -250 428 -30 o
ATOM 1580 N GLY B 184 6.416 -4. 282 -26 .267 1 .00 44 .34 N
ANISOU 1580 N GLY B 18 5261 5884 5705 -305 461 -43 N
ATOM 1581 CA GLY B 184 6.016 -5. 629 -25 .903 1 .00 40 .12 C
ANISOU 1581 CA GLY B 184 4726 5345 5174 -281 454 -37 C
ATOM 1582 C GLY B 184 5.021 -5. 636 -24 .761 1 .00 34 .74 C
ANISOU 1582 C GLY B 184 4062 4645 4491 -270 436 -35 C
ATOM 1583 o ARG B 185 1.781 -4. 071 -24 .592 1 .00 18 .52 o
ANISOU 1583 o ARG B 185 1894 2484 2659 -231 43 106 o
ATOM 1584 N ARG B 185 4.600 -4. 446 -24 .345 1 .00 28 .25 N
ANISOU 1584 N ARG B 185 3037 3888 3810 -314 49 167 N
ATOM 1585 CA ARG B 185 3.721 -4. 310 -23 .189 1 .00 31 .87 C
ANISOU 1585 CA ARG B 185 3529 4267 4313 -296 46 189 C
ATOM 1586 C ARG B 185 2.252 -4. 510 -23 .538 1 .00 17 .52 C
ANISOU 1586 C ARG B 185 1739 2364 2554 -225 58 135 C
ATOM 1587 CB ARG B 185 3.902 -2. 938 -22 .533 1 .00 48 .57 C ANISOU 1587 CB ARG B 185 5757 6356 6343 -37 -2 238 C
ATOM 1588 CG ARG B 185 5.087 -2. ,848 -21. ,587 1 .00 60. 39 C
ANISOU 1588 CG ARG B 185 7243 7905 7798 -424 -11 293 C
ATOM 1589 CD ARG B 185 5.486 -1. 402 -21. 323 1 .00 66. 70 C
ANISOU 158 CD ARG B 185 8155 8699 8488 -524 -66 341 C
ATOM 1590 NE ARG B 185 4.393 -0. 595 -20. 786 1 .00 68. 14 N
ANISOU 1590 NE ARG B 185 8457 8770 8663 -515 -90 341 N
ATOM 1591 CZ ARG B 185 3.703 0. 294 -21. 493 1 .00 67. 96 C
ANISOU 1591 CZ ARG B 185 8528 8697 8596 -525 -122 320 C
ATOM 1592 NHl ARG B 185 3.988 0. 492 -22. 774 1 .00 68. 69 N
ANISOU 1592 NHl ARG B 185 8605 8842 8651 -563 -134 304 N
ATOM 1593 NH2 ARG B 185 2.729 0. 988 -20. 919 1 .00 66. 40 N
ANISOU 1593 NH2 ARG B 185 8442 8398 8388 -493 -143 313 N
ATOM 1594 N TRP B 186 1-531 -5. 162 -22. 633 1 .00 14. 57 N
ANISOU 1594 N TRP B 186 1357 1928 2250 -162 84 124 N
ATOM 1595 CA TRP B 186 0.086 -5. 310 -22. 771 1 .00 12. 87 C
ANISOU 1595 CA TRP B 186 1166 1639 2086 -101 95 78 C
ATOM 1596 C TRP B 186 -0.607 -3. 993 -22. 446 1 .00 10. 63 C
ANISOU 1596 C TRP B 186 996 1296 1749 -118 56 . 87 C
ATOM 1597 o TRP B 186 -0.318 -3. 360 -21. 424 1 .00 13. 64 o
ANISOU 1597 o TRP B 186 1437 1655 2090 -148 37 129 o
ATOM 1598 CB TRP B 186 -0.426 -6. 398 -21. 828 1 .00 13. 29 C
ANISOU 1598 CB TRP B 186 1203 1652 2195 -38 127 64 C
ATOM 1599 CG TRP B 186 -0.110 -7. 777 -22. 276 1 .00 14. 55 C
ANISOU 1599 CG TRP B 186 1353 1845 2329 -2 139 34 C
ATOM 1600 CD1 TRP B 186 0.856 -8. 610 -21. 777 1 .00 16. 33 C
ANISOU 1600 CD1 TRP B 186 1563 2108 2535 3 144 48 C
ATOM 1601 CD2 TRP B 186 -0.773 -8. 503 -23. 314 1 .00 12. 85 C
ANISOU 1601 CD2 TRP B 186 1146 1626 2110 30 143 -9 C
ATOM 1602 NE1 TRP B 186 0.833 -9. 812 -22. 452 1 .00 21. 45 N
ANISOU 1602 NE1 TRP B 186 2207 2773 3170 38 151 1.7 N
ATOM 1603 CE2 TRP B 186 -0.157 -9. 769 -23. 401 1 .00 18. 12 C
ANISOU 1603 CE2 TRP B 186 1802 2326 2756 50 151 -14 C
ATOM 1604 CE3 TRP B 186 -1.825 -8. 202 -24. 181 1 .00 13. 73 c
ANISOU 1604 CE3 TRP B 186 1272 1711 2235 41 139 -41 c
ATOM 1605 CZ2 TRP B 186 -0.563 -10. 734 -24. 319 1 .00 21. 03 c
ANISOU 1605 CZ2 TRP B 186 2176 2697 3118 75 155 -42 c
ATOM 1606 CZ3 TRP B 186 -2.230 -9. 159 -25. 087 1 .00 12. 38 c
ANISOU 1606 CZ3 TRP B 186 1107 1546 2052 62 143 -70 c
ATOM 1607 CH2 TRP B 186 -1.597 -10. 409 -25. 154 1 .00 20. 53 c
ANISOU 1607 CH2 TRP B 186 2130 2606 3066 76 151 -67 c
ATOM 1608 N LEU B 187 -1.507 -3. 568 -23. 326 1 .00 9. 98 N
ANISOU 1608 N LEU B 187 947 1186 1659 -95 41 48 N
ATOM 1609 CA. LEU B 187 -2.249 -2. 331 -23. 131 1 .00 14. 27 C
ANISOU 1609 CA LEU B 187 1602 1669 2148 -94 1 50 C
ATOM 1610 C LEU B 187 ■ -3.695 -2. 530 -23. 515 1 .00 11. 43 C
ANISOU 1610 C LEU B 187 1238 1268 1837 . -19 11 -4 c
ATOM 1611 o LEU B 187 -3.994 -3. 231 -24. 480 1 .00 10. 38 o
ANISOU 1611 o LEU B 187 1039 1158 ! 1,746 5 29 -43 o
ATOM 1612 CB' LEU B 187 -1.690 -1. 238 -24. 030 1 .00 10. 37 c
ANISOU 1612 CB LEU B 187 1180 1198 1562 -163 -48 65 c
ATOM. 1613 CG LEU B 187 -0.298 -0. 711 -23. 706 1 .00 13. 36 c
ANISOU 1613 CG LEU B 187 1586 1625 1867 -257 -72 124 c
ATOM 1614 CD1 LEU B 187 0.075 0. 318 -24. 769 1 .00 17. 56 c
ANISOU 1614 CD1 LEU B 187 2190 2176 2306 -328 -120 131 c
ATOM 1615 CD2 LEU B 187 -0.260 -0. 111 -22. 311 1 .00 23. ,33 c
ANISOU 1615 CD2 LEU B 187 2927 2838 3099 -270 -88 165 c
ATOM 1616 N TRP B 188 -4.598 -1. 912 -22. 767 1 .00 8. 57 N
ANISOU 1616 N TRP B 188 945 847 1464 21 -2 -8 N
ATOM 1617 CA TRP B 188 -5.975 -1. 812 -23. 219 1 .00 6. 90 C
ANISOU 1617 CA TRP B 188 739 605 1275 89 -4 -58 C ATOM 1618 C T P B 188 -6.014 -0.832 -24.384 1.00 12.88 C
ANISOU 1618 C TRP B 188 1567 1361 1966 70 -54 -69 C
ATOM 1619 o TRP B 188 -5.044 -0. 097 -24 .624 1 .00 11. 92 o
ANISOU 1619 o TRP B 188 1507 1251 1771 -1 -89 -34 o
ATOM 1620 CB TRP B 188 -6.895 -1. 317 -22 .099 1 .00 10. 88 C
ANISOU 1620 CB TRP B 188 1302 1058 1775 142 -6 -59 C
ATOM 1621 CG TRP B 188 -6.978 -2. 248 -20 .958 1 .00 9. 00 c
ANISOU 1621 CG TRP B 188 1002 819 1600 159 42 -50 c
ATOM 1622 CD1 TRP B 188 -6.466 -2. 053 -19 .703 1 .00 13. 15 c
ANISOU 1622 CD1 TRP B 188 1565 1324 2107 136 46 -8 c
ATOM 1623 CD2 TRP B 188 -7.595 -3. 535 -20 .943 1 .00 8. 34 c
ANISOU 1623 CD2 TRP B 188 819 756 1594 192 89 -81 c
ATOM 1624 NE1 TRP B 188 -6.739 -3. 133 -18 .910 1 .00 12. 81 N
ANISOU 1624 NE1 TRP B 188 1449 1283 2134 160 94 -13 N
ATOM 1625 CE2 TRP B 188 -7.417 -4. 068 -19 .652 1 .00 10. 47 C
ANISOU 1625 CE2 TRP B 188 1082 1027 1870 178 115 -54 C
ATOM 1626 CE3 TRP B 188 -8.272 -4. 301 -21 .905 1 .00 .9. 87 C
ANISOU 1626 CE3 TRP B 188 974 1000 1776 189 95 ■113. C
ATOM 1627 CZ2 TRP B 188 -7.901 -5. 327 -19 .283 1 .00 10. 06 C
ANISOU 1627 CZ2 TRP B 188 994 1014 1814 161 139 -61 c
ATOM 1628 CZ3 TRP B 188 -8.761 -5. 539 -21 .537 1 .00 13. 99 c
ANISOU 1628 CZ3 TRP B 188 1470 1554 2290 172 117 ■113 c
ATOM 1629 CH2 TRP B 188 -8.571 -6. 043 -20 .237 1 .00 10. 17 c
ANISOU 1629 CH2 TRP B 188 987 1061 1814 159 ' 137 -90 c
ATOM 1630 N GLN B 189 -7.134 -0. 808 -25 .094 1 .00 11. 15 N
ANISOU 1630 N GLN B 189 1341 1129 1766 .128 -61 ■116 N
ATOM 1631 C GLN B 189 -7.404 1. 512 -25 .982 1 .00 13. 81 c
ANISOU 1631 C GLN B 189 1890 1407 1950 115 -169 ■113 • c
ATOM 1632 o GLN B 189 -7.497 2. 325 -26 .898 1 .00 13. 34 o
ANISOU 1632 o GLN B 189 1906 1334 1830 102 -218 122 o
ATOM 1633 CA AGLN B 189 -7.263 0. 029 -26 .287 0 .50 9. 47 c
ANISOU 1633 CA AGLN B 189 1192 911 1494 115 -110 132 c
ATOM 1634 CB AGLN B 189 -8.410 -0. 454 -27 .177 0 .50 9. 58 c
ANISOU 1634 CB AGLN B 189 1150 933 1558 178 -101 189 c
ATOM 1635 CG AGLN B 189 -8.022 -1. 611 -28 .079 0 .50 11. 46 c
ANISOU 1635 CG AGLN B 189 1281 1221 1851 154 -66 208 c
ATOM 1636 CD AGL B 189 -9.140 -2. 030 -29 .001 0 .50 14. 29 c
ANISOU 1636 CD AGLN B. 189 1593 1585 2250 205 -63 262 c
ATOM 1637 OEIAGLN B 189 -10.192 -2. 482 -28 .549 0 .50 19. 50 o
ANISOU 1637 OEIAGLN B 189 2216 2242 2950 258 -40 282 o
ATOM 1638 NE2AGLN B 189 -8.919 -1. 887 -30 .307 0 .50 10. 72 N
ANISOU 1638 NE2AGLN B 189 1148 1152 1772 177 -86 277 N
ATOM 1639 CA BGLN B 189 -7.239 0. 028 -26 .284 0 .50 9. 65 c
ANISOU 1639 CA BGLN B 189 1215 934 1516 114 -110 131 c
ATOM 1640 CB BGLN B 189 -8.334 -0. 499 -27 .206 0 .50 10. 41 c
ANISOU 1640 CB BGLN B 189 1251 1040 1663 174 -99 188 c
ATOM 1641 CG BGLN B 189 -8.050 -1. 925 -27 .637 0 .50 12. 75 c
ANISOU 1641 CG BGLN B 189 1422 1384 2038 162 -48 204 c
ATOM 1642 CD BGLN B 189 -6.605 -2. 108 -28 .073 0 .50 13. 04 c
ANISOU 1642 CD BGLN B 189 1441 1464 2049 84 -45 174 c
ATOM 1643 OEIBGLN B 189 -6.218 -1. 665 -29 .154 0 .50 17. 18 o
ANISOU 1643 OEIBGLN B 189 1995 2008 2526 44 -74 178 o
ATOM 1644 NE2BGLN B 189 -5.795 -2. 752 -27 .228 0 .50' 10. 90 N
ANISOU 1644 NE2BGLN B 189 1123 1212 1804 61 -11 143 N
ATOM 1645 0 ASP B 190 -5.763 4. 808 -23 .458 1 .00 15. 28 o
ANISOU 1645 o ASP B 190 2472 1462 1873 -13 -290 28 o
ATOM 1646 N ASP B 190 -7.413 1. 869 -24 .701 1 .00 11. 97 N
ANISOU 1646 N ASP B 190 1707 1140 1702 127 -167 -86 . N
ATOM 1647 CA ASP B 190 -7.399 .3. 285 -24 .344 1 .00 15. 23 c
ANISOU 1647 CA ASP B 190 2276 1496 2017 119 -226 -63 ■ c
ATOM 1648 C ASP B 190 -5.987 3. 693 -23 .897 1 .00 17. 32 c ANISOU 1648 C ASP B 190 2595 1769 2219 14 -243 -1 C
ATOM 1649 CB ASP B 190 -8.466 3 625 -23.291 1.00 10.43 C
ANISOU 1649 CB ASP B 190 1711 840 1414 210 -222 -78 , C
ATOM 1650 CG ASP B 190 -8.279 2 842 -22.002 1.00 12.28 C
ANISOU 1650 CG ASP B 190 1877 1084 1705 211 -168 -58 C
ATOM 1651 ODl ASP B 190 -7.195 2 256 -21.831 1.00 13.45 O
ANISOU 1651 ODl ASP B 190 1972 1267 1872 138 -146 -24 0
ATOM 1652 OD2 ASP B 190 -9.211 2 814 -21.170 1.00 14.54 0
ANISOU 1652 OD2 ASP B 190 2161 1348 2014 287 -149 -77 0
ATOM 1653 0 GLY B 191 -2.114 3 025 -21.856 1.00 15.14 O
ANISOU 1653 o GLY B 191 2250 1606 1897 -241 -207 162 O
ATOM 1654 N GLY B 191 '■ -5.033 2 776 -24.021 1.00 13.77 N
ANISOU 1654 N GLY B 191 2039 1382 1810 -45 -205 17 N
ATOM 1655 CA GLY B 191 -3.651 3 072 -23.692 1.00 11.52 C
ANISOU 1655 CA GLY B 191 1784 1127 1468 -148 -220 74 C
ATOM 1656 C GLY B 191 -3.263 2 829 -22.232 1.00 11.04 C
ANISOU 1656 C GLY B 191 1714 - 1056 ' 1423 -156 -196 - 112 C
ATOM 1657 o SER B 192 -3.561 -0 180 -20.742 1.00 13.91 O
ANISOU 1657 o · SER B 192 1800 1470 2014 -59 -55 89 O
ATOM 1658 N SER B 192 -4.227 2 444 -21.402 1.00 11.00 N
ANISOU 1658 N SER B 192 1689 1014 1478 -71 -165 89 N
ATOM 1659 CA SER B 192 -3.933 2 078 -20.016 1.00 13.52 C
ANISOU 1659 CA SER B 192 1990 1324 1821 -74 -137 120 C
ATOM 1660 C SER B 192 -3.348 0 676 -19.879 1.00 13.04 · C
ANISOU 1660 C SER B 192 1787 1324 1845 -84 -82 124 C
ATOM 1661 CB SER B 192 -5.179 2 194 -19.141 1.00 14.61 C
ANISOU 1661 CB SER B 192 2162 1405 1986 18 -123 94 C
ATOM 1662 OG SER B 192 -6.160 1 230 -19.489 1.00 12.80 O
ANISOU 1662 OG SER B 192 1826 1193 1845 92 -79 42 O
ATOM 1663 o SER B 193 -3.415 -1 236 -16.620 1.00 18.78 O
ANISOU 1663 o SER B 193 2403 2024 •2709 -30 19 162 O
ATOM 1664 N SER B 193 -2.620 0 454 -18.777 1.00 10.86 N
ANISOU 1664 N SER B 193 1504 1054 1569 -119 -70 168 N
ATOM 1665 CA SER B 193 -1.991 -0 840 -18.498 1.00 15.56 C
ANISOU 1665 CA SER B 193 1977 1700 . 2236 -125 -24 177 C
ATOM 1666 C SER B 193 -2.871 -1 711 -17.611 1.00 18.22 C
ANISOU 1666 C SER B 193 2265 2004 2653 -53 22 154 C TOM 1667 CB SER B 193 -0.651 -0 634 -17.781 1.00 20.38 C
ANISOU 1667 CB SER B 193 2602 2342 2798 -205 -39 240 C
ATOM 1668 OG SER B 193 0.303 -0 006 -18.621 1.00 23.63 O
ANISOU 1668 OG SER B 193 3037 2806 3137 -286 -76 264 O
ATOM 1669 o PRO B 194 -1.709 -3 996 -15.686 1.00 13.88 O
ANISOU 1669 o PRO B 194 2057 1818 1400 5 134 -26 O
ATOM 1670 N PRO B 194 -2.998 -3 001 -17.948 1.00 10.28 N
ANISOU 1670 N PRO B 194 1764 1349 794 12 138 ' 7 N
ATOM 1671 CA PRO B 194 -3.724 -3 865 -17.013 1.00 10.25 C
ANISOU 1671 CA PRO B 194 1684. 1348 862 39 61 45 C
ATOM 1672 C PRO B 194 -2.937 -4 082 -15.717 1.00 14.40 C
ANISOU 1672 C PRO B 194 2132 1881 1458 33 76 32 C
ATOM 1673 CB PRO B 194 -3.855 -5 189 -17.777 1.00 13.25 C
ANISOU 1673 CB PRO B 194 2043 1724 1267 42 8 7 C
ATOM 1674 CG RO B 194 -2.703 -5 194 -18.718 1.00 20.91 C
ANISOU 1674 CG PRO B .19 3046 2692 2205 10 68 -71 C
ATOM 1675 CD PRO B 194 -2.472 -3 748 -19.104 1.00 12.89 · C
ANISOU 1675 CD PRO B 194 2111 1678 1110 -5 141 -60 C
ATOM 1676 o SER B 195 -2.706 -6 909 -14.357 1.00 11.72 O
ANISOU 1676 o SER B 195 1601 1545 1306 . 56 -39■ 7 O
ATOM 1677 N SER B 195 -3.663 -4 380 -14.656 1.00 11.21 N
ANISOU 1677 N SER B 195 1672 1483 1105 58 22 86 N
ATOM 1678 CA SER B 195 -3.038 -4 731 -13.388 1.00 11.51 C
ANISOU 1678 CA SER B 195 1627 1533 1214 56 24 79 c' ATOM 1679 C SER B 195 -2.415 -6..123 -13.452 1.00 14.,09 C
ANISOU 1679 C SER B 195 1889 1858 1607 52 -4 28 C
ATOM 1680 CB SER B 195 -4.068 -4. ,674 -12. ,270 1 .00 14. ,94 C
ANISOU 1680 CB SER B 195 2019 1975 1680 86 -28 155 C
ATOM 1681 OG SER B 195 -4.888 -5. ,830 -12. 299 1 .00 15. 86 0
ANISOU 1681 OG SER B 195 2094 2088 1843 110 -109 178 o
ATOM 1682 .0 PRO B 196 -1.597 -10. 064 -12. 480 1 .00 14. 20 o
ANISOU 1682 o PRO B 196 1674 1861 1858 74 -128 -37 0
ATOM 1683 N PRO B 196 -1.542 -6. 439 -12. 488 1 .00 11. 22 N
ANISOU 1683 N PRO B 196 1'455 1507 1302 44 13 6 N
ATOM 1684 CA PRO B 196 -0.938 -7. 775 -12. 458 1 .00 9. 58 C
ANISOU 1684 CA PRO B. 196 1180 1296 1164 44, -13 -38 c
ATOM 1685 C PRO B 196 -1.959 -8. 907 -12. 308 1 .00 11. 24 c
ANISOU 1685 C PRO B 196 1346 1497 1429 73 -102 -1 c
ATOM 1686 CB PRO B 196 -0.006 -7. 705 -11. 235 1 .00 11. 46 c
ANISOU 1686 CB PRO B 196 1349 1556 1450 36 18 -48 c
ATOM 1687 CG PRO B 196 0.390 -6. 250 -11. 174 1 .00 11. 94 c
ANISOU 1687 CG " PRO B 196 1465 1626 1445 14 91 -50 c
ATOM 1688 CD PRO B 196 -0.858 -5. 491 -11. 584 1 .00 9. 17 c
ANISOU 1688 CD PRO B 196 1183 1264 1035 30 71 8 c
ATOM 1689 o GLY B 197 -5.766 -10. 762 -13. 423 1 .00 13. 53 o
ANISOU 1689 o GLY B 197 1647 1752 1742 141 -326 116 o
ATOM 1690 N GLY B 197 -3.207 -8. 583 -11. 986 1 .00 9. 16 N
ANISOU 1690 N GLY B 197 1095 1235 "1152 97 -146 71 N
ATOM 1691 CA GLY B 197 -4.263 -9. 579 -11. 955 1 .00 10. 19 C
ANISOU 1691 CA GLY B 197 1191 1356 1326 122 -230 108 C
ATOM 1692 C GLY B 197 -4.958 -9. 828 -13. 293 1 .00 13. 14 C
ANISOU 1692 C GLY B 197 1624 1712 1655 124 -258 97 C
ATOM 1693 o LEU B 198 -4.541 -8. 533 -17. 782 1 .00 14. 61 o
ANISOU 1693 o LEU B 198 2076 1883 1593 69 -153 -29 o
ATOM 1694 N LEU B 198 -4.658 -8. 990 -14.283 1 .00 9. 34 N
ANISOU 1694 N LEU B 198 1225 1230 1095 105 -205 67 N
ATOM 1695 CA LEU B 198 -5.311 -9. 097 -15. 580 1 .00 14. 38 C
ANISOU 1695 CA LEU B 198 1926 1859 1680 105 -227 58 C
ATOM 1696 C LEU B 198 -4.314 -9. 134 -16. 732 1 .00 12. 52 C
ANISOU 1696 C LEU B 198 1735 1618 1406 77 -174 -22 C
ATOM 1697 CB LEU B 198 -6.272 -7. 926 -15. 746 1 .00 16. 53 C
ANISOU 1697 CB LEU B 198 2264 2139 1879 117 -224 120 C
ATOM 1698 CG LEU B 198 "7.388 -7. 974 -14. 697 1 .00 16. 86 C
ANISOU 1698 CG LEU B 198 2262 2186 1958 148 -284 201 C
ATOM 1699 CD1 LEU B 198 -8.052 -6. 622 -14. 516 1 .00 15. 93 C
ANISOU 1699 CD1 LEU B 198 2199 2077 1777 159 -262 261 C
ATOM 1700 CD2 LEU B 198' -8.404 -9. 049 -15. 078 1 .00 22.27 c
ANISOU 1700 CD2 LEU B 198 2925 2865 2672 166 -366 218 c
ATOM 1701 o LEU B 199 -2.725 -12. 415 -17. 556 1 .00 17. 71 o
ANISOU 1701 o LEU B 199 2275 2241 2215 58 -214 185 o
ATOM 1702 N LEU B 199 -3.203 -9. 830 -16. 523 1 .00 13. 15 N
ANISOU 1702 N LEU B 199 1764 1'693 1539 62 -152 -81 N
ATOM 1703 ' CA LEU B 199 -2.234 -10. 065 -17. 584 1 .00 12. 65 C
ANISOU 1703 CA LEU B 199 1731 1624 1452 37 -107 162 C
ATOM 1704 C LEU B 199 -2.525 -11. 406 -18. 241 1 .00 15. 62 C
ANISOU 1704 C LEU B 199 2084 1985 1867 42 -163 196 C
ATOM 1705 CB LEU B 199 -0.816 -10. 075 -17. 039 1 .00 18. 71 C
ANISOU 1705 CB LEU B 199 2458 2397 2256 18 -49 212 c
ATOM 1706 CG LEU B 199 -0.381 -8. 770 -16. 384 1 .00 22. 18 c
ANISOU 1706 CG LEU B 199 2917 2851 2659 7 12 189 c
ATOM 1707 CD1 LEU B 199 1.013 -8. 920 -15. 7.95 1 .00 22. 12 c
ANISOU 1707 CD1 LEU B 199 2858 2853 2695 -12 63 ■240 c
ATOM 1708 CD2 LEU B 199 -0.427 -7. 656 -17. 403 1 .00 26. 52 c
ANISOU 1708 CD2 LEU B 199 3564 3402 3110 -10 63 ■195 c
ATOM 1709 N PRO B 200 -2.557 -11. 428 -19. 572 1 .00 13. 39 N ANISOU 1709 N PRO B 200 1862 1699 1525 29 -153 ■238 N
ATOM 1710 CA PRO B 200 -2.831 -12. 722 -20. 193 1 .00 16 .20 C
ANISOU 1710 CA PRO B 200 2194 2041 1920 32 -205 •275 C
ATOM 1711 C PRO B 200 -1.718 -13. 717 -19. 891 1 .00 21 .09 C
ANISOU 1711 C PRO B 200 2746 2647 2619 24 -190 •338 C
ATOM 1712 0 PRO B 200 -0.550 -13. 332 -19. 798 1 .00 21 .85 o
ANISOU 1712 o PRO B 200 r 2842 2750 2709 6 -124 •379 o
ATOM 1713 CB PRO B 200 -2.847 -12. 390 -21. 683 1 .00 17 .05 C
ANISOU 1713 •CB PRO B 200 2384 2155 1940 15 -180 316 C TOM 1714 CG PRO B 200 -3.256 -10. 947 -21. 739 1 .00 13 .33 C
ANISOU 1714 CG PRO B 200 1981 1701 1385 16 -148 263 C
ATOM 1715 CD PRO B 200 -2.526 -10. 338 -20. 562 1 .00 11 .25 C
ANISOU 1715 CD PRO B 200 1684 1440 1152 13 -103 245 C
ATOM 1716 N ALA B 201 -2.081 -14. 986 -19. 735 1 .00 17 .42 N
ANISOU 1716 N ALA B 201 2224 2164 2231 37 -251 345 N
ATOM 1717 CA ALA B 201 ' -1.084. -16. 041 -19. 564 1 .00 24 .03 C
ANISOU 1717 CA ALA B 201 2999 2986 3147 32 -241 406 C
ATOM 1718 C ALA B 201 -0.235 -16. 199 -20. 829 1 .00 27 .26 C
ANISOU 1718 C ALA B 201 3451 3391 3515 7 -194 496 C
ATOM 1719 o ALA B 201 -0.696 -15.' 924 -21. 931 1 .00 36 .06 o
ANISOU 1719 o ALA, B 201 4632 4511 4557 · -3 -195 511 ; o
ATOM 1720 CB ALA B 201 -1.758 -17. 350 -19. 211 1 .00 25 .86 < c
ANISOU 1720 CB ALA B 201 , 3166 3193 3466 52 -317 391 c
ATOM 1721 o GLU B 202 0.727 -18. 889 -22. 156 1 .00 70 .61 o
ANISOU 1721 o GLU B 202 8880 8833 9117 -5 -218 655 o
ATOM 1722 N GLU B 202 1.008 -16. 634 -20. 665 1 .00 46 .66 N
ANISOU 1722 N GLU B 202 5868 5843 6018 -4 -152 - 554 N
ATOM 1723 CA GLU B 202 1.899 -16. 823 -21. 805 1 .00 65 .22 C
ANISOU 1723 CA GLU B 202 8253 8191 8336 -27 -104 - 642 C
ATOM 1724 C GLU B 202 1.565 -18. 094 -22. 580 1 .00 68 .50 C
ANISOU 1724 C GLU B 202 8656 8584 8789 -25 -150 689 C
ATOM 1725 CB GLU B 202 3.353 -16. 854 -21. 342 1 .00 74 .11 C
ANISOU 1725 CB GLU B 202 9337 9321 9500 -39 -42 688 C
ATOM 1726 CG GLU B 202 3.820 -15. 551 -20. 724 1 .00 81 .64 C
ANISOU 1726 CG GLU B 202 10310 10300 10408 -49' 14 - 656 C
ATOM 1727 CD GLU B 202 4.628 -15. 762 -19. 461 1 .00 87 .25 c
ANISOU 1727 CD GLU B 202 10940 11016 11196 -42 30 - 649 c
ATOM 1728 OEl GLU B 202 4.975 -16. 925 -19. 165 1 .00 87 .18 o
ANISOU 1728 OEl GLU B 202 10863 10990 11273 -30 5 - 675 o
ATOM 1729 OE2 GLU B, 202 4.912 -14. 766 -18. 762 1 .00 90 .05 o
ANISOU 1729 OE2 GLU B 202 11298 11391 11524 -48 67 - 616 o
ATOM 1730 o ASN B 208 3.271 -10. 283 -33. 949 1 .00 64 .81 o
ANISOU 1730 o ASN B 208 8950 8337 7337 -195 289 - 869 o
ATOM 1731 N ASN B 208 1.948 -12. 736 -35. 874 1 .00 67 .54 N
ANISOU 1731 N ASN B 208 9305 8698 7661 -196 170 - 983 N
ATOM 1732 CA ASN B 208 2.592 -11. 429 -35. 948 1 .00 68 .55 C
ANISOU 1732 CA ASN B 208 9458 8838 7750 - -195 235 - 937 C
ATOM 1733 C ASN B 208 2.341 -10. 596 -34. 694 1 .00 62 .36 C
ANISOU 1733 C ASN B 208 8673 8042 6978 -191 246 - 872 C
ATOM 1734 CB ASN B 208 2.129 -10. 674 -37. 198 1 .00 76 .15 C
ANISOU 1734 CB ASN B 208 10481 9839 8616 -192 242 - 909 C
ATOM 1735 CG AS B 208 2.783 -9. 311 -37. 335 1 .00 81 .74 C
ANISOU 1735 CG ASN B 208 11210 10558 9290 -193 307 - 862 C
ATOM 1736 ODl ASN B 208 3.973 -9. 207 -37. 632 1 .00 82 .38 o
ANISOU 1736 ODl ASN B 208 11274 10637 9391 -201 354 - 890 o
ATOM 1737 ND2 ASN B 208 2.001 -8. 256 -37. 131 1 .00 83 .86 N
ANISOU 173 ND2 ASN B 208 11516 10837 9511 -186 307 786 N
ATOM 1738 o GLN B 209 -0.871 -11. 237 -32. 800 1 .00 27 .48 o
ANISOU 1738 0. GLN B 209 4221 3598 2620 -144 63 732 o
ATOM 1739 N GLN B 209 1.082 -10. 237 -34. 468 1 .00 47 .11 N
ANISOU 1739 N GLN B 209 6781 6117 5003 -184 205 817 N ATOM 1740 CA GLN B 209 0.702 -9.481. -33.280 1.00 35.44 C
ANISOU 1740 CA GLN B 209 5309 4624 3532 -177 209 ■749 C
ATOM 1741 C GLN B 209 -0.142 -10. 348 -32. 351 1 .00 31. 57 C
ANISOU 1741 C GLN B 209 4754 4114 3128 -152 127 •717 C
ATOM 1742 CB GLN B 209. -0.053 -8. 209 -33. 662 1 .00 36. 83 c
ANISOU 1742 CB GLN B 209 5545 4821 3628 -164 216 •666 c
ATOM 1743 CG GLN B 209 -0.469 -7. 344 -32. 485 1 .00 43. 74 c
ANISOU 1743 CG GLN B 209 6429 5681 4510 -152 221 589 c
ATOM 1744 CD GLN B 209 -1.359 -6. 184 -32. 905 1 .00 49. 66 c
ANISOU 1744 CD GLN B 20 7231 6449 5190 -135 219 •504 c TOM 1745 OEl GLN B 209 -1.304 -5. 729 -34. 047 1 .00 56. 79 o
ANISOU 1745 OEl GLN B 209 8159 7376 ' 6043 -138 240 502 o
ATOM 1746 NE2 GLN B 209 -2.191 -5. 709 -31. 985 1 .00 45. 98 N
ANISOU 1746 NE2 GLN B 209 6776 5970 4724 -115 193 ■431 N
ATOM 1747 N VAL B 210 -0.038 -10. 079 -31. 054 1 .00 21. 76 N
ANISOU 1747 N VAL B 210 2524 2760 2986 -103 92 •305 N
ATOM 1748 CA VAL B 210 -0.687 -10. 899 -30. 047 1 .00 15. 65 C
ANISOU 1748 CA VAL B 210 1741 1979 2227 -75 81 288 C
ATOM ' 1749 C VAL B 210 -1.754 -10. 112 -29. 284 1 .00 10. 80 C
ANISOU 1749 c VAL B 210 1163 1369 1570 -58 60 268 C
ATOM 1750 o VAL B 210 -1.556 -8. 947 -28. 926 1 .00 15. 74 o
ANISOU 1750 o VAL B 210 1816 1996 2168 -61 41 261 o
ATOM 1751 CB VAL B 210 0.341 -11. 474 -29. 067 1 .00 29. 47 C
ANISOU 1751 CB VAL B 210 3461 3719 4018 -67 69 277 C
ATOM 1752 CGI VAL B 210 -0.359 -12. 227 -27. 963 1 .00 24. 62 C
ANISOU 1752 CGI VAL B 210 2842 3099 3414 -42 54 254 C
ATOM 1753 CG2 VAL B 210 1.320 -12. 384 -29. 810 1 .00 29. 58 C
ANISOU 1753 CG2 VAL B 210 3433 3727 4080 -80 91 301 C
ATOM 1754 N CYS B 211 -2.895 -10. 754 -29. 058 1 .00 12. 71 N
ANISOU 1754 N CYS B 211 1406 1613 1809 -39 62 260 N
ATOM 1755 CA CYS B 211 -4.034 -10. 106 -28. 424 1 .00 12. 58 C
ANISOU 1755 CA CYS B 211 1420 1605 1754 -21 46 245 C
ATOM 1756 C CYS B 211 -4.604 -10. 955 -27. 317 1 .00 12. 01 C
ANISOU 1756 C CYS B 211 1334 1536 .1692 1 38 225 C
ATOM 1757 o CYS B 211, -4.352 -12. 155 -27. 240 1 .00 15. 43 o
ANISOU 1757 o CYS B 211 1736 1961 2164 2 45 221 o
ATOM 1758 CB CYS B 211 -5.139 -9. 869 -29. 456 1 .00 11. 95 C
ANISOU 1758 CB CYS B 211 1360 1532 1649 -25 59 256 C
ATOM 1759 SG CYS B 211 -4.688 -8. 660 -30. 714 1 .00 20. 67 s
ANISOU 1759 SG CYS B 211 2489 2637 2728 -54 63 274 s
ATOM 1760 N GLY B 212 -5.394 -10. 330 -26. 460 1 .00 13. 90 N
ANISOU 1760 N GLY B 212 1597 1786 1897 19 21 211 N
ATOM 1761 CA GLY B 212 -5.904 -11. 028 -25. 297 1 .00 11. 11 C
ANISOU 1761 CA GLY B 212 1232 1441 1547 37 11 188 C
ATOM 1762 C GLY B 212 -7.355 -11. 464 -25. 439 1 .00 9. 54 c
ANISOU 1762 C GLY B 212 1036 1255 1334 49 20 183 c
ATOM 1763 o GLY B 212 -8.170 -10. 811 -26. 107 1 .00 9. 4.9 o
ANISOU 1763 o GLY B 212 1051 1254 1299 51 26 195 o
ATOM 1764 N TYR B 213 -7.675 -12. 580 -24. 789 1 .00 6. 91 N
ANISOU 1764 N TYR B 213 680 925 1021 56 19 164 N
ATOM 1765 CA TYR B 213 -9.048 -13. 053 -24. 742 1 .00 8. 23 c
ANISOU 1765 CA TYR B 213 847 1107 1175 67 25 155 c
ATOM ' 1766 C TYR B 213 -9.378 -13. 656 -23. 401 1 .00 9. 15 c
ANISOU 1766 C TYR B 213 949 1237 1289 78 12 125 c
ATOM 1767 o TYR B 213 -8.484 -14. 027 -22. 653 ' 1 .00 8. 08 o
ANISOU 1767 o TYR B 213 800 1095 1175 75 -1 •111 o
ATOM 1768 CB TYR B 213 -9.338 -14. 069 -25. 855 1 .00 8. 28 c
ANISOU 1768 CB TYR B 213 834 1100 1211 57 45 164 c
ATOM 1769 CG TYR B 213 -8.603 -15. 400 -25. 807 1 .00 12. 48 c
ANISOU 1769 CG TYR B 213 1332 1614 1797 49 47 ■158 c
ATOM 1770 CD1 TYR B 213 -9.253 -16. 564.-25. 383 1 .00 11. 63 c ANISOU 1770 CD1 TYR B 213 1203 1507 1709 54 45 -137 C
ATOM 1771 CD2 TYR B 213 -7.295 -15. 508 -26. 250 1. 00 10. 51 C
ANISOU 1771 CD2 TYR B 213 1068 1345 1580 37 51 -174 C
ATOM 1772 CE1 TYR B 213 -8.599 -17. 793 -25. 377 1. 00 16. 62 C
ANISOU 1772 CE1 TYR B 213 1803 2117 2396 47 43 -132 C
ATOM 1773 CE2 TYR B 213 -6.622 -16. 739 -26. 242 1. 00 11. 13 C
ANISOU 1773 CE2 TYR B 213 1113 1404 1713 32 51 -172 c
ATOM •1774 CZ TYR B 213 -7.282 -17. 877 -25. 802 1. 00 17. 34 c
ANISOU 1774 CZ TYR B 213 1881 2187 2521 38 46 -151 c
ATOM 1775 OH TYR B 213 -6.624 -19. 101 -25. 813 1. 00 13. 29 o
ANISOU 1775 OH TYR B 213 1334 1650 2066 .35 42 -149 o
ATOM 1776 N VAL B 214 -10.673 -13. 734 -23. 092 1. 00 9. 10 N
ANISOU 1776 N VAL B 214 948 1253 1257 90 14 -115 N
ATOM 1777 C VAL B 214 -11.706 -15. 764 -22. 195 1. 00 11. 45 C
ANISOU 1777 C VAL B 214 1205 1564 1580 90 12 -69 C
ATOM 1778 o VAL B 214 -12.402 -15. 935 -23. 197 1. 00 8. 60 o
ANISOU 1778 o VAL B 214 846 1200 1223 88 27 -83 o
ATOM 1779 CA AVAL B 214 -11.120 -14. 397 -21. 872 0. 50 13. 42 C
ANISOU 1779 CA AVAL B 214 1481 1820 1800 97 3 -84 C
ATOM 1780 CB AVAL B 214 -12.192 -13. 577 -21. 137 0. 50 14. 12 C
ANISOU 1780 CB AVAL B 214 1589 1943 1835 114 -2 -80 C
ATOM 1781 CG1AVAL B 214 -12.550 -14. 236 -19. 801 0. 50 11. 41 . c
ANISOU 1781 CG1AVAL B 214 1229 1624 1482 117 ■12 -47 c
ATOM 1782 CG2AVAL B 214 -11.714 -12. 158 -20. 913 0. 50 21. 32 c
ANISOU 1782 CG2AVAL B 214 2528 2856 2716 .122 ■12 ,-99 c
ATOM 1783 CA BVAL B 214 ' -11.119 -14. 396 -21. 874 0. 50 13. 43 c
ANISOU 1783 CA BVAL B 214 1482 1820 1800 3 -84 c
ATOM 1784 CB BVAL B 214 -12.172 -13. 559 -21. 114 0. 50 14. 25 c
ANISOU 1784 CB BVAL B 214 , 1604 1958 1850 114 -2 -80 c TOM 1785 CG1BVAL B 214 -11.571 -12. 262 -20. 620 0. 50 20. 19 c
ANISOU 1785 CG1BVAL B 214 2381 2714 2574 122 ■15 -93 c
ATOM 1786 CG2BVAL B 214 -13.384 -13. 277 -21. 985 0. 50 7. 02 c
ANISOU 1786 CG2BVAL B 214 699 1051 916 121 13 -95 c
ATOM 1787 N LYS B 215 -11.418 -16. 744 -21. 350 1. 00 11. 04 N
ANISOU 1787 N LYS B 215 1130 1511 1553 85 -1 -41 N
ATOM 1788 CA LYS B 215 -12.018 -18. 068 -21. 480 1. 00 11. 77 c
ANISOU 1788 CA LYS B 215 1198 1598 1676 7.9 2 -22 C
ATOM 1789 C LYS B 215 -12.267 -18. 559 -20. 061 1. 00 18. 24 C
ANISOU 1789 C LYS B 215 2005 2439 2484 79 ■16 18 C
ATOM 1790 O LYS B 215 -11.332 -18. 653 -19. 268 1. 00 15. 79 o
ANISOU 1790 o LYS B 215 1689 2123 2187 75 ■34 33 o
ATOM 1791 CB LYS B 215 -11.080 -19. 027 -22. 208 1. 00 7. 75 c
ANISOU 1791 CB LYS B 215 667 1050 1228 67 4 -30 c
ATOM 1792 CG LYS B 215 -11.692 -20. 381 -22. 490 1. 00 10. 84 c
ANISOU 1792 CG LYS B 215 1034 1429 1654 60 5 -16 c
ATOM 1793 CD LYS B 215 -10.639 -21. 339 -23. 049 1. 00 17. 29 c
ANISOU 1793 CD LYS B 215 1827 2206 2536 50 2 -26 c
ATOM 1794 CE LYS B 215 -11.289 -22. 610 -23. 604 1. 00 18. 54 c
ANISOU 1794 CE LYS B 215 1965 2347 ' 2732 44 5 -21 c
ATOM 1795 NZ LYS B 215 -12.127 -22. 315 -24. 826 1. 00 14. 99 N
ANISOU 1795 NZ LYS B 215 1530 1903 2264 44 29 -50
ATOM 1796 o SER B 216 -13.895 -17. 018 -17. 431 1. 00 17. 85 o
ANISOU 1796 o SER B 216 1984 2492 2306 102 •30 51 o
ATOM 1797 N SER B 216 -13.528 -18. 830 -19. 734 1. 00 13. 86 N
ANISOU 1797 N SER B 216 1448 1913 1905 81 •13 36 N
ATOM 1798 CA SER B 216 -13.884 -19. 220 -18. 380 r. 00 16. 17 C
ANISOU 1798 CA SER B 216 1731 2235 2179 78 ■29 75 C
ATOM 179 C SER B 216 -13.418 -18. 145 -17. 396 1. 00 18. 56 C
ANISOU 1799 C SER B 216 2052 2561 2440 87 ■39 73 C
ATOM 1800 CB SER B 216 -13.272 -20. 576 -18. 036 1. 00 21. 31 C
ANISOU 1800 CB SER B 216 2354 2861 2883 62 ■46 106 C ATOM 1801 OG SER B 216 -13.805 -21.583 -18.883 1..00 30.50 o
ANISOU 1801 OG SER B 216 3501 4004 4083 55 -39 107 o
ATOM 1802 0 ASN B 217 -10.098 -16. 273 -14 .902 1. ,00 20. 14 o
ANISOU 1802 0 ASN B 217 2281 2760 2611 87 -95 77 o
ATOM 1803 N ASN B 217 -12.483 -18. 487 -16 .519 1. ,00 17. 29 . N
ANISOU 1803 N ASN B 217 1881 2393 2294 79 -60 96 N
ATOM 1804 CA ASN B 217 -12.051 -17. 533 -15 .500 1. ,00 12. 96 C
ANISOU 1804 CA ASN B 217 1350 1868 1706 85 -72 96 C
ATOM 1805 C ASN B 217 -10.692 -16. 914 -15 .782 1. 00 20. 15 C
ANISOU 1805 C ASN B 217 2272 2750 2637 86 -79 74 C
ATOM 1806 CB ASN B 217 -11.955 -18. 244 -14 .149 1. 00 17. 33 C
ANISOU 1806 CB ASN B 217. 1888 2443 2254 73 -94 140 C
ATOM 1807 CG ASN B 217 -13.309 -18. 639 -13 .599 1. 00 30. 91 C
ANISOU 1807 CG ASN B 217 3599 4206 3940 71 -89 165 C
ATOM 1808 ODl ASN B 217 -14.249 -17. 854 -13 .614 i: 00 29. 31 o
ANISOU 1808 ODl ASN B 217 3410 4036 3690 85 -73 147 o
ATOM 1809 ND2 ASN B 217 -13.411 -19. 869 -13 .107 1. 00 40. 25 N
ANISOU 1809 ND2 ASN B 217 4758 5389 5148 , 52 - 103 208 N
ATOM 1810 N SER B 218 -10.184 -17. 129 -16 .989 1. 00 15. 76 N
ANISOU 1810 N SER B 218 1710 2156 2122 82 -68 51 N
ATOM 1811 CA SER B 218 -8.801 -16. 787 -17 .298 1. 00 16. 56 C
ANISOU 1811 CA SER B 218 1813 2227 2252 78 -75 34 C
ATOM 1812 C SER B 218 -8.668 -15. 825 -18 .478 1. 00 18. 73 C
ANISOU 1812 C SE B 218 210'8 2489 2519 83 -57 -7 C
ATOM 1813 o SER B 218 -9.454 -15. 874 -19 .420 1. 00 14. 91 o
ANISOU 1813 o SER B 218 1628 2004 2034 86 -38 -22 o
ATOM 181 CB SER B 218 -8.013 -18. 052 -17 .619 1. 00 24. 25 C
ANISOU 1814 CB SER B 218 < 2755 3165 3292 66 -81 46 C
ATOM 1815 OG SER B 218 -7.928 -18. 904 -16 .489 1. 00 24. 13 o
ANISOU 1815 OG SER B 218 2722 3156 3289 58 105 88 o
ATOM 1816 N LEU B 219 -7.652 -14. 970 -18 .405 1. 00 16. 90 N
ANISOU 1816 N LEU B 219 1889 2248 2284 82 -65 -21 N
ATOM 1817 CA LEU B 219 -7.203 -14. 157 -19 .527 1. 00 12. 41 C
ANISOU 1817 CA LEU B 219 1336 1662 1718 80 -52 -56 C
ATOM 1818 C LEU B 219 -6.016 -14. 846 -20 .180 1. 00 17. 12 C
ANISOU 1818 C LEU B 219 1908 2226 2372 67 -50 -63 C
ATOM 1819 o LEU B 219 -5.036 -15. 189 -19 .506 1. 00 15. 76 o
ANISOU 1819 o LE'U B 219 1720 2043 2225 62 -67 -48 o
ATOM 1820 CB LEU B 219 -6.792 -12. 760 -19 .068 "1. 00 17. 93 C
ANISOU 1820 CB LEU B 219 2062 2370 2379 86 -65 -68 C
ATOM 1821 CG LEU B 219 -7.904 -11. 738 -18 .861 1. 00 24. 68 C
ANISOU 1821 CG LEU B 219 2947 3251 3178 102 -63 -78 C
ATOM 1822 CDl LEU B 219 -7.329 -10. 443 -18 .323 1. 00 ,23. 11 C
ANISOU 1822 CDl LEU B 219 2774 3056 2950 106 -81 -90 C
ATOM 1823 CD2 LEU B 219 -8.586 -11. 481 -20 .179 1. 00 24. 54 C
ANISOU 1823 CD2 LEU B; 219 2939 3226 3158 103 -43 ■101 C
ATOM 1824 N LEU B 220 -6.138 -15. 075 -21 .486 1. 00 14. 85 N
ANISOU 1824 N LEU B 220 1615 1923 2103 61 -28 -86 N
ATOM 1825 CA LEU B 220 -5.149 -15. 788 -22 .276 1. 00 7. 06 C
ANISOU 1825 CA LEU B 220 603 909 1171 50 -20 100 C
ATOM 1826 C LEU B 220 -4.706 -14. 960 -23 .475 1. 00 14. 87 c
ANISOU 1826 C LEU B 220 1605 1890 2153 41 -3 •134 c
ATOM 1827 o LEU B 220 -5.292 -13. 923 -23 .790 1. 00 12. 50 o
ANISOU 1827 o LEU B 220 1335 1604 1810 44 2 145 o
ATOM 1828 CB LEU B 220 -5.739 -17. 126 -22 .763 1. 00 10. 22 c
ANISOU 18-28 CB LEU B 220 979 1297 1606 49 -10 -96 c
ATOM 1829 CG LEU B 220 -6.304 -18. 009 -21 .643 1. 00 16. 82 c
ANISOU 1829 CG LEU B 220 1802 2141 2448 53 -27 -58 c
ATOM 1830 CDl LEU B 220 -7.059 -19. 203 -22 .223 1. 00 17. 62 c
ANISOU 1830 CDl LEU B 220 1884 2232 2580 51 -18 -55 c
ATOM 1831 CD2 LEU B 220 -5.209 -18. 482 -20 .691 1. 00 18. 66 c ANISOU 1831 CD2 LEU B 220 2015 2361 2715 50 -52 -35 C
ATOM 1832 N SER B 221 -3.675 -15. 451 -24. 152 1 .00 12 .90 N
ANISOU 1832 N SER B 221 1332 1620 1948 30 6 151 N
ATOM 1833 CA SER B 221 -3.043 -14. 749 -25. 243 1 .00 13 .59. C
ANISOU 1833 CA SER B 221 1428 1704 2033 16 21 181 C
ATOM 1834 C SER B 221 -3.178 -15. 570 -26. 525 1 .00 12 .00 C
ANISOU 1834 C SER B 221 1207 1491 1861 8 47 205 C
ATOM 1835 0 SER B 221 -3.132 -16. 799 -26. 484 1 .00 15 .60 o
ANISOU 1835 o SER B 221 1634 1933 2360 11 48 201 o
ATOM 1836 CB SER B 221 -1.564 -14. 535 -24. 906 1 .00 20 .75 C
ANISOU 1836 CB SER B 221 2319 2600 2965 8 10 182 C
ATOM 1837 OG SER B 221 -0.867 -13. 993 -26. 003 1 .00 33 .00 o
ANISOU 1837 OG SER B 221 3871 4148 4518 -8 27 211 o
ATOM 1838 SER B' 222 -3.360 -14. 889 -27. 655 " 1 .00 12 .95 N
ANISOU 1838 N SER B 222 1344 1618 1956 -3 66 230 , N
ATOM 1839 CA SER B 222 -3.496 -15. 572 -28. 937 1 .00 14 .55 C
ANISOU 1839 CA SER B 222 1533 1815 2181 -13 91 256 C
ATOM 1840 C SER B 222 -3.017 -14. 680 -30. 073 1 .00 17 .45 C
ANISOU 1840 C SER B 222 1914 2190 2528 -34 , 108 284 C
ATOM 1841 o SER B 222 -3.039 -13. 452 -29. 960 1 .00 15 .79 o
ANISOU 1841 o SER B 222 1732 1989 2276 -38 99 - 279 o TOM 1842 CB SER B 222 -4.963 -15. 921 -29. 186 1 .00 24 .13
ANISOU 1842 CB SER B 222 2759 3036 3375 -5 97 251 C
ATOM 1843 OG SER B 222 -5.123 -16. 502 -30. 468 1 .00 21 .37 o
ANISOU 1843 OG SER B 222 2398 2681 3041 -17 120 278 o
ATOM 1844 N ASN B 223 -2.590 -15. 292 -31. 175 1 .00 14 .23 N
ANISOU 1844 N ASN B 223 1483 1777 2145 -47 131 - 313 N
ATOM 1845 CA ASN B 223 -2.399 -14. 531 -32. 401 1 .00 13.68 C
ANISOU 1845 CA ASN B 223 1429 1720 2049 -70 150 337 C
ATOM 1846 C ASN B 223 -3.691 -13. 784 -32. 717 1 .00 13 .85 C
ANISOU 1846 C ASN B 223 1489 1753 2020 -70 147 - 330 C
ATOM 1847 O' ASN B 223 -4.779 -14. 359 -32. 635 1 .00 13 .29 o
ANISOU 1847 o · ASN B 223 1421 1682 1948 -57 147 322 o
ATOM 1848 CB ASN B 223 -2.048 -15. 459 -33. 562 1 .00 20 .04 c
ANISOU 1848 CB ASN B 223 2205 2522 2886 -83 178 372 c
ATOM 1849 CG ASN B 223 -1.514 -14. 706 -34. 754 1 .00 29 .33 c
ANISOU 1849 CG ASN B 223 3389 3715 4038 -111 197 397 c
ATOM 1850 ODl ASN B 223 -0.310 -14. 712 -35. 020 1 .00 42 .52 o
ANISOU 1850 ODl ASN B 223 5037 5389 5731 -125 208 415' o
ATOM 1851 ND2 ASN B 223 -2.403 -14. 021 -35. 466 1 .00 21 .15 N
ANISOU 1851 ND2 ASN B 223 2387 2693 2957 -123 202 398 N
ATOM 1852 N CYS B 224 -3.577 -12. 506 -33. 064 1 .00 13 .69 N
ANISOU 1852 N CYS B 224 1497 1743 1961 -84 143 330 N
ATOM 1853 CA CYS B 224 -4.747 -11. 667 -33. 306 1 .00 16 .52 C
ANISOU 1853 CA CYS B 224 1893 2111 2273 -82 136 322 C
ATOM 1854 C CYS B 224 -5.553 -12. 107 -34. 522 1 .00 13 .19 C
ANISOU 1854 C CYS B 224 1474 1695 1843 -93 156 339 C
ATOM 1855 o CYS B 224 -6.757 -11. 810 -34. 634 1 .00 14 .48 o
ANISOU 1855 o CYS B 224 1659 1863 1978 -85 150 330 o
ATOM 1856 CB CYS B 224 -4.313 -10. 208 -33. 478 1 .00 17 .30 C
ANISOU 1856 CB CYS B 224 2021 2215 2337 -98 123 320 C
ATOM 1857 SG CYS B 224 -3.429 -9. 558 -32. 032 1 .00 25 .64 S
ANISOU 1857 SG CYS B 224 3080 3264 3397 -86 96 300 S
ATOM 1858 N ASP B 225 -4.881 -12. 803 -35. 440 1 .00 13 .68 N
ANISOU 1858 N ASP B 225 1510 1758 1930 -111 180 ■365 N
ATOM 1859 CA ASP B 225 -5.492 -13. 240 -36. 697 1 .00 17 .08 C
ANISOU 1859 CA ASP B 225 1941 2195 2352 -126 200 •386 C
ATOM 1860 C ASP B 225 -6.077 -14. 639 -36. 568 1 .00 19 .94 C
ANISOU 1860 c ASP B 225 2279 2547 2750 -109 207 •390 C
ATOM 1861 o ASP B 225 -5.773 -15. .540 -37. 364 1 .00 18 .49 o
ANISOU 1861 o ASP B 225 2072 2362 2593 -119 228 ■417 o ATOM 1862 CB ASP B 225 -4.465 -13.210 -37.831 1.00 18.61 C
ANISOU 1862 CB ASP B 225 2121 2399 2550 -157 224 ■417 C
ATOM 1863 CG ASP B 225 -5.086 -13. 529 -39 .183 1 .00 32 .09 C
ANISOU 1863 CG ASP B 225 3832 4118 4241 -176 245 440 C
ATOM 1864 ODl ASP B 225 -6.302 -13. 288 -39 .356 1 .00 27 .91 o
ANISOU 1864 ODl ASP B 225 3329 3591 3685 -171 236 427 o
ATOM 1865 OD2 ASP B 225 -4.355 -14. 027 -40 .065 1 .00 33 .37 o
ANISOU 1865 OD2 ASP B 225 3971 4289 4418 -196 270 471 o
ATOM 1866 N THR B 226 -6.918 -14. 807 -35 .557 1 .00 12 .01 N
ANISOU 1866 N THR B 226 1282 1537 1746 -84 189 363 N
ATOM 1867 CA THR B 226 -7.646 -16. 049 -35 .336 1 .00 13 .03 C
ANISOU 1867 CA THR B 226 1392 1656 1903 -70 190 359 C
ATOM 1868 C THR B 226 -9.098 -15. 718 -35 .033 1.00 11 .53 C
ANISOU 1868 C THR B 226 1226 1474 1680 -57 178 336 C
ATOM 1869 o THR B 226 -9.401 -14. 616 -34 .596 1 .00 11 .62 o
ANISOU 1869 o THR B 226 1265 1495 1656 -51 163 321 o
ATOM 1870 CB THR B 226 -7.035 -16. 875 -34 .166 1 .00 16 .16 CANISOU 1870 CB THR B 226 1759 2036 2344 -52 178. - 344 C
ATOM 1871 OGl THR B 226 -6.989 -16. 079 -32 .970 1 .00 17 .59 o
ANISOU 1871 OGl THR B 226 1956 2222 2505 -39 156 315 o
ATOM 1872 CG2 THR B 226 -5.630 -17. 312 -34 .517 1 .00 18 .87 c
ANISOU 1872 CG2 THR B 226 2073 2371 .2727 -62 191 369 c
ATOM 1873 N TRP B 227 -9.993 -16. 675 -35 .248 1 .00 10 .13 N
ANISOU 1873 N TRP B 227 1040 1293 1517 -51 182 336 N
ATOM 1874 CA TRP B 227 -11.428 -16. 447 -35 .048 1 .00 8 .17 c
ANISOU 1874 CA TRP B 227 810 1055 1240 -40 172 316 C
ATOM 1875 C TRP B 227 -11.835 -16. 830 -33 .633 1 .00 13 .63 c
ANISOU 1875 C TRP B 227 1492 1745 1941 -17 . 155 - 285 c
ATOM 1876 0 TRP B 227 -11.653 -17. 978 -33 .215 1 .00 13 .35 o
ANISOU 1876 0 TRP B 227 1430 1697 1946 -12 154 279 o
ATOM 1877 CB TRP B 227 -12.245 -17. 288 -36 .045 1 .00 10 .53 c
ANISOU 1877 CB TRP B 227 1103 1352 1547 -49 185 331 c
ATOM 1878 CG TRP B 227 -12.415 -16. 668 -37 .400 1 .00 14 .67 c
ANISOU 1878 CG TRP B 227 1647 1886 2040 -72 198 353 c
ATOM 1879 CD1 TRP B 227 -12.101 -15. 397 -37 .760 1 .00 17 .35 c
A ISOU 1879 CD1 TRP B 227 2012 2236 2344 -83 195 356 c
ATOM 1880 CD2 TRP B 227 -12.947 -17. 300 -38 .575 1 .00 14 .47 c
ANISOU 1880 CD2 TRP B 227 1619 1861 2017 -87 212 374 c
ATOM 1881 NE1 TRP B 227 -12.407 -15. 190 -39 .087 1 .0013 .72 N
ANISOU 1881 NE1 TRP B 227 1566 178.4 1863 -107 207 376 N
ATOM 1882 CE2 TRP B 227 -12.930 -16. 343 -39 .607 1 .00 15 .16 C
ANISOU 1882 CE2 TRP B 227 1732 1963 2067 -109 218 388 C
ATOM 1883 CE3 TRP B 227 -13.444 -18. 578 -38 .849 1 .00 17 .87 C
ANISOU 1883 CE3 TRP B 227 2029 2281 2479 -86 218 382 C
ATOM 1884 CZ2 TRP B 227 -13.383 -16. 625 -40 .898 1 .00 20 .52 c
ANISOU 1884 CZ2 TRP B 227 2415 2647 2734 -130 231 410 c
ATOM 1885 CZ3 TRP B 227 -13.899 -18. 854 -40 .132 1 .00 21 .37 c
ANISOU 1885 CZ3 TRP B 227 2478 2729 2912 -105 232 - 406 c
ATOM 1886 CH2 TRP B 227 -13.862 -17. 881 -41 .136 1 .00 21 .20 c
ANISOU 1886 CH2 TRP B 227 2481 2724 2850 -127 239 419 c
ATOM 1887 N LYS B 228 -12.372 -15. 859 -32 .894 1 .00 9 .79 N
ANISOU 1887 N LYS B 228 1028 1273 1419 -4 140 265 N
ATOM 1888 CA LYS B 228 -12.941 -16. 104 -31 .574 1 .00 9 .17 C
ANISOU 1888 CA LYS B 228 944 1202 1340 17 125 235 C
ATOM 1889 C LYS B 228 -14.209 -15. 294 -31 .451 1 .00 8 .80 C
ANISOU 188 C LYS B 228 919 1174 1249 28 118 226 C
ATOM 1890 o LYS B 228 -14.469 -14. 413 -32 .270 1 .00 9 .35 o
ANISOU 1890 o LYS B 228 1013 1248 1292 21 120 240 o
ATOM 1891 CB LYS B 228 -11.960 -15. 680 -30 .473 1 .00 6 .96 c
ANISOU 1891 CB LYS B 228 662 921 1062 24 112 •224 c
ATOM 1892 CG LYS B 228 -10.616 -16. 396 -30 .543 1 .00 7 .70 c ANISOU 1892 CG LYS B 228 730 995 1201 15 116 •234 C
ATOM 1893 CD LYS B 228 -9.758 -15. 986 -29. 363 1 .00 13. 04 c
ANISOU 1893 CD LYS B 228 1405 1671 1878 23 99 ■218 c
ATOM 1894 CE LYS B 228 -8.380 -16. 608 -29. 447 1 .00 16. 06 c
ANISOU 1894 CE LYS B 228 1762 2035 2307 13 103 ■229 c
ATOM 1895 NZ LYS B 228 -8.445 -18. 047 -29. 702 1 .00 23. 34 N
ANISOU 1895 NZ LYS B 228 2653 2939 3275 .12 109 ■231 N
ATOM 1896 N TYR B 229 -15.021 -15. 572 -30. 438 1 .00 7. 45 N
ANISOU 1896 N TYR B 229 743 1017 1072 45 108 ■201 N
ATOM 1897 CA TYR B 229 -16.123 -14. 658 -30. 160 1 .00 6. 04 C
ANISOU 1897 CA TYR B 229 584 859 851 60 100 •194 • C
ATOM 1898 C TYR B 229 -15.509 -13. 369 -29. 607 1 .00 6. 02 c
ANISOU 1898 c TYR B 229 604 860 823 68 88 ■197 c
ATOM 1899 o TYR B 229 -14.292 -13. 254 -29. 496 1 .00 8. 55 o
ANISOU 1899 o TYR B 229 923 1169 1159 60 87 203 o
ATOM 1900 CB TYR B 229 -17.074 -15. 258 -29. 137 1 .00 6. 09 c
ANISOU 1900 CB TYR B 229 575 884 854 75 94 167 c TOM 1901 CG TYR B 229 -17.658 -16. 569 -29. 593 1 .00 9. 26 c
ANISOU 1901 CG TYR B 229 955 1280 1284 66 103 161 c
ATOM 1902 CD1 TYR B 229 -17.038 -17. 782 -29. 291 1 .00 8. 94 c
ANISOU 1902 CD1 TYR B 229 889 1224 1286 58 103 150 c
ATOM . 1903 CD2 TYR B 229 -18.825 -16. 592 -30. 329 1 .00 10. 80 c
ANISOU 1903 CD2 TYR B 229 1155 1483 1466 64 108 164 c
ATOM 1904 CEl TYR B 229 -17.582 -19. 001 -29. 740 1 .00 8. 47 c
ANISOU 1904 CEl TYR B 229 809 1154 1254 49 107 145 c
ATOM 1905 CE2 TYR B 229 -19.377 -17. 782 -30. 768 1 .00 11. 73 c
ANISOU 1905 CE2 TYR B 229 1253 1594 1610 55 114 159 c
ATOM 1906 CZ TYR B 229 -18.766 -18. 980 -30. 457 1 .00 9. 51 c
ANISOU 1906 CZ TYR B 229 947 1296 1370 47 113 149 c
ATOM 1907 OH TYR B 229 -19.347 -20. 163 -30. 921 1 .00 10. 34 o
ANISOU 1907 OH TYR B 229 1034 1392 1504 37 116 144 o
ATOM 1908 N PHE B 230 -16.351 -12. 393 -29. 289 1 .00 7. 00 N
ANISOU 1908 N PHE B 230 749 1001 . 912 84 78 196 N
ATOM 1909 CA PHE B 230 -15.862 -11. 142 -28. 706 1 .00 6. 69 c
ANISOU 1909 CA PHE B 230 730 963 847 94 63 200 c
ATOM 1910 C PHE B 230 -16.886 -10. 544 -27. 755 1 .00 9. 83 c
ANISOU 1910 C PHE B 230 1137 1386 1214 120 52 192 c
ATOM 1911 o PHE B 230 -18.046 -10. 911 -27. 781 1 .00 9. 32 o
ANISOU 1911 o PHE B 230 1063 1336 1142 129 56 185 o
ATOM 1912 CB PHE B 230 -15.466 -10. 129 -29. 794 1 .00 6. 45 c
ANISOU 1912 CB PHE B 230 726 919 806 . 80 61 - 220 c
ATOM 1913 CG PHE B 230 ' -16.559 -9. 797 -30. 749 1 .00 8. 49 c
ANISOU 1913 CG PHE B 230 998 1181 1048 79 62 228 c
ATOM 1914 CD1 PHE B 230 -17.423 -8. 755 -30. 492 1 .00 8. 92 c
ANISOU 1914 CD1 PHE B 230 1072 .1244 1071 98 47 - 229 c
ATOM 1915 CD2 PHE B 230 -16.709 -10. 518 -31. 920 1 .00 9. 08 c
ANISOU 1915 CD2 PHE B 230 1064 1247 1137 59 78 - 235 c
ATOM 1916 CEl PHE B 230 -18.440 -8. 430 -31. 396 1 .00 11. 65 c
ANISOU 1916 CEl PHE B 230 1430 1592 1405 97 45 235 c
ATOM 1917 CE2 PHE B 230 -17.733 -10. 211 -32. 830 1 .00 10. 45 c
ANISOU 1917 CE2 PHE B 230 1251 1425 1296 56 77 240 c
ATOM 1918 CZ PHE B 230 =-18.595 -9. 175 -32. 567 1 .00 9. 49 c
ANISOU 1918 CZ PHE B 230 1148 1311 1145 75 60 239 c
ATOM 1919 N ILE B 231 -16.441 -9. 646 -26. 881 1 .00 7. 35 N
ANISOU 1919 N ILE B 231 836 1075 880 132 37 - 193 N
ATOM 1920 CA ILE B 231 -17.366 -8. 941 -26. 007 1 .00 13. 08 c
ANISOU 1920 CA ILE B 231 1571 1826 1574 ' 159 26 192 c
ATOM 1921 C ILE B 231 -17.146 -7. 449 -26. 202 1 .00 10. 90 c
ANISOU 1921 C ILE B 231 1327 1538 1276 166 9 212 c
ATOM 1922 o ILE B 231 -15.999 -7. 001 -26. 227 1 .00 10. 61 o
ANISOU 1922 o ILE B 231 1301 1484 1245 154 1 •217 o ATOM 1923 CB .ILE B 231 -17.119 -9.293 -24.,523 1.00 13..25 C
ANISOU 1923 CB ILE B 231 1578 1867 1589 170 21 -176 C
ATOM 1924 CGI ILE B 231 -17.386 -10 .781 -24. .281 1 .00 10. ,68 C
ANISOU 1924 CGI ILE B 231 1220 1550 1286 161 34 ■152 C
ATOM 1925 CG2 ILE B 231 -18.011 -8 .453 -23. .623 1 .00 8. 14 C
ANISOU 1925 CG2 ILE B 231 940 1248 904 197 11 ■181 C
ATOM 1926 CD1 ILE B 231 -16.709 -11 .332 -23. ,007 1 .00 6. 94 C
ANISOU 1926 CD1 ILE B 231 731 1087 818 160 27 -132 C
ATOM 1927 N CYS B 232 -18.232 -6 .698 -26. 358 1 .00 9. 73 N
ANISOU 1927 N CYS B 232 1192 1400 1105 184 1 ■222 N
ATOM 1928 CA CYS B 232 -18.144 -5 .247 -26. 480 1 .00 10. 10 C
ANISOU 1928 CA CYS B 232 1270 1434 1134 194 -20 ■240 C
ATOM 1929 C CYS B 232 -18.647 -4 .615 -25. 196 1 .00 10. 97 C
ANISOU 1929. C CYS B 232 1383 1567 1217 225 -32 ■245 C
ATOM 1930 o CYS B 232 -19.406 -5 .225 -24. 436 1 .00 13. 27 o
ANISOU 1930 o CYS B 232 1654 1889 1501 240 -23 •236 o
ATOM 1931 CB CYS B 232 -18.996 -4 .707 -27. 644 1 .00 13. 77 C
ANISOU 1931 CB CYS B 232 1749 1888 1593 193 -25 ■250 C
ATOM 1932 SG CYS B 232 -18.726 .-5 .426 -29. 271 1 .00 16. 94 S
ANISOU 1932 SG CYS B 232 2148 2272 2018 156 -8 •246 S
ATOM 1933 N GLU B 233 -18.233 -3 .371 -24. 980 1 .00 11. 63 N
ANISOU 1933 N GLU B 233 1494 1637 1288 234 -55 ■261 N
ATOM 1934 CA GLU B 233 -18.589 -2 .612 -23. 796 1 .00 9. 57 C
ANISOU 1934 CA GLU B 233 1240 1394 1002 264 -69 •273 C
ATOM 1935 C GLU B 233 ,-18.820 -1 .155 -24. 186 1 .00- 11. 60 C
ANISOU 1935 C GLU B 233 1528 1630 1248 278 -96 296 C
ATOM 1936 o GLU B 233 -18.043 -0 .592 -24. 957 1 .00 15. 01 o
ANISOU 1936 o GLU B 233 19.82 2031 1691 258 -109 299 o
ATOM 1937 CB GLU B 233 -17.439 -2 .661 -22. 793 1 .00 9. 77 c
ANISOU 1937 CB GLU B 233 1265 1421 1026 259 -75 268 c
ATOM 1938 CG GLU B 233 -17.718 -1 .863 -21. 532 1 .00 16. 47 c
ANISOU 1938 CG ■ GLU B 233 2122 2290 1845 288 -91 284 c
ATOM 1939 CD GLU B 233 -16.575 -1 .875 -20. 528 1 .00 13. 72 c'
ANISOU 1939 CD GLU' B 233 1776 1944 1494 282 -99 278 c
ATOM 1940 OEl GLU B 233 -16.736 -1 .216 -19. 483 1 .00 15. 38 o
ANISOU 1940 OEl GLU B 233 1994 2171 1678 304 -112 292 o
ATOM 1941 OE2 GLU B 233. -15.522 -2 .517 -20. 779 1 .00 12. 09 o
ANISOU 1941 OE2 GLU B 233 1561 1721 1310 255 -93 - 261 o
ATOM 1942 N LYS B 234 -19.871 -0 .544 -23. 648 1 .00 15. 43 N
ANISOU 1942 N LYS B 234 2015 2133 1713 311 -105 313 N
ATOM 1943 CA LYS B 234 -20.038 0 .908 -23. 755 1 .00 18. 63 c
ANISOU 1943 CA LYS B 234 2451 2517 2111 329 -135 338 C
ATOM 1944 C LYS B 234 -20.684 1, .472 -22. 502 1 .00 24. 40 C
ANISOU 1944 C LYS B 234 - 3179 3275 2817 368 -144 - 359 C
ATOM 1945 0 LYS B 234 -21.281 0, .726 -21. 720 1 .00 18. 49 o
ANISOU 1945 0 LYS B 234 2404 2567 2055 381 -124 - 354 o
ATOM 1946 CB LYS B 234 -20.876 1, .287 -24. 978 1 .00 19. 52 c
ANISOU 1946 CB LYS B 234 2573 2613 2232 329 -143 - 342 c
ATOM 1947 CG LYS B 234 -22.289 0. .750 -24. 967 1 .00 16. 78 c
ANISOU 1947 ' CG LYS B 234 2201 2295 1879 349 -127 - 342 c
ATOM 1948 CD LYS B 234 -23.004 1, .213 -26. 244 1 .00 21. 72 c
ANISOU 1948 CD LYS B 234 2839" 2899 2516 346 -140 - 345 c
ATOM 1949 CE LYS B 234 -24.226 0, .377 -26. 555 1 .00 32. 39 c
ANISOU 1949 CE LYS B 234 4163 4275 3868 352 -120 - 336 c
ATOM 1950 NZ LYS B 234 -24.938 0, .930 -27. 747 1 .00.34. 99 N
ANISOU 1950 NZ LYS B 234 4506 4583 4206 350 -137 - 340 N
ATOM 1951 o TYR B 235 -23.268 3, .579 -22. 360 1 .00 20. 48 o
ANISOU 1951 o TYR B 235 2697 2786 2300 451 -183 - 422 o
ATOM 1952 N TYR B 235 -20.567 2 .790 -22. 321 1 .00 23. 18 N
ANISOU 1952 N TYR B 235 3053 3099 2656 386 -175 - 384 N
ATOM 1953 CA TYR B 235 -21.180 3 .463 -21. 183 1 .00 27. 04 c ANISOU 1953 CA TYR B 235 3542 3612 3121 425 -186 •412 C
ATOM 1954 C TYR B 235 -22.693 3. 347 -21 .294 1 .00 25. 34 C
A ISOU 1954 C TYR B 235 3306 3422 2900 453 -176 ■423 C
ATOM 1955 CB TYR B 235 -20.804 4. 952 -21 .141 1 .00 29. 51 C
A ISOU 1955 CB TYR B 235 3888 3887 3436 437 -225 •436 C
ATOM 1956 CG TYR B 235 -19.342 · 5. 283 -20 .882 1 .00 27. 82 C
A ISOU 1956 CG TYR B 235 3696 3649 3224 416 -241 ■433 C
ATOM 1957 CD1 TYR B 235 -18.768 5. 067 -19 .633 1 .00 30. 95 C
ANISOU 1957 CD1 TYR B 235 4086 4070 3605 419 -236 ■434 C
ATOM 1958 CD2 TYR B 235 -18.548 5. 853 -21 .879 1 .00 27. 41 C
ANISOU 1958 CD2 TYR B 235 3671 3551 3191 389 -2.63 ■426 C
ATO 1959 CEl TYR B 235 -17.440 5. 385 -19 .388 1 .00 30. 97 C
ANISOU 1959 CEl TYR B 235 4108 4050 3609 400 -252 432 C
ATOM 1960 CE2 TYR B 235 -17.219 6. 175 -21 .646 1 .00 33. 44 C
ANISOU 1960 CE2 TYR B 235 4452 4294 3959 367 -277 421 C
ATOM 1961 CZ TYR B 235 -16.667 5. 940 -20 .394 1 .00 34. 80 C
ANISOU 1961 CZ TYR B 235 4617 4489 4116 373 -273 424 C
ATOM 1962 OH TYR B 235 -15.340 6. 258 -20 .143 1 .00 31. 19 0
ANISOU 1962 OH TYR B 235 4176 4011 3663 352 -289 418 0
ATOM 1963 0 ALA B 236 -24.585 5. 343 -19 .773 1 .00 29. 69 o
ANISOU 1963 0 ALA B 236 3846 3996 3437 525 -206 472 o
ATOM 1964 N ALA B 236 -23.344 · 2. 986 -20 .195 1 .00 23. 56 N
ANISOU 1964 N ALA B 236 3055 3243 2653 474 -159 427 N
ATOM 1965 CA ALA B 236 -24.797 2. 986 -20 .158 1 .00 27. 31 C
ANISOU 1965 CA ALA B 236 3506 3744 3126 497 -151 434 c
ATOM 1966 C ALA B 236 -25.282 4. 431 -20 .220 1 .00 30. 45 c
ANISOU 1966 C ALA B 236 3920 4115 3532 522 -183 459 c
ATOM 1967 CB ALA B 236 -25.304 2. 302 -18 .894 1 .00 35. 59 c
ANISOU 1967 CB ALA B 236 4523 4851 4151 508 -126 429 c
ATOM 1968 o LEU B 237 -28.928 6. 342 -19 .478 1 .00 62. 10 o
ANISOU 1968 o LEU B 237 7886 8168 7542 616 -209 521 o
ATOM 1969 N LEU B 237 -26.466 4. 632 -20 .785 1 .00 37. 28 N
ANISOU 1969 N LEU B 237 4775 4984 4407 539 -186 467 N
ATOM 1970 CA LEU B 237 -27.038 5. 967 -20 .904 1 .00 49. 29 C
ANISOU 1970 CA LEU B 237 6309 6480 5939 566 -218 492 C
ATOM 1971 C LEU B 237 -27.707 6. 402 -19 .605 1 .00 56. 42 C
ANISOU 1971 C LEU B 237 7192 7420 6826 595 -217 - 512 C
ATOM 1972 CB LEU B 237 -28.038 6. 023 -22 .061 1 .00 49. 10 C
ANISOU 1972 CB LEU B 237 6282 6444 5931 573 -225 493 ■ C
ATOM 1973 CG LEU B 237 -27.434 6. 044 -23 .466 1 .00 55. 57 C
ANISOU 1973 CG LEU B 237 7130 7216 6767 547 -240 - 481 C
ATOM 1974 CD1 LEU B 237 -28.524 6. 005 -24 .526 1 .00 60. 44 C
ANISOU 1974 CD1 LEU B 237 7742 7826 7397 556 -245 - 483 C
ATOM 1975 CD2 LEU B 237 -26.548 7. 268 -23 .648 1 .00 57. 04 C
ANISOU 1975 CD2 LEU B 237 7355 7351 6965 542 -279 - 490 c
TER 1977 LEU B 237
ATOM 1976 CA CA C 1 -8.408 2. 090 -3 .842 1 .00 12. 32 Ca
TER 1979 CA C 1
ATOM 1977 CA CA D 2 -14.603 -1. 857 -18 .394 1 .00 12. 54 Ca
TER 1981 CA D ,2
ATOM 1978 o HOH E 1 -4.283 11. 822 12 .141 1 .00 15. 39 o
ATOM 1979 o HOH E 2 -7.351 -22. 194 -24 .770 1 .00 30. 07 o
ATOM 1980 o HOH E 3 -16.590 -5. 168 -39 .571 1 .00 48. 94 o
ATOM 1981 o HOH E. 4 -23.321 -11. 715 -32 .400 1 .00 15. 46 o
ATOM 1982 o HOH E 5 -10.986 2. 157, -25 .099 1 .00 22. 26 o
ATOM 1983 o HOH E 6 -17.430 2. 305 -21 .797 1 .00 41. 51 o
ATOM 1984 o HOH E 7 -10.999 10. 163 8 .190 1 .00 28. 36 o
ATOM 1985 o HOH E 8 -6.892 0. 112 -3 .508 1 .00 15. 35 o
ATOM 1986 o HOH E 9 -0.188 -4. 038 8 .805 1 .00 20. 14 o
ATOM 1987 o HOH E 10 -18.065 4. 152 -33 .171 1 .00 21. 47 o
ATOM 1988 o HOH E 11 -13.911 0. 160 -19 .739 1 .00 15. 94 o ATOM 1989 o HOH E 12 -9.365 -19.114 -32.037 1.00 18.43 o
ATOM 1990 o HOH E 13 -7. 717 19.747 3. 265 1 .00 20. 67 o TOM 1991 o HOH E 14 -0. 604 1.902 -0. 948 1 .00 15. 58 o
ATOM 1992 o HOH E 15 -21. 527 -16.634 -27. 122 1 .00 16. 73 o
ATOM 1993 o HOH E 16 -11. 267 -1.671 -26. 008 1 .00 16. 98 o
ATOM 1994 o HOH E 17 -11. 102 2..080 -7. 592 1 .00 19. 64 o
ATOM 1995 o HOH E 18 -6. 861 16.789 7. 222 1· .00 16. 96 o
ATOM 1996 o HOH E 19 5. 152 19.275 1. 864 1 .00 16. 12 o
ATOM 1997 o HOH E 20 -25. 008 -10.010 -24. 717 1 .00 24. 97 o
ATOM 1998 o HOH E 21 3. 517 17.863 -9. 433 1 .00 26. 89 o
ATOM 1999 o HOH E 22 -14. 758 4.769 -12. 658 1 .00 17. 65 o
ATOM 2000 o HOH E 23 -15. 327 -0.276 -16. 689 1 .00 17. 02 o
ATOM 2001 o HOH E 24 -7. 371 0.769 -17. 223 1 .00 39. 61 o
ATOM 2002. o HOH E 25 -0. 997 -1.914 -1. 830 1 .00 20. 20 o
ATOM 2003 o HOH E 26 -13. 708 -1.853 -13. 789 1 .00 17. 61 o
ATOM 2004 o HOH E 27 -4. 386 15.200 -11. 674 1 .00 30. 39 o
ATOM 2005 o HOH E 28 -4. 262 -0.755 -9. 872 1 .00 35. 49 o
ATOM 2006 o HOH E 29 -8. 235 4.662 -14. 193 1 .00 19. 25 o
ATOM 2007 o HOH E 30 -15. 657 -18.409 -21. 509 1 .00 16. 09 o
ATOM 2008 o HOH E 31 5. 074 19.723 -2. 410 1 .00 23. 08 o
ATOM 2009 o HOH E 32 -7. 573 -13.141 -14. 121 1 .00 43. 00 o
ATOM 2010 O HOH E 33 2. 985 20.477 -10. 913 1 .00 56. 36 o
ATOM 2011 o HOH E 34 -9. 003 20.934 -11. 793 1 .00 30. 35 o
ATOM 2012 o HOH E 35 -21. 611 -4.573 -35. 084 1 .00 31. 16 o
ATOM 2013 o HOH E 36 -19. 228 -19.689 -23. 727 1 .00 18. 61 o
ATOM 2014 o HOH E 37 -o. 904 20.491 3. 342 1 .00 16. 16 o
ATOM 2015 o HOH E 38 -2. 365 -18.111 -31. 199 1 .00 33. 00 o
ATOM 2016 o HOH E 39 -3. 011, 12.086 -15. 374 1 .00 17. 16 o
ATOM 2017 o HOH E 40 -17. 552 8.319 -6. 826 1 .00 25. 63 o
ATOM 2018 o HOH E 41 -2. 232 -17.689 -23. 132 1 .00 26. 96 o
ATOM 2019 o HOH E 42 -8. 861 21.022 8. 103 1 .00 31. 07 o TOM 2020 o HOH E 43 7. 043 -2.300 6. 733 1 .00 35. 46 o
ATOM 2021 o HOH E 44 -7. 043 -4.505 -11. 303 1 .00 19. 66 o
ATOM 2022 o HOH E 45 3. 893 10.041 12. 364 1 .00 19. 12 o
ATOM 2023 o HOH E 46 -8. 427 16.873 -12. 169 1 .00 34. 94 o
ATOM 2024 o HOH E 47 -17. 194 8.839 -11. 197 1 .00 31. 25 o
ATOM 2025 o HOH E 48 -3. 441 -3.301 -1. 382 1 .00 27. 76 o
ATOM 2026 o HOH E 49 -30. 239 -1.957 -18. 196 1 .00 42. 96 o
ATOM 2027 o HOH E 50 6. 841 -0.004 4. 222 1 .00 46. 92 o
ATOM 2028 o HOH E 51 -10. 057 0.509 -4. 472 1 .00 17. 08 o
ATOM 2029 o HOH E 52 -12. 045 -9.735 -41. 179 1 .00 33. 47 o
ATOM 2030 o . HOH E 53 -8. 659 -7.538 -4. 448 1 .00 15. 21 o
ATOM 2031 o HOH E 54 -7. 158 -1.937 0. 588 1 .00 47. 41 o
ATOM 2032 o HOH E 55 -9. 121 -4.762 5. 641 1 .00 40. 65 o
ATOM 2033 0 HOH E 56 2. 424 -2.856 -13. 317 1 .00 41. 38 o
ATOM 2034 o HOH E 57 1. 055 -4.242 -1. 747 1 .00 18. 37 o
ATOM 2035 o HOH E 58 -18. 761 -8.012 -9. 655 1 .00 24. 12 o
ATOM 2036 o HOH E 59 -2. 388 16.808 -11. 815 1 .00 30. 70 o
ATOM 2037 o HOH E 60 4. 809 -1.264 2. 983 1 .00 24. 16 o
ATOM 2038 o HOH E 61 2. 989 -6.165 -20. 176 1 .00 26. 35 o
ATOM 2039 o HOH E 62 -6. 984 18.921 8. 976 1 .00 23. 40 o
ATOM 2040 o HOH E 63 -9. 517 16.268 8. 244 1 .00 30. 02 o
ATOM 2041 o HOH E 64 9. 555 9.360 2. 589 1 .00 26. 22 o
ATOM 2042 o HOH E 65 -1. 691 3.030 -17. 513 1 .00 24. 49 o
ATOM 2043 o HOH E 66 -10. 072 21.992 -1. 960 1 .00 38. 42 o
ATOM 2044 o HOH E 67 . -6. 907 -9.099 -35. 898 1 .00 23. 11 o
ATOM 2045 o HOH E 68 -14. 178 2.468 -37. 533 1 .00 53. 02 o
ATOM 2046 o HOH E 69 5. 207 -3.916 -10. 883 1 .00 26. 60 o
ATOM 2047 o HOH E 70 13. 522 11.929 6. 636 1 .00 33. 30 o
ATOM 2048 o HOH E 71 -1. 022 4.862 12. 732 1 .00 25. 60 o
ATOM 2049 o HOH E 72 -23. 941 -16.190 -25. 793 1 .00 37. 53 o ATOM 2050 o HOH E 73 -6/825 18.467 -0.891 1.00 15.61 0
ATOM 2051 o HOH E 74 7 .553 11 .718 9. 157 1 .00 18. 00 o
ATOM 2052 o HOH E 75 -18 .419 -18 .886 -21. 301 1 .00 36. 78 o
ATOM 2053 o HOH E 76 -7 .064 13 .205 -11. 657 1 .00 42. 26 o
ATOM . 2054 o HOH E 77 4 .447 19 .417 -6. 283 1 .00 39. 94 o
ATOM 2055 o HOH E 78 -8 .507 -17 .007 -12. 737 1 .00 30. 71 o
ATOM 2056 o HOH E 79 -2 .739 -1 .249 -13. 649 1 .00 20. 46 o
ATOM 2057 o HOH E 80 -12 .553 -4 .508 -7. 696 1 .00 17. 55 o
ATOM 2058 o HOH E 81 -10 .796 -14 .306 -12. 347 1 .00 43. 69 o TOM 2059 o HOH E 82 -9 .640 4 .368 -26. 648 1 .00 17. 84 o
ATOM 2060 o HOH E 83 -20 .434 5 .545 -34. 109 1 .00 42. 48 o
ATOM 2061 o HOH E 84 -4 .804 -16 .552 -16. 977 1 .00 30. 97 o
ATOM 2062 o HOH E 85 -0 .273 2 .946 -19. 744 1 .00 33. 63 o
ATOM 2063 o HOH E 86 -5 .492 23 .528 -4. 615 1 .00 23. 66 o
ATOM 2064 o HOH E 87' -9 .465 23 .932 -6. 243 1 .00 34. 72 o
ATOM 2065 o HOH E 88 -4 .847 3 .939 -11. 170 1 .00 23. 93 - o
ATOM 2066 o HOH E 89 -0 .142 4 .122 -23. 415 1 .00 26. 70 o
ATOM 2067 o HOH E 90 -22 .119 -20 .194 -30. 562 " 1 .00 21. 94 o
ATOM 2068 o HOH E 91 -12 .881' -11 .538 -38. 957 1 .00 16. 33 o
ATOM 2069 o HOH E 92 -4 .821 20 .453 10. 082 1 .00 22. 51 o
ATOM 2070 o ' HOH E 93 • 9 .835 13 .106 9. 687 1 .00 28. 41 o
ATOM 2071 o HOH E 94 -13 .525 1 .871 -16. 400. 1 .00 32. 10 o
ATOM 2072 o HOH E 95 -17 .932 -8 .195 -6. 796 1 .00 53. 47 o
ATOM 2073 o HOH E '96 -17 .851 -9 .897 -38. 446 1 .00 21. 96 o
ATOM 2074 o HOH E 97 -16 .405 6 .690 -13. 295 1 .00 36. 32 o
ATOM 2075 o HOH E 98 -6 .755 -3 .886 -15. 823 1 .00 19. 46 o
ATOM 2076 o HOH E 99 1 .430 -13 .282 -17. 163 1 .00 46. 19 o
ATOM 2077 o HOH E 100 -20 .566 -22 .137 -18. 737 1 .00 35. 16 o
ATOM 2078 o HOH E 101 -1 .239 -13 .751 -15.. 836 1 .00 26. 31 o
ATOM 2079 o HOH E 102 -7 .223 -22 .034 -19. 269 1 .00 '24. 27 o
ATOM 2080 o HOH E 103 -22 .174 -12 .207 -13. 267 1 .00 40. 25 o
ATOM 2081 o HOH E 104 6 .347 11 .518 11. 662 1 .00 32. 52 o
ATOM 2082 o HOH E 105 5 .774 0 .217 -0. 265 1 .00 33. 84 o
ATOM 2083 o HOH E 106 -22 .766 -18 .808 -28. 147 1 .00 26. 46 o
ATOM 2084 o HOH E 107 -2 .564 -11 .758 -14. 333 1 .00 19. 55 o
ATOM 2085 o HOH E 108 -0 .086 -2 .765 -13. 807 1 .00 26. 57 o
ATOM 2086 o HOH E 109 -0 .924 13 .933 -15. 311 1 .00 23. 06 o
ATOM 2087 o HOH E 110 -12 .080 13 .805 -9. 257 1 .00 27. 65 o
ATOM 2088 o HOH E 111 -18 .129 -25 .815 -20. 818 1 .00 32. 23 o
ATOM 2089 o HOH E 112 -16 .484 17 .321 -5. 274 1 .00 46. 57 o
ATOM 2090 o HOH E 113 -13 .419 -13 .769 -11. 912 1 .00 28. 91 o
ATOM 2091 o HOH E 114 -6 .280 -19 .616 -28. 973 1 .00 24. 0 0
ATOM 2092 o HOH E 115 -3 .602 1 .491 -15. 660 1 .00 20. 66 o
ATOM 2093 o HOH E 116 -6 .834 -11 .868 -41. 290 1 .00 40. 67 o
ATOM 2094 o HOH E 117 -26 .502 -4 .984 -30. 666 1 .00 39. 80 o
ATOM 2095 o HOH E 118 -25 .606 -12 .299 -29. 119 1 .00 45. 10 o
ATOM 2096 o HOH E 119 9 .978 6 .319 7. 718 1 .00 43. 04 o
ATOM 2097 o HOH E 120 -11 .735 -13 .007 -40. 925 1 .00 29. 30 o
ATOM 2098 o HOH E 121 0 .697 -3 .965 -17. 048 1 .00 30. 65 o
ATOM 2099 o HOH E 122 -21 .165 -7 .914 -35. 128 1 .00 32. 98 o
ATOM 2100 o HOH E 123 -10 .049 -23 .452 -30. 500 1 .00 32. 57 o
ATOM 2101 o HOH E 124 3 .954 5 .907 -15. 205 1 .00 21. 87 o
ATOM 2102 o HOH E 125 3 .451 -12 .228 -25. 589 1 .00 43. 61 o
ATOM 2103 o HOH E 126 -8 .785 19 .432 0. 179 1 .00 39. 89 o
ATOM 2104 o HOH E 127 3 .862 12 .986 12. 557 1 .00 24. 67 o
ATOM 2105 o HOH E 128 -17 .845 -12 .850 -38. 682 1 .00 24. 80 o
ATOM 2106 o HOH E 129 1 .614 13 .593 -16. 152 1 .00 36. 42 o
ATOM 2107 o HOH E 130 -4 .922 . -l .095 -12. 430 1 .00 30. 89 o
ATOM 2108 o HOH E 131 -10 .672 18 .728 -1. 841 1 .00 41. 69 o
ATOM 2109 o HOH E 132 5 .294 5 .701 12. 230 1 .00 38. 41 o TOM 2110 o HOH E 133 -9 .850 -0 .229 -34. 526 1 .00 38. 37 o ATOM 2111 o HOH E 134 9.726 9.926 10.441 1.00 32.78 0
ATOM 2112 o HOH E 135 -6. 106 -3. 723 -3. 223 1 .00 32. 43 0
ATOM 2113 o HOH E 136 1. 427 4. 133 -15. 807 1 .00 35. 19 0
ATOM 2114 o HOH E 137 9. 473 6. 655 12. 177 1 .00 41. 18 o
ATOM 2115 o HOH E 138 -9. 876 -21. 525 -19. 491 1 .00 32. 21 o
ATOM 2116 o HOH E 139 -28. 785 1. 756 -24. 707 1 .00 37. 01 o
ATOM 2117 o HOH E 140 -21. 065 5. 538 -28. 394 1 .00 43. 85 o
ATOM 2118 o HOH E 141 -5. 232 -12. 525 -15. 893 1 .00 39. 69 o
ATOM 2119 o HOH E 142 7. 619 4. 690 11. 385 1 .00 37. 17 o
ATOM 2120 o HOH E 143 2. 457 -11. 733 -20. 179 1 .00 50. 63 o
ATOM 2121 o HOH E 144 1. 937 -17. 297 -33. 471 1 .00 36. 75 o
ATOM 2122 o HOH E 145 -23. 131 -19. 075 -32. 878 1 .00 32. 29 o
ATOM 2123 o HOH E 146 -10. 216 0. 717 -12. 983 1 .00 47. 56 o
ATOM 2124 o HOH E .147 1. 998 -8. 127 -30. 378 1 .00 52. 52 o
ATOM 2125 o HOH E 148 -23. 045 3. 918 -33. 946 1 .00 36. 78 o
ATOM 2126 o HOH E 149 -9. 864 2. 474 -18. 734 1 .00 27. 41 o
ATOM 2127 o HOH E 150 -3. 849 19. 234 12. 367 1 .00 27. 85 o
ATOM 2128 o HOH E 151 -11. 580 22. 125 -4. 391 1 .00 33. 72 o
ATOM 2129 o HOH E 152 3. 653 2. 664 -14. 507 1 .00 30. 62 o
ATOM 2130 o HOH E 153 4. 612 12. 375 -14. 182 1 .00 39. 75 o
ATOM 2131 o HOH E 154 -9. 277 -4. 669 -8. 691 1 .00 23. 95 o
ATOM 2132 o HOH E 155 -12. 214 -13. 548 -8. 396 1 .00 27. 35 o TOM 2133 o HOH E 156 -25. 379 -20. 061 -27. 553 1 .00 61. 42 o TOM 2134 o HOH E 157 -9. 065 -1. 655 -6. 030 1 .00 34. 59 o
ATOM 2135 o HOH E 158 -2. 730 22. 304 -8. 399 1 .00 25. 88 o
ATOM 2136 o HOH E 159 -0. 867 25. 033 -0. 412 1 .00 25. 86 o
ATOM 2137 o HOH E 160 -6. 592 2. 980 -12. 883 1 .00 30. 55 o
ATOM 2138 o HOH E 161 -17. 181 -17. 836. -17. 310 1 .00 33. 29 o
ATOM 2139 o HOH E 162 -4. 309 21. 759 -6. 443 1 .00 34. 80 o
ATOM 2140 o HOH E 163 -6. 646 26. 837 5. 731 1 .00 38. 79 o
ATOM 2141 o HOH E 164 -8. 725 -2. 096 -8. 831 1 .00 32. 14 o
ATOM 2142 o HOH E 165 -10. 186 19. 946 1. 979 1 .00 41. 20 o
ATOM 2143 o HOH E 166 -18. 846 6. 182 5. 967 1 .00 45. 26 o TOM 2144 o HOH E 167 -17. 077 2. 537 -25. 038 1 .00 38. 70 o TOM 2145 o HOH E 168 -1. 612 7. 830 12. 380 1 .00 35. 28 o TOM 2146 o HOH E 169 -9. 277 25. 860 0. 520 1 .00 40. 19 o
ATOM 2147 o HOH E 170 -19. 722 4. 315 -24. 545 1 .00 40. 66 o
ATOM 2148 o HOH E 171 2. 895 -2. 496 11. 378 1 .00 41. 62 o TOM 2149 o HOH E 172 -25. 220 ' -1. 431 -36. 540 1 .00 42. 83 o
ATOM 2150 o HOH E 173 -17. 827 -3. 637 -11. 975 1 .00 39. 68 o
ATOM 2151 o HOH E 174 8. 552 3. 383 -7. 273 1 .00 38. 48 ■ o
ATOM 2152 ■ o HOH E 175 -18. 130 2. 561 -14. 246 1 .00 51. 97 o
ATOM 2153 o HOH E 176 0. 604 -3. 284 13. 375 1 .00 39. 35 o
ATOM 2154 o HOH E 177 -10. 092 14. 255 -10. 757 1 .00 40. 94 o
ATOM 2155 o HOH E 178 -15. 884 -13. 360 -6. 652 1 .00" 51. 66 o
ATOM 2156 o HOH E 179 -14. 005 13. 829 -12. 502 1 .00 30. 24 o TOM 2157 o HOH E 180 -25. 105 -5. 852 -33. 123 1 .00 52. 36 o
ATOM 2158 o HOH E 181 -10. 119 3. 862 14. 539 1 .00 40. 86 o
ATO I 2159 o HOH E 182 8. 355 14. 428 -11. 136 1 .00 46. 95 o
ATOM 1 2160 o HOH E 183 -4. 849 -2. 163 -7. 113 1 .00 27. 57 o
ATOM 2161 o HOH E 184 -28. 574 -6. 878 -17. 423 1 .00 40. 84 o
ATOM 2163 o HOH E 185 -20. 311 -5. 213 -9. 751 1 .00 39. 56 o
ATOM 2164 o HOH E 186 -11. 493 -0. 328 -8. 551 1 .00 35. 82 o
ATOM 2165 o HOH E 187 -21. 316 -27. 121 -23. 136 1 .00 57. 01 o
ATOM 2166 o HOH E 188 -15. 085 -16. 317 -11. 107 1 .00 45. 68 o
ATOM 2167 o HOH E 189 -25. 065 -11. 215 -16. 273 1 .00 41. 39 o
ATOM 2168 o HOH E 190 -15. 604 -11. 624 -39. 618 1 .00 32. 03 o
ATOM 2169 o HOH E 191 -23. 472 1. 686 -37. 307 1 .00 48. 61 o
ATOM 2170 o HOH E 192 -6. 450 -14. 917 -15. 892 1 .00 32. 39 o
ATOM 2171 o HOH E 193 -6. 653 -2. 560 -13. 542 1 .00 36. 66 o
ATOM 2172 o HOH E 194 1. 499 -2. 765 -19. 561 1 .00 33. 36 o ATOM 2173 0 HOH E 1 5 -6.103 1.364 -14.990 1.00 31.46 0
ATOM 2174 0 HOH E 196 -11. 373 19. 529 -4. ,511 1 .00 32 .15 0
ATOM 2175 o HOH E 197 -20. 656 -10. 233 -10. 372 1 .00 38 .78 0
ATOM 2176 o HOH E 198 -15. 104 1. 650 -22. 308 1 .00 52 .01 o
ATOM 2177 o HOH E 199 -25. 012 -13. 669 -18. 911 1 .00 45 .41 o
ATOM 2178 o HOH E 200 -5. 011 -9. 740 -37. 592 1 .00 47 .16 o
ATOM 2179 o HOH E 201 -16. 181 -19. 233 -15. 316 1 .00 44 .55 o
ATOM 2180 o HOH E 202 -17. 656 -0. 509 -15. 387 1 .00 45 .21 o
ATOM 2181 o HOH E 203 -7. 328 23. 160 '9. 243 1 .00 32 .06 o
ATOM 2182 o HOH E 204 -13. 182 1. 748 -5. 598 1 .00 56 .52 o
ATOM 2183 o HOH E 205 -23. 289 -22. 986 -28. 047 1 .00 29 .74 o
ATOM 2184 o HOH E 206 8. 081 2. 839 -9. 821 1 .00 36 .48 o
ATOM 2185 o HOH E 207 -18. 660 -3. 909 -14. 523 1 .00 29 .79 o
ATOM 2186 o HOH E 208 -8. 068 -3. 458 9. 508 1 .00 50 .40 o
ATOM 2187 o HOH E 209 11. 389 -1. 019 -29. 514 1 .00 42 .43 o
ATOM 2188 o HOH E 210 -6. 032 -12. 983 -11. 746 1 .00 41 .56 o
ATOM 2189 o HOH E 211 -13. 834 -5. 059 0. 958 1 .00 37 .04 o
ATOM 2190 o HOH E 212 . -23. 088 -5. 545 -31. 465 1 .00 41 .70 o
ATOM 2191 o HOH E 213 -12. 826 11. 703 12. 837 1 .00 50 .79 o
ATOM 2192 o HOH E 214 { 9. 021 4. 091 9. 106 1 .00 62 .30 o
ATOM 2193 o HOH E 215 -13. '511 3. 865 -20. 435 1 .00 33 .41 o
ATOM 2194 o HOH E 216 -26. 071 -12. 132 -23. 326 1 .00 38 .57 o
ATOM 2195 o HOH E 217 -12. 717 12. 260 7. 620 1 .00 45 .46 o
ATOM 2196 o HOH E 218 6. 287 2. 358 2. 262 1 .00 47 .77 o
ATOM 2197 o HOH E 219 -5. 023 -13. 767 -42. 743 1 .00 55 .79 o
ATOM 2198 o HOH E 220 -6. 148 -4. 993 7. 933 1 .00 33 .52 o
ATOM 2199 o HOH E 221 7. 914 -6. 810 -23. 708 1 .00 41 .38 o
ATOM 2200 o HOH E 222 ' -3. 350 -2. 875 -31. 283 1 .00 53 .21 o
ATOM 2201 o. HOH E 223 -10. 270 -20. 435 -16. 344 1 .00 32 .88 o
ATOM 2202 o HOH E 224 -9. 878 20. 921 11. 090 1 .00 47 .59 o
ATOM 2203 o HOH E 225 -21. 005 3. 245 -0. 603 1 .00 49 .98 o
ATOM 2204 o HOH E 226 -20. 042 -26. 791 -26. 406 1 .00 41 .76 o
ATOM 2205 o HOH E 227 -22. 855 5· 155 -24. 592 1 .00 50 .20 o
ATOM 2206 o HOH E 228 5. 658 -3. 184 •9. 118 1 .00 56 .35 o TOM 2207 o HOH E 229 -3. 873 25. 259 -0. 282 1 .00 25 .56 o
ATOM 2208 o HOH E 230 -11·. 351 -22. 344 -15. 183 1 .00 45 .45 o
ATOM 2209 o HOH E 231 0. 699 16. 760 -16. 740 1 .00 44 .93 o
ATOM 2210 o HOH E 232 11. 654 19. 136 -1. 534 1 .00 36 .04 o
ATOM 2211 o■ HOH E 233 -34. 462 5. 711 -22. 5,16 1 .00 46 .97 o
ATOM 2212 o HOH E 234 -2. 670 20. 743 14. 231 1 .00 35 .43 o
ATOM 2214 o HOH E 235 -8. 534 -5. 746 -6. 411 1 .00 28 .01 o
ATOM 2215 o HOH E 236 8. 352 1. 828 11. 119 1 .00 34 .09 o
ATOM 2216 o HOH E 237 2. 184 15. 335 -19. 338 1 .00 52 .00 o
ATOM 2217 o HOH E 238 -14. 648 1. 705 -7. 840 1 .00 43 .88 o
ATOM 2218 o HOH E 239 -21. 144 3. 448 2. 128 1 .00 52 .50 o
ATOM 2219 o HOH E 240 -11. 602 2. 443 -36. 867 1 .00 39 .21 o
ATOM 2220 o HOH E 241 1. 455 19. 674 4. 009 1 .00 30 .27 o
ATOM 2221 o HOH E 242 -11. 213 2. 037 -27. 966 1 .00 28 .39 o
ATOM 2222 o HOH E 243 1. 187 22. 080 -4. 319 1 .00 33 .37 o
ATOM 2223 o HOH E 244 -13. 314 -8. 935 -4. 776 1 .00 32 .84 o
ATOM 2224 o HOH E 245 -11. 995 4. 875 -14. 291 1 .00 22 .79 o
ATOM 2225 o HOH E 246 -14. 527 -20. 240 -28. 743 1 .00 17 .23 o
ATOM 2226 o HOH E 247 -6. 884 20. 918 -8. 198 1 .00 28 .32 o
ATOM 2227 o HOH E 248 -11. 937 -23. 337 -19, 801 1 .00 26 .28 o
ATOM 2228 o HOH E 249 -9. 317 -22. 287 -26. 967 1 .00 44 .07 o
ATOM 2229 o HOH E 250 -11. 323 4. 665 -21. 647 1 .00 32 .31 o
ATOM 2230 0 HOH E 251 -16. 752 -22. 346 -18. 137 1 .00 44 .95 o
ATOM 2231 o HOH E 252 -17. 649 -7. 850 -40. 217 1 .00 38 .75' o
ATOM 2232 o HOH E 253 -7. 080 -4. 008 3. 600 1 .00 33 .82 o
ATOM 2233 o HOH E 254 -10. 462 1. 487 -32. 600 1 .00 25 .71 o
ATOM 2234 o HOH E 255 -15. 330 -8. 715 -6. 899 1 .00 34 .12 o ATOM 2235 o HOH E 256 -5.052 -4.992 -35.21 1.00 41.16 o
ATOM 2236 o HOH E 257 -3 .800 12. 715 -12. 371 1 .00 34.20 o
ATOM 2237 o HOH E 258 -18 .450 5. 776 -5. 668 1 .00 37.70 o
"ATOM 2238 o HOH E 259 -7 .381 -0. 076 -30. 955 1 .00 37.56 o
ATOM 2239 o HOH E 260 -4 .365 1. 286 -11. 397 1 .00 44.64 o
ATOM 2240 o HOH E 261 -4 .651 0. 318 -1. 489 1 .00 43.09 o
ATO 2241 o HOH E 262 -19 .432 -1. 497 -13. 852 1 .00 55.91 o
ATOM 2242. o HOH E 263 -7 .693 4. 649 13. 136 1 .00 31.52 o
ATOM 2243 o HOH E 264 . -7 .839 -21. 949 -29. 057 1 .00 51.30 o
ATOM 2244 o HOH E 265. -31 .166 -1. 579 -20. 675 1 .00 55.00 o TOM 2245 o HOH E 266 -25 .692 1. 899 -24. 034 1 .00 39.20 o
ATOM 2246 o HOH E 267 -5 .026 -2. 261 2. 838 1 .00 27.34 o
ATOM 2247 o HOH E 268 -6 .341 1. 945 -29. 466 1 .00 38.43 o
ATOM 2248 o HOH E 269 14 .703 16. 412 0. 953 1 .00 46.71 o
ATOM 2249 o HOH E 270 7 .284 2. 587 4. 685 1 .00 47.25 o
ATOM 2250 o HOH E 271 5 .678 14. 535 -11. 931 1 .00 49.48 o
ATOM 2251 o HOH E 272 -13 .765 -0. 357 -22. 884 1 .00 34.33 o
ATOM 2252 o HOH E 273 -23 .390 5. 882 -15. 485 1 .00 47.65 0
ATOM 2253 :0 HOH E 274 -21 .205 -6. 438 -33. 135 1 .00 49.18 o
ATOM 2254 o HOH E 275 -30 .243 8. 685 -19. 421 1 .00 53.12 o
ATOM 2255 o HOH E 276 -9 .955 13. 017 9. 400 1 .00 35.90 o
ATOM 2256 o HOH E 277 2 .471 22. 332 -1. 499 1 .00 42.38 o
ATOM 2257 o HOH E 278 -13 .339 -2. 122 -7. 356 1 .00 42.37 o
ATOM 2258 o HOH E 279 -10 .390 -0. 309 -10. 771 1 .00 40.06 o
ATOM 2259 o HOH E 280 -6 .881 18. 860 -11. 849 1 .00 53.98 o
ATOM 2260 o HOH E 281 -17 .705 -14. 647 -10. 725 1 .00 33.45 o
ATOM 2261 o HOH E 282 -35 .035 -9. 407 -26. 046 1 .00 59.07 o
ATOM 2262 0 HOH E 283 -15 .704 0. 063 -37. 583 1 .00 32.85 o
ATOM 2263 o HOH E 284 -13 .278 -19. 487 -30. 916 1 .00 26.45 o
ATOM 2264 o HOH E 285 -11 .550 4. 865 -18. 617 1 .00 29.78 o
ATOM 2265 o HOH E 286 -14 .074 -25. 157 -24. 018 1 .00 26.17 o
ATOM 2266 o HOH E 287 -1 .931 16. 330 -14. 927 1 .00 37.37 o
ATOM 2267 o HOH E 288 -13 .819 -6. 723 -6. 098 1 .00 37.27 o
ATOM 2268 o HOH E 289 -11 .115 -20. 702 -30. 282 1 .00 32.10 o
ATOM 2269 o HOH E 290 -17 .657 1. 384 -19. 408 1 .00 42.85 o
ATOM 2270 o HOH E 291 2 .485 21. 098 1. 976 1 .00 30.08 o
ATOM 2271 o HOH E 292 -23 .215 -20. 998 -26. 084 ■ 1 .00 33.91 o
ATOM 2272 o HOH E 293 4 .712' -9. 099 -21. 642 1 .00 46.40 o
ATOM 2273 o HOH E 294 -9 .630 -1. 101 Γ -0. 501 1 .00 38.76 o
ATOM 2274 o HOH E 295 -26 .206 -20. 060 -23. 679 1 .00 40.52 o
ATOM 2275 o HOH E 296 -17 .510 -20. 003 -18. 597 1 .00 48.53 o
ATOM 2276 o HOH E 297 6 .656 -2. 928· -13. 196 1 .00 48.42 o
ATOM 2277 o HOH E 298 -23 .296 -8. 102 -33. 976 1 .00 47.01 o
ATOM 2278 o HOH E 299 5 .352 1. 628 5. 833 1 .00 43.42 o
ATOM 2279 o HOH E 300 -26 .314 2. 952 -26. 308 1 .00 34.07 o
ATOM 2280 o HOH E 301 -0 .680 -3. 417 -28. 334 1 .00 50.49 o
ATOM 2281 o HOH E 302 -12 .289 -2. 226 8. 151 1 .00 45.99 o
ATOM 2282 o HOH E 303 -27 .948 8. 745 -18. 050 1 .00' 58.40 o
ATOM 2283 o HOH E 304 -25 .317 -12. 598 -26. 200 1 .00 42.65 o
ATOM 2284. o HOH E 305 -17 .557 12. 190 -1. 880 1 .00 34.94 o
ATOM 2285 o HOH E 306 -16 .461 -8. 470 3. 655 1 .00 63.59 o
ATOM 2286 o HOH E 307 -25 .985 -15. 326 -20. 825 1 .00 51.90 o
ATOM 2287 o HOH E 308 -6 .064 -2. 546 -9. 583 1 .00 24.58 o
ATOM 2288 o HOH E 309 -8 .714 2. 760 -16. 255 1 .00 25.20 o
ATOM 2289 o HOH E 310 -13 .021 5. 911 -16. 711 1 .00 28.68 o
ATOM 2290 o HOH E 311 -6 .362 -4. 740 -5. 686 1 .00 31.41 o
ATOM 2291 o HOH E 312 7 .138 19. 284 -4. 617 1 .00 35.62 o
ATOM 2292 o. HOH E 313 9 .474 18. 234 -3. 728 1 .00 29.06 o
ATOM 2293 o HOH E 314 -1 .820 -15. 871 -38. 727 1 .00 48.15 o
ATOM 2294 o HOH E 315 -18 .612 -17. 282 -11. 308 1 .00 49.07 o
ATOM 2295 o HOH E 316 -17 .693 -4'. 761 -5. 957 1 .00 52.94, o ATOM 2296 0 HOH E 317 5.830 -0.572 -3.142 1.00 45.66 0
ATOM 2297 o HOH E 318 -0 .790 -4 .926 11. 365 1 .00 42. 27 0
ATOM 2298 o HOH E 319 4 .801 . -1 .725 -14. 572 1 .00 50. 32 o
ATOM 2299 o HOH E 320 -10 .570 13 .166 12. 168 1 .00 50. 99 o TOM 2300 o HOH E 321 11 .122 4 .228 -6. 685 1 .00 46. 96 o
ATOM 2301 o HOH E 322 -15 .581 -2 .550 -5. 440 1 .00 45. 61 o
ATOM 2302 o HOH: E 323 1 .840 22 .169 -6. 755 1 .00 31. 22 o
ATOM 2303 o HOH E 324 -23 .761 -20 .617 -34. 866 1 .00 30. 03 o
ATOM 2304 o HOH E 325 -14 .373 7 .783 -17. 777 1 .00 14. 86 o
ATOM 2305 o HOH E 326 13 .953 17 .283 -5. 040 1 .00 27. 93 o
ATOM 2306 o HOH E 327 -14 .053 4 .309 -3. 703 1 .00 28. 55 o
ATOM 2307 o HOH E 328 -16 .758 7 .762 -16. 225 1 .00 35. 57 o
ATOM 2308 o HOH E 329 7 .234 18 .589 -8. 903 1 .00 39. 60 o
ATOM 2309 o HOH E 330 -11 .254 -7 .472 -3. 694 1 .00 30. 58 o
ATOM 2310 o HOH E 331 10 .687 0 .027 11. 600 1 .00 37. 96 o
ATOM 2311 o HOH E 332 -15 .550 4 .278 -38. 917 1 .00 48. 17 o
TER 2314 HOH E 332
END
APPENDIX II
CRYSTl 42. ,170 53, .824 57. 446 90.00 109.35 90.00 P 1. 21 1
SCALE1 0.023714 0.000000 0.008329 0.00000
SCALE2 0.0,00000 0.018579 0.000000 0.00000
SCALE3 0.000000 0.000000 0.018450 0.00000
ATOM 1 o SER A 111 -17.288 -3 .124 10.455 1.00 61. 64 0
ATOM •2 N SER A 111 -19.432 -0 .525 11.490' 1.00 68. 71 N
ATOM 3 CA SER A 111 -19.061 -1 .936 11.544 1.00 68. 78 C TOM 4 C SER A 111 -18.252 -2 .371 10.320 1.00 59. 72 c
ATOM 5 CB SER A 111 -20.301 -2 .819 11.718 1.00 76. 12 c
ATOM 6 OG SER A 111 -19.939 -4 .155 12.025 1.00 78. 16 o
ATOM 7 o PRO A 112 -15.448 -2 .005 7.665 1.00 33. 81 o
ATOM 8 N PRO A 112 -18.647 -1 .914 9.118 1.00 51. 08 N
ATOM 9 CA PRO A 112 -17.823 -2 .172 7.932 1.00 44. 85 c
ATOM 10 C PRO A 112 -16.482 -1 .446 8.029 1.00 35. 83 C
ATOM 11 CB PRO A 112 -18.656 -1 .579 6.789 1.00 46. 14 c
ATOM 12 CG PRO A 112 -20.050 -1 .569 7.297 1.00 49. 25 c
ATOM 13 CD PRO A 112 -19.931 -1 .285 8.763 1.00 54. 73 c
ATOM 14 N CYS A 113 -16.512 -0 .212 8.521 1.00 34. 90 N
ATOM 15 CA CYS A 113 -15.302 0 .588 8.687 1.00 38. 20 C
ATOM 16 C CYS A 113 -15.063 0 .932 10.153 1.00 40. 83 C
ATOM 17 o CYS A 113 -15.998 0 .950 10.950 1.00 41. 15 o
ATOM 18 CB CYS A 113 -15.394 1 .876 7.865 1.00 39. 53 c
ATOM 19 SG CYS A 113 -15.396 1 .625 6.072 1.00 42. 14 S
ATOM 20 N PRO A 114 -13.801 1 .204 10.513 1.00 38. 90 N
ATOM 21 CA PRO A 114 -13.459 1 .642 11.870 1.00 36. 60 c
ATOM 22 C PRO A 114 · -14.060 3 .005 12.193 1.00 41. 47 C
ATOM 23 o PRO A 114 -14.465 3 .730 11.285 1.00 36. 92 o
ATOM 24 CB PRO A 114 -11.932 1 .768 11.821 1.00 28. 77 ' C
ATOM 25 CG PRO A 114 -11.506 0 .924 10.682 1.00 30. 80 c
ATOM 26 CD PRO A 114 -12.604 1 .026 9.674 1.00 36. 78 c
ATOM 27 N ASN A 115 -14.110 3 .349 13.476 1.00 39. 29 N
ATOM 28 CA ASN A 115 -14.482 4 .696 13.884 1.00 40. 69 C
ATOM 29 . C ASN A 115 -13.490 5 .713 13.340 1.00 36. 96 C
ATOM ' 30 o ASN A 115 -12.284 5 .469 13.344 1.00 34. 78 o
ATOM 31 CB ASN A 115 -14.540 4 .798 15.413 1.00 50. 66 c
ATOM 32 CG ASN A 115 -15.960 4 .778 15.950 1.00 59. 51 c
ATOM 33 ODl ASN A 115 -16.917 4 .544 15.208 1.00 63. 21 o
ATOM 34 ND2 ASN A 115 -16.103 5 .019 17.248 1.00 62. 55 N
ATOM 35 N . ASN A 116 -14.001 6 .851 12.875 1.00 35^ 33 N
ATOM 36 CA ASN A 116 -13.165 7 .922 12.345 1.00 33. 49 C
ATOM 37 C ASN A 116 -12.830 7 .706 10.869 1.00 30. 07 C
ATOM 38 o ASN A 116 -12.246 8 .579 10.224 1.00 31. 62 o
ATOM 39 CB ASN A 116 -11.884 ' 8 .070 13.178 1.00 39. 49 c
ATOM 40 CG ASN A 116 -11.147 9 .369 12.904 1.00 51. 57 c
ATOM 41 ODl ASN A 116 -11.139 9 .873 11.781 1.00 54. 77 o
ATOM 42 ND2 ASN A 116 -10.517 9 .919 13.941 1.00 53. 29 N
ATOM 43 N TRP A 117 -13.212 6 .547 10.335 1.00 26. 80 N
ATOM 44 CA TRP A 117 -12.942 6 .227 8.933 1.00 23. 63 C
ATOM 45 C TRP A 117 -14.207 6 .426 8.110 1.00 23. 64 c
ATOM 46 o TRP A 117 -15.314 6 .327 8.632 1.00 26. 81 o
ATOM 47 CB TRP A 117 -12.462 4 .779 8.790 i . oo 19. 21 c
ATOM 48 CG TRP A 117 -11.107 4 .523 9.367 1.00 20. 69 c
ATOM 49 CD1 TRP A 117 -10.711 4 .746 10.656 1.00 23. 29 c
ATOM 50 CD2 TRP A 117 -9.967 3 .974 8.685 1.00 15. 11 c
ATOM 51 NE1 TRP A 117 -9.389 4 .385 10.817 1.00 21. ,27 N ATOM 52 CE2 TRP A 117 -8.913 3.902 9.625 1.00 17.68 C
ATOM 53 CE3 TRP A 117 . ' -9.734 3. 545 7.374 1 .00 16. 74 C
ATOM 54 CZ2 TRP A 117 -7.647 3. 421 9.295 1 .00 18. 68 C
ATOM 55 CZ3 TRP A 117 -8.472 3. 067 7.044 1 .00 19. 29 C
ATOM 56 CH2 TRP A 117 -7.445 3. 005 8.007 1 .00 17. 86 C
ATOM 57 N ILE A 118 -14.043 6. 703 6.824 1 .00 18. 49 N
ATOM 58 CA ILE A 118 -15.191 6. 895 5.942 1 .00 19. 57 C
ATOM 59 C ILE A 118 -15.277 5. 763 4.913 1 .00 23. 96 C
ATOM 60 0 ILE A 1Ϊ8 -14.259 5. 342 4.363 1 .00 20. 09 o
ATOM 61 CB ILE A 118 -15.122 8. 265 5.234 1 .00 23. 51 c
ATOM 62 CGI ILE A 118 -13.878 8. 360 4.355 1 .00 26. 48 c
ATOM 63 CG2 ILE A 118 -15.093 9. 386 6.252 1 .00 29. 28 c
ATOM 64 CD1 ILE A 118 -13.818 9. 642 3.547 1 .00 36. 82 c
ATOM 65 N GLN A 119 -16.483 5. 263 4.655 1 .00 23. 47 N
ATOM 66 CA GLN A 119 -16.641 4. 182 3.679 1 .00 19. 30 c
ATOM 67 C GLN A 119 ' -17.063 4. 651 2.285 1 .00 27. 17 c
ATOM 68 0 GLN A 119 -17.817 5. 615 2.121 1 .00 21. 74 0
ATOM 69 CB GLN A 119 -17.640 3. 124 4.174 1 .00 22. 05 c
ATOM 70 CG GLN A 119 -17.846 1. 966 3.190 1 .00 26. 74 c
ATOM 71 CD GLN A 119 -18.907 0. 972. 3.644 1 .00 34. 07 c
ATOM 72 OEl GLN A 119 -19.603 1. 193 4.635 1 .00 36. 63 o
ATOM 73 NE2 GLN A 119 -19.031 -0. 134 2.915 1 .00 37. 84 N
ATOM 74 N ASN A 120 -16.556 3. 951 1.280 1 .00 24. 13 N
ATOM 75 CA ASN A 120 -17.098 4. 036 -0.063 1 .00 24. 87 c
ATOM 76 C ASN A 120 -17.034 2. 655 -0.692 1 .00 26. 36 c
ATOM 77 0 ASN A 120 -15.956 2. 152 -1.009 1 .00 24. 29 o
ATOM 78 CB ASN A 120 -16.366 5. 083 -0.914 1 .00 27. 46 C
ATOM 79 CG ASN A 120 -17.036 5. 304 -2.267 1 .00 38. 36 C
ATOM 80 ODl ASN A 120 -18.059 : 5. 984 -2.370 1 .00 42. 56 o
ATOM 81 ND2 ASN A 120 -16.464 4. 721 -3.306 1 .00 35. 28 N
ATOM 82 N ARG A 121 -18.202 2. 035 -0.833 1 .00 31. 08 N TOM 83 CA ARG A 121 -18.309 0. 684 -1.373 1 .00 30. 19 C
ATOM 84 C ARG A 121 -17.433 -0. 307 -0.597 1 .00 29. 82 C
ATOM 85 o ARG A 121 -17.659 -0. 536 0.591 1 .00 37. 94 o
ATOM ^86 CB ARG A 121 -17.988 0. 675 -2.870 1 .00 29. 78 C
ATOM 87 CG ARG A 121 -18.707 1. 778 -3.644 1 .00 31. 69 C
ATOM 88 CD ARG A 121 -18.743 1. 488 -5.136 1 .00 31. 36 C
ATOM 89 NE ARG A 121 -19.329 0. 179 -5.384 1 .00 33. 01 N
ATOM 90 CZ ARG A 121 -18.898 -0. 666 -6.311 1 .00 35. 36 C
ATOM 91· NH1 ARG A 121 -17.881 -0. 328 -7.091 1 .00 41. 19 N
ATOM 92 NH2 ARG A 121 -19.488 -1. 843 -6.458 1 .00 41. 25 N
ATOM 93 N GLU A 122 -16.430 -0. 882 -1.252 1 .00 25. 87 N
ATOM 94 CA GLU A 122 -15.628 -1. 923 -0.607 1 .00 31. 35 C
ATOM 95 C GLU A 122 -14.455 -1. 374 0.207 1 .00 26. 29 C
ATOM 96 o GLU A 122 -13.727 -2. 141 0.836 1 .00 28. 86 o
ATOM 97 CB GLU A 122 -15.112 -2. 939 -1.638 1 .00 34. 59 C
ATOM 98 CG GLU A 122 -16.174 -3. 864 -2.206 1 .00 54. 47 C
ATOM 99 CD GLU A 122 -15.665 -4. 676 -3.384 1 .00 73. 23 C
ATOM 100 OEl GLU A 122 -14.543 -4. 399 -3.861 1 .00 77. 66 o
ATOM 101 OE2 GLU A 122 -16.385 -5. 592 -3.835 1 .00 80. 44 o
ATOM 102 N SER A 123 -14.274 -0. 056 0.196 1 .00 22. 21
ATOM 103 CA SER A 123 -13.106 0. 551 0.830 1 .00 19. 16 C
ATOM 104 C SER A 123 -13.455 1. 404 2.052 1 .00 22. 12 C
ATOM . 105 0 SER A 123 -14.549 1. 969 2.136 1 .00 23. 53 o
ATOM 106 CB SER A 123 -12.341 1. 412 -0.177 1 .00 22. 28 c
ATOM 107 OG SER A 123 -11.797 0. 628 -1.221 1 .00 27. 64 ' o
ATOM 108. N CYS A 124 -12.509 1. 492 2.990 1 .00 18. 93 N ATOM • 109 CA CYS A 124 -12.588 2.427 4.117 1.00 18.34 C
ATOM 110 C CYS A 124 -11. 385 3. 349 4. 041 1 .00 19 .83 C
ATOM 111 o CYS A 124 -10. 277 2. 894 3. 754 1 .00 20 .58 o
ATOM 112 CB CYS A 124 -12. 509 1. 702 5. 459 1 .00 19 .40 c
ATOM 113 SG CYS A 124 -13. 772 0. 456 5. 712 1 .00 30 .28 S
ATOM 114 TYR A 125 -11. 595 4. 629 4. 326 1 .00 15 .49 N
ATOM 115 CA TYR A 125 -10. 515 5. 607 4. 232 1 .00 15 .24 C
ATOM 116 C TYR A 125 -10. 311 6. 386 5. 523 1 .00 -13 .75 C
ATOM 117 o TYR A 125 -11. 258 6. 681 6. 251 1 .00 15 .04 o
ATOM 118 CB TYR A 125 -10. 791 6. 604 3. 114 1 .00 11 .86 C
ATOM 119 CG TYR A 125 -11. 098 5. 966 1. 781 1 .00 13 .11 c
ATOM 120 CDl TYR A 125 -12. 410 5. 751 1. 383 1 .00 20 .20 c
ATOM 121 CD2 TYR A 125 -10. 082 5. 582 0. 922 1 .00 14 .53 c
ATOM 122 CEl TYR A 125 -12. 702 . 5. 171 0. 163 1 .00 18 .26 c
ATOM 123 CE2 TYR A 125 -10. 370 4. 992 -0. 306 1 .00 14 .14 c
ATOM 124 CZ TYR A 125 -11. 683 4. 801 -0. 677 1 .00 17 .00 c
ATOM 125 OH TYR A 125 -11. 997 4. 221 -1. 886 1. .00 17 .10 o
ATOM 126 N TYR A 126 -9. 065 6. 748 5. 784 1 .00 11 .80 N
ATOM 127 CA TYR A 126 -8. 770 7. 614 6. 912 1 .00 11 .21 C
ATOM 128 C TYR A 126 -7. 983 8. 806 6. 435 1 .00 13 .21 C
ATOM 129 o TYR A 126 •-6. 939 8. 654 5. 795 1 .00 13 .54 o
ATOM 130 CB TYR A 126 -7. 973 6. 862 7. 975. 1 .00 10 .72 C
ATOM 131 CG TYR A 126 -7. 533 7. 737 9. 117 1 .00 12 .99 c
ATOM 132 CDl TYR A 126 -8. 397 8. 016 10. 166 1 .00 18 .84 c
ATOM 133 CD2 TYR A 126 -6. 257 8. 278 9. 151 1 .00 15 .43 c
ATOM 134 CEl TYR A 126 -8. 000 8. 806 11. 224 1 .00 22 .28 c
ATOM 135 CE2 TYR A 126 -5. 847 9. 078 10. 219 1 .00 14 .55 c
ATOM 136 CZ TYR A 126 -6. 726 9. 331 11. 248 1 .00 21 .14 c
ATOM 137 OH TYR A 126 -6, 342 10. 121 12. 316 1 .00 25 .27 o
ATOM 138 N VAL A 127 -8. 472 10. 001 6. 750 1 .00 16 .98 N
ATOM 139 CA VAL A 127 -7. 765 11. 209 6. 365 1 .00 14 .27 c
ATOM 140 C VAL A 127 -7. 037 11. 788 7. 568 1 .00 12 .23 c
ATOM 141 o VAL A 127 -7. 669 12. 148 8. 566 1 .00 15 .90 o
ATOM 142 CB VAL A 127 -8. 734 12. 269 5. 797 1 .00 13 .41 c
ATOM 143 CGI VAL A 127 -7. 950 13. 507 5. 340 1 .00 16 .39 c
ATOM 144 CG2 VAL A 127 -9. 556 11. 685 4. 645 1 .00 15 .00 c
ATOM 145 N SER A 128 -5. 711 11. 887 7. 482 1 .00 8 .57 N
ATOM 146 CA SER A 128 -4. 916 12. 309 8. 634 1 .00 8 .10 C
ATOM 147 C SER A 128 -5. 067 13. 777 8. 934 1 .00 17 .50 c
ATOM 148 o SER A 128 -5. 435 14. 580 8. 068 1 .00 14 .78 o
ATOM 149 CB SER A 128 -3. 438 11. 981 8. 419 1 .00 11 .31 c
ATOM 150 OG SER A 128 -2. 789 13. 028 7. 707 1 .00 10 .20 o
ATOM 151 N GLU A 129 -4. 767 14. 109 10. 184 1 .00 15 .40 N
ATOM 152 CA GLU A 129 -4. 697 15. 480 10. 650 1 .00 14 .71. C
ATOM 153 C GLU A 129 -3. 286 15. 775 11. 138 1 .00 12 .06 c
ATOM 154 o GLU A 129 -3. 044 16. 814 11. 743 1 .00 16 .79 o
ATOM 155' CB GLU A 129 -5. 696 15. 703 11. 787 1 .00 18 .15 c
ATOM 156 CG GLU A 129 -7. 142 15. 442 11. 390 1 .00 33 .28' c
ATOM 157 CD GLU A 129 -8. 081 15. 394 12. 58.5 1 .00 51 .34 c
ATOM 158 OEl GLU A 129 -7. 967 14. 451 13. 397 1 .00 56 .81 o
ATOM 159 OE2 GLU A 129 -8. 933 16. 300 12. 713 1 .00 52 .40 o
ATOM 160 N ILE A 130 -2. 356 14. 866 10. 871 1 .00 12 .56 N
ATOM 161 CA ILE A 130 -0. 943 15. 092 11. 155 1 .00 11 .32 C
ATOM 162 C ILE A 130 -0. 126 14. 957 9. 878 1 .00 11 .25 c
ATOM 163 0 ILE A 130 -0. 574 14. 345 8. 892 1 .00 16 .50 o
ATOM 164 CB ILE A 130 -0. 388 14. 070 12. 172 1 .00 12 .06 c
ATOM 165 CGI ILE A 130 -0. 592 12. 643 11. 646 1 .00 17 .95 c ATOM 166 CG2 ILE A 130 -1.051 14.251 13.535 1.00 16.29 C
ATOM 167 CD1 ILE A 130 0. 136 11. 596 12 .447 1. 00 22 .76 C
ATOM 168 N TRP A 131 1. 079 15. 515 9 .899 1. 00 9 .89 N
ATOM 169 CA TRP A 131 1. 997 15. 411 8 .775 1. 00 9 .58 C
ATOM 170 ■C TRP A 131 2. 988 14. 323 9 .050 1. 00 14 .30 C
ATOM 171 0 TRP A 131 3. 326 14. 050 10 .203 1. 00 15 .66 o
ATOM 172 CB TRP A 131 2. 832 16. 682 8 .603 1. 00 13 .78 c
ATOM 173 CG TRP A 131 2. 125 17. 937 8 .207 1. 00 9 .71 c
ATOM 174 CD1 TRP A 131 0. 893 18. 075 7 .636 1. 00 12 .40 c TOM 175 CD2 TRP A 131 2. 635 19. 253 8 .393 1. 00 9 .68 c
^ ATOM 176 NEl TRP A 131 0. 602 19. 408 7 .449 1. 00 13 .58 N
ATOM 177 CE2 TRP A 131 1. 662 20. 154 7 .903 1. 00 11 .67 c
ATOM 178 CE3 TRP A 131 3. 827 19. 759 8 .926 1. 00 13 .62 c
ATOM 179 CZ2. TRP A 131 1. 843 21. 535 7 .935 1. 00 16 .41 c
ATOM 180 CZ3 TRP A 131 4. 012 21. 130 8 .950 1. 00 13 .53 c
ATOM 181 CH2 TRP A 131 3. 024 22. 005 8 .449 1. 00 13 .26 c
ATOM 182 N SER A 132 3. 485 13. 728 7 .976 1. 00 10 .68 N
ATOM 183 CA SER A 132 4. 650 12. 859 8 .038 1. 00 9 .25 C
ATOM 184 C SER A 132 5. 250 12. 768 6 .641 1. 00 14 .99 C
ATOM 185 0 SER A 132 4. 641 13. 223 5 .665 1. 00 10 .93 o
ATOM 186 CB SER A 132 4. 309 11. 480 8 .601 1. 00 16 .24 C
ATOM 187 OG SER A 132 3. 034 11. 030 8 .187 1. 00 20 .49 o
ATOM 188 N ILE A 133 6. 461 12. 240 6 .548 1. 00 9 .85 N
ATOM 189 CA ILE A 133 7. 042 11. 976 5 .243 1. 00 7 .25 C
ATOM 190 C ILE A 133 6. 349 10. 757 4 .651 1. 00 9 .80 C
ATOM 191 o ILE A 133 5. 549 10. 087 5 .330 1. 00 12 .55 o
ATOM 192 CB ILE A 133 8. 565 11. 750 5 .344 1. 00 12 .07 C
- ATOM 193 CGI ILE A 133 8. 841 10. 495 6 .178 1. 00 13 .82 C
ATOM 194 CG2 ILE A 133 9. 244 12. 991 5 .946 1. 00 18 .49 C
ATOM 195 CD1 ILE A 133 10. 298 10. 098 6 .228 1. 00 18 .73 C
ATOM 196 N TRP A 134 6. 645 10. 454 3 .397 1. 00 11 .00 N
ATOM 197 CA TRP A 134 5. 852 . 9. 455 2 .676 1. 00 10 .42 C
ATOM 198 C TRP A 134 6. 012 8. 079 3 .314 1. 00 12 .15 C
ATOM 199 o TRP A 134 5. 024 7. 399 3 .606 1. 00 15 .00 o
ATOM 200 CB TRP A 134 6. 250 9. 425 1 .207 1. 00 10 .48 C
ATOM 201 CG TRP A 134 5. 414 8. 517 0 .356 1. 00 9 .32 C
ATOM 202 CD1 TRP A 134 4. 261 8. 835 -0 .274 1. 00 9 .91 C
ATOM 203 CD2 TRP A 134 5. 719 7. 178 -o .009 1. 00 13 .13 C
ATOM 204 NEl TRP A 134 3. 801 7. 765 -0 .999 1. 00 10 .99 N
• ATOM 205 CE2 TRP A 134 4. 678 6. 728 -0 .843 1. 00 13 .66 C
ATOM 206 CE3 TRP A 134 6. 750 6. 302 0 .316 1. 00 11 .30 C
ATOM 207 CZ2 TRP A 134 4. 652 5. 450 -1 .381 1'. 00 14 .85 C
ATOM 208 CZ3 TRP A 134 6. 727 5. 033 -0 .213 1. 00 22 .69 C
ATOM 209 CH2 TRP A 134 5. 685 4. 615 -1 .055 1. 00 18 .62 C
ATOM 210 N HIS A 135 7. 252 7. 669 3 ,515 1. 00 14 .45 N
ATOM 211 CA HIS A 135 7. 529 6. 372 4 .111 1. 00 17 .69 C
ATOM 212 C HIS A 135 6. 902 6. 204 5 .491 1. 00 12 .38 c
ATOM 213 o HIS A 135 6. 361 5. 174 5 .797 1. 00 16 .40 o
ATOM 214 CB HIS A 135 9. 036 6. 106 4 .165 1. 00 19 .59 c
ATOM 215 CG HIS A 135 9. 389 4. 698 4 .532 1. 00 35 .63 c
ATOM 216 ND1 HIS A 135 . 9. 198 4. 184 5 .797 1. 00 36 .25 N
ATOM 217 CD2 HIS A 135 9. 925 3. 696 3 .798 1. 00 3 .23 c
ATOM 218 CE1 HIS A 135 9. 598 2. 927 5 .827 1. 00 33 .41 C
ATOM 219 NE2 HIS A 135 10. 047 2. 608 4 .627 1. 00 38 .09 N
ATOM 220 N THR A 136 6. 965 7. 237 6 .318 1. 00 10 .88 N
ATOM 221 CA THR A 136 6. 447 7. 132 7 .678 1. 00 14 .15/ C
ATOM 222 C THR A 136 4. 924 7. 125 7 .640 1. 00 13 .44 C ATOM 223 o THR A 136 4.279 6.487 8.469 1.00 11.47 o
ATOM 224 CB THR A 136 6. 948 8. 305 8. 533 1 .00 12 .18 C
ATOM 225 OGl THR A 136 8. 377 8. 218 8. 668 1 .00 15 .49 o
ATOM 226 CG2 THR A 136 6. 294 8. 311 9. 915 1 .00 15 .98 c
ATOM 227 N SER A 137 4. 354 7. 841 6. 668 1 .00 11 .52 N
ATOM 228 CA SER A 137 2. 910 7. 833 6. 468 1 .00 11 .40 C
ATOM 229 C SER A 137 2. 444 6. 416 6. 143 1 .00 14 .79 C
ATOM 230 o SER A 137 1. 438 5. 939 6. 672 1 .00 12 .41 o
ATOM 231 CB SER A 137 2. 498 8. 799 5. 337 1 .00 10 .43 c
ATOM 232 OG SER A 137 2. 855 10. 138 5. 691 1 .00 12 .55 o
ATOM 233 N GLN A 138 3. 193 5. 736 5. 282 1 .00 15 .89 N
ATOM 234 CA GLN A 138 2. 880 4. 348 4. 965 1 .00 14 .81 c
ATOM 235 C GLN A 138 2. 955 3. 490 6. 236 1 .00 14 .13 c
ATOM 236 0 GLN A 138 2. 081 2. 659 6. 476 1 .00 18 .36 o
ATOM 237 CB GLN A 138 3. 820 3. 812 3. 885 1 .00 15 .61 C
ATOM 238 CG GLN A 138 3. 643 2. 319 3. 582 1 .00 18 .43 C
ATOM 239 CD GLN A 138 - 2. 251 1. 952 3. 098 1 .00 22 .43 C
ATOM 240 OEl GLN A 138 1. 44 2. 816 2. 762 1 .00 14 .96 o
ATOM 241 NE2 GLN A 138 1. 965 0. 649 3. 060 1 .00 23 .31 N
ATOM 242 N GLU A 139 3. 982 3. 708 7. 060 1 .00 13 .26 N
ATOM 243 CA GLU A 139 4. 103 2. 943 8. 309 1 .00 15 .10 C
ATOM 244 C GLU A 139 2. 936 3. 234 9. 251 1 .00 18 .86 C
ATOM 245 o GLU A 139 2. 456 2. 342 9. 953 1 .00 21 .52 o
ATOM 246 CB GLU A 139 5. 435 ' 3. 219 9. 003 1 .00 22 .60 C
ATOM 247 CG GLU A 139 6. 616 2. 529 8. 354 1 .00 35 .86 C
ATOM 248 CD GLU A 139 6. 495 1. 013 8. 380 1 .00 55 .46 C
ATOM 249 OEl GLU A 139 5. 871 0. 475 9. 323 1 .00 62 .60 o
ATOM 250 OE2 GLU A 139 7. 023 0. 358 7. 453 1 .00 57 .28 o
ATOM 251 N ASN A 140 2. 476 4. 482 9. 256 1 .00 16 .40 N
ATOM 252 CA ASN A 140 1. 298 4. 844 10. 029 1 .00 15 .26 C
ATOM 253 C ASN A 140 0. 067 4. 069 9. 547 1 .00 "17 .95 C
ATOM 254 o ASN A 140 -0. 707 3. 558 10. 348 1 .00 18 .70 o
ATOM 255 CB ASN A 140 1. 042 6. 360 9. 982 1 .00 18 .19 C
ATOM 256 CG ASN A 1'40 2. 094 ' 7,. 165 10. 749 1 .00 20 .68 C
ATOM 257 ODl ASN A 140 2. 783 6. 638 11. 632 1 .00 20 .08 o
ATOM 258 ND2 ASN A 140 2. 224 8. 439 10. 406 1 .00 20 .34 N
ATOM 259 N CYS A 141 -0. 106 3. 966 8. 233 1 .00 15 .41 N
ATOM 260 CA CYS A 141 -1. 212 3. 180 7. 699 1 .00 15 .45 C
ATOM 261 C CYS A 141 -1. 065 1. 708 8. 090 1 .00 15 .27 C
ATOM •262 o · CYS A 141 -2. 033 1. 059 8. 485 1 .00 18 .16 o
ATOM 263 CB CYS A 141 -1. 303 3. 342 6. 181 1 .00 15 .92 C
ATOM 264 SG CYS A 141 -1. 692 5. 045 5. 656 1 .00 15 .42 S
ATOM 265 N LEU A 142 0. 152 1. 187 7. 980 1 .00 13 .99 N
ATOM 266 CA LEU A 142 0. 385 -0. 222 8. 280 1 .00 19 .50 C
ATOM 267 C LEU A 142 0. 027 -0. 563 9. 726 1 .00 22 .44 C
ATOM 268 o LEU A 142 -0. 412 -1. 681 10. 014 1 .00 24 .89 o
ATOM 269 CB LEU A 142 1. 838 -0. 603 7. 986 1 .00 17 .10 C
ATOM 270 CG LEU A 142 2. 217 -0. 616 6. 499 1 .00 17 .68 C
ATOM 271 GDI LEU A 142 3. 712 -0. 816 6. 342 1 .00 26 .03 C TOM 272 CD2 LEU A 142 1. 448 -1. 713 5. 750 1 .00 23 .45 . c
ATOM 273 N LYS A 143 0. 209 0. 397 10. 629 1 .00 18 .72 N
ATOM 274 CA LYS A 143 -0. 078 0. 160 12. 042 1 .00 22 .66 c
ATOM 275 C LYS A 143 -1. 574 -0. 024 12. 246 1 .00 30 .61 c
ATOM 276 o LYS A 143 -2. 021 -0. 610 13. 235 1 .00 27 .31 o
ATOM 277, CB LYS A 143 0. 437 1. 314 12. 912 1 .00 21 .16 c
ATOM 278 CG LYS A 143 1. 949 1. 422 12. 973 1 .00 31 .52 c
ATOM 279 CD ■LYS A 143 2. 367 2. 638 13. 786 1 .00 34 .07 c ATOM 280 CE LYS A 143 3.871 2.674 14.003 1.00 42.74 C
ATOM 281 NZ LYS A 143 4. 264 3 .833 14. 866 1 .00 50. 90 N
ATOM 282 N GLU A 144 -2. 346 0 .481 11. 295 1 .00 25. 92 N
ATOM 283 CA GLU A 144 -3. 795 0 .393 11. 364 1 .00 24. 25 C
ATOM 284 C GLU A 144 -4. 325 -0 .701 10. 442 1 .00 21. 06 C
ATOM 285 0 GLU A 144 -5. 515 -0 .736 10. 141 1 .00 27. 02 Q
ATOM 286 CB GLU A 144 -4. 426 1 .737 11. 003 1 .00 29. 42 c
ATOM 287 CG GLU A 144 -5. 379 2 .275 12. 057 1 .00 49. 43 c
ATOM 288 CD GLU A 144 -4. 720 2 .431 13. 417 1 .00 60. 66 c
ATOM 289 OEl GLU A 144 -3. 625 3 .029 13. 485 1 .00 67. 77 o
ATOM 290 OE2 GLU A 144 -5. 294 1 .948 14. 417 1 .00 62. 93 o
ATOM 291 N GLY A 145 -3. 445 -1 .591 9. 995 1 .00' 18. 20 N TOM 292 CA GLY A 145 -3. 845 -2 .639 9. 071 1 .00 19. 96 C
ATOM 293 C GLY A 145 -4. 324 -2 .099 7. 733 1 .00 15. 92 C
ATOM 294 0 GLY A 145 -5. 209 -2 .667 7. 091 1 .00 21. 72 o
ATOM 295 N SE A 146 -3. 734 -0 .986 7. 316 1 .00 15. 84 N
ATOM 296 CA SER A 146 -4. 110 -0 .346 6. 068 1 .00 16. 51 ' C
ATOM 297 C SER A 146 -2. 865 0 .036 5. 278 1 .00 15. 03 C
ATOM 298 o SER A 146 , -1. 741 -0 .288 5. 668 1 .00 16. 42 o
ATOM 299 CB SER A 146 -4. 993 0 .885 6. 316 1 .00 16. 41 C
ATOM 300 OG. SER A 146 -4. 270 1 .922 6. 973 1 .00 17. 74 o
ATOM 301 N THR A 147 -3. 077 0 .729 4. 166 1 .00 14. 06 N
ATOM 302 CA THR A 147 -1. 981 1 .153 3. 312 1 .00 14. 64 C
ATOM 303 C THR A 147 -2. 305 2 .541 2. 765 1 .00 13. 94 C
ATOM 304 o THR A 147 -3. 445 2 .998 2. 849 1 .00 12. 01 o
ATOM 305 CB THR A 147 -1. 806 0 .156 2. 143 1 .00 23. 13 C
ATOM 306 OGl THR A 147 -0. 548 0 .377 1. 495 1 .00 25. 00 o
ATOM 307 CG2 THR A 147 -2. 958 0 .282 1. 141 1 .00 20. 01 C
ATOM 308 N LEU A 148 -1. 314 3 .244 2. 236 1 .00 11. 00 N
ATOM 309 CA LEU A 148 -1. 638 4 .551 1. 677 1 .00 11. 85 C
ATOM 310 C LEU A 148 -2. 698 4 .404 0. 575 1 .00 11. 34 C
ATOM 311 0 LEU A 148 -2. 719 3 .410 -0. 164 1 .00 10. 46 o
ATOM 312 CB LEU A 148 -0. 385 5 .267 Ϊ. 166 1 .00 8. 37 C
ATOM 313 CG LEU A 148 0. 501 5 .923 2. 227 1 .00 11. 73 C
ATOM 314 CD1 LEU A 148 1. 784 6 .413 1. 598 1 .00 12. 57 C
ATOM 315 CD2 LEU A 148 -0. 234 7 .076 2. 894 1 .00 13. 49 C
ATOM 316 N LEU A L49 -3. 595 5 .380 0. 492 1 .00 9. 35 N
ATOM 317 CA LEU A 149 -4. 632 5 .415 -0. 544 1 .00 10. 45 C
ATOM 318 C LEU A 149 -4. 107 5 .033 -1. 923 1 .00 10. 59 C
ATOM 319 0 LEU A 149 -3. 093 5 .575 -2. 379 1 .00 11. 11 o
ATOM 320 CB LEU A 149 -5. 195 6 .841 -0. 622 1 .00 9. 74 c
ATOM 321 CG LEU A 149 -6. 231 7 .136 -1. 713 1 .00 12. 18 c
ATOM 322 CD1 LEU A 149 -7. 471 6 .261 -1. 583 1 .00 15. 79 c
ATOM 323 CD2 LEU A 149 -6. 602 8 .608 -1. 660 1 .00 14. 05 c
ATOM 324 N GLN* A 150 -4. 796 4 .105 -2. 585 1 .00 7. 87 N
ATOM 325 CA GLN A 150 -4. 542 3 .841 -3. 991 1 .00 10. 45 c
ATOM 326 C GLN A 150 -5. 817 4 .163 -4. 744 1 .00 13. 43 c
ATOM 327 o GLN A 150 -6. 915 3 .836 -4. 298 1 .00 11. 64 o
ATOM 328 CB GLN A 150 -4. 112 2 .398 ^4. 210 1 .00 14. 78 c
ATOM 329 CG GLN A 150 -2. 879 2 .083 -3. 404 1 .00 14. 94 c
ATOM 330 CD GLN A 150 -2. 392 0 .666 -3. 585 .1 .00 16. 35 c
ATOM 331 OEl GLN A 150 -1. 348 0 .301 -3. 063 1 .00 21. 47 o
ATOM 332 NE2 GLN A 150 -3. 144 -0 .137 -4. 334 1 .00 15. 90 N
ATOM 333 • N ILE A 151 -5. 679 4 .860 -5. 858 1 .00 10. 48 N
ATOM 334 CA ILE A 151 -6. 840 5 .311 -6. 595 1 .00 9. 56 C
ATOM 335 C ILE A 151 -6. 836 4 .563 -7. 921 1 .00 13. 50 C
ATOM 336 o ILE A 151 -6. 019 4 .833 -8. 805 1 .00 13. 85 o ATOM 337 CB ILE A 151 -6.782 6.814 -6.808 1.00 14.28 ' C
ATOM 338 CGI ILE A 151 -6. 546 7. 514 -5. 454 1 .00 15 .75 c
ATOM 339 CG2 ILE A 151 -8. 056 7. 302 -7. 491 1 .00 15 .10 . c
ATOM 340 CD1 ILE-, A 151 -6. 203 8. 986 -5. 565 1 .00 19 .49 c
ATOM 341 N GLU A 152 -7. 730 3. 595 -8. 041 1 .00 13 .30 N
ATOM 342 CA GLU A 152 -7. 695 2. 697 -9. 196 1 .00 15 .23 c
ATOM 343 C GLU A 152 -8. 753 3. 004 -10. 245 1 .00 18 .76 c
ATOM 344 0 GLU A 152 -8. 873 2. 279 -11. 230 1 .00 21 .08 0 TOM 345 CB GLU A 152 -7. 824 1. 237 -8. 755 1 .00 21 .85 c
ATOM 346 CG GLU A 152 -6. 561 0. 652 -8. 121 1 .00 26 .64 c
ATOM 347 CD GLU A 152 -6. 602 0. 665 -6. 605 1 .00 26 .04 c
ATOM 348 OEl GLU A 152 -7. 515 1. 305 -6. 051 1 .00 19 .07 o
ATOM 349 OE2 GLU A 152 -5. 737 0. 022 -5. 972 1 .00 27 .13 o
ATOM 350 N SER A 153 -9. 524 4. 067 -10. 040 1 .00 15 .00 N
ATOM 351 CA SE A 153 -10. 635 4. 358 -10. 936 1 .00 13 .96 C
ATOM 352 C SER A 153 -11. 060 5. 801 -10. 827 1 .00 14 .85 C
ATOM 353 0 SER A 153 -10. 773 6. 468 -9. 830 1 .00 13 .19 o
ATOM 354 CB SER A 153 -11. 842 3. 467 -10. 628 1 .00 13 .59 C
ATOM 355 OG SER A 153 -12. 509 3. 885 -9. 448 1 .00 18 .38 o
ATOM 356 N LYS A 154 -11. 748 6. 283 -11. 857 1 .00 11 .68 N
ATOM 357 CA LYS A 154 -12. 279 7. 632 -11. 828 1 .00 12 .44 C
ATOM 358 C LYS A 154 -13. 323 7. 740 -10. 731 1 .00 16 .46 C
ATOM 359 0 LYS A 154 -13. 418 8. 770 -10. 046 1 .00 14 .68 o
ATOM 360 CB LYS A 154 -12. 884 8. 015 rl3. 182 1 .00 10 .26 C
ATOM 361 CG LYS A 154 -13. 539 9. 377 -13. 166 1 .00 14 .31 c
ATOM 362 CD LYS A 154 -14. 043 9. 757 -14. 548 1 .00 15 .03 c
ATOM 363 CE LYS A 154 -14. 560 11. 183 -14. 569 1 .00 18 .54 c
ATOM 364 NZ LYS A 154 -15. 797 11. 335 -13. 758 1 .00 18 .21 N
ATOM 365 N GLU A 155 -14. 094 6. 672 -10. 547 1 .00 13 .30 N
ATOM 366 CA GLU A 155 -15. 113 6. 671 -9. 502 1 .00 15 .36 c
ATOM 367 C GLU A 155 -14. 488 6. 962 -8. 131 1 .00 16 .54 c
ATOM 368 0 GLU A 155 -14. 998 7. 778 -7. 362 1 .00 15 .99 o
ATOM 369 CB GLU A 155 -15. 877 5. 346 -9. 480 1 .00 22 .40 c
ATOM 370 CG GLU A 155 -17. 009 5. 319 -8. 473 1 .00 34 .59 c
ATOM 371 CD GLU A 155 -17. 646 3. 950 -8. 351 1 .00 46 .61 c
ATOM 372 OEl GLU-, A 155 -18. 599 3. 805 -7. 555 1 .00 50 .27 o
ATOM 373 OE2 GLU A 155 -17. 188 3. 019 -9. 049 1 .00 49 .01 o
ATOM 374 N GLU A 156 . -13. 368 6. 309 -7. 842 1 .00 12 .60 N
ATOM 375 CA GLU A 156 -12. 710 6. 478 -6. 547 1 .00 11 .81 C
ATOM 376 C GLU A 156 -12. 068 7. 863 -6. 432 1 ;00 15 .15 C
ATOM 377 0 GLU A 156 -12. 112 8. 501 -5. 367 1 .00 12 .45 o
ATOM 378 CB GLU A 156 .-11. 667 5. 383 -6. 336 1 .00 13 .28 . C
ATOM 379 CG. GLU A 156 -10. 970 5. 472 -4. 998 1 .00 13 .76 C
ATOM 380 CD GLU A 156 -10. 208 4. 211 -4. 650 1 .00 19 .46 C
ATOM 381 OEl GLU A 156 -10. 072 3. 918 -3. 447 1 .00 15 .53 o
ATOM 382 OE2 GLU A 156 -9. 742 3. 504 -5. 571 1 .00 15 .19 o
ATOM 383 N MET A 157 -11. 481 8. 333 -7. 531 1 .00 13 .02 N
ATOM 384 CA MET A 157 -10. 893 9. 670 -7. 544 1 .00 13 .33 C
ATOM 385 C MET A 157 -11. 962 10. 710 -7. 233 1 .00 14 .86 C
ATOM 386 0 MET A 157 -11. 744 11. 610 -6. 421 1 .00 15 .02 o
ATOM 387 CB MET A 157 -10. 223 9. 978 -8. 886 1 .00 13 .68 C
ATOM 388 CG MET A 157 -9. 495 11. 313 -8. 928 1 .00 16 .36 C
ATOM 389 SD MET A 157 -8. 093 11. 368 -7. 794 1 .00 16 .66 s
ATOM 390 CE MET A 157 -7. 415 12. 991 -8. 154 1 .00 17 .91 C TOM 391 N ASP A 158 -13. 118 10. 571 -7. 874 1 .00 12 .37 . N
ATOM 392 CA ASP A 158 -14. 244 11. 465 -7. 636 1 .00 14 .56 C
ATOM 393 C ASP A 158 -14. 709 11. 407 -6. 184 1 .00 16 .58 C ATOM 394 0 ASP A 158 -15.084 12.429 -5.601 1.00 17.61 o
ATOM 395 CB ASP A 158 -15. 401 11. 138 -8. 581 1 .00 19 .09 C
ATOM 396 CG ASP A 158 -15. 093 11. 503 -10. 025 1 .00 21 .15 C
ATOM 397 ODl ASP A 158 -14. 147 12. 290 -10. 257 1 .00 22 .77. o
ATOM 398 OD2 ASP A 158 -15. 798 11. 012 -10. 933 1 .00 24 .40 o
ATOM 399 N PHE A 159 -14. 688 10. 218 -5. 591 1 .00 13 .94 r N
ATOM 400 CA PHE A 159 .-15. 054 10. 125 -4. 181 1 .00 15 .61 c
ATOM 401 C PHE A 159 -14. 044 10. 831 -3. 296 1 .00 19 .31 c
ATOM 402 o PHE A 159 -14. 409 11. 570 -2. 379 1 .00 20 .22 o
ATOM 403 CB PHE A 159 -15. 185 8. 678 -3. 719 1 .00 17 .37 c
ATOM 404 CG PHE A 159 -15. 489 8. 558 -2. 258 1 .00 19 .22 c
ATOM 405 CD1 PHE A 159 -16. 787 8. 677 -1. 801 1 .00 22 .86 c
ATOM 406 CD2 PHE A 159 -14. 472 8. 363 -1. 341 1 .00 22 .52 c
ATOM 407 CE1 PHE A 159 -17. 073 8. 583 -0. 455 1 .00 25 .63 c
ATOM 408 CE2 PHE A 159 -14. 751 8. 272 0. 012 1 .00 22 .06 c
ATOM 409 CZ PHE A 159 -16. 055 8. 382 0. 450 1 .00 23 .64 c
ATOM 410 N ILE A 160 -12. 767 10. 594 -3. 561 1 .00 16 .21 N
ATOM 411 CA ILE A 160 -11. 724 11. 183 -2. 740 1 .00 20. .49 c
ATOM 412 C ILE A 160 -11. 744 12. 702 -2. 856 1 .00 24 .31 c
ATOM 413 o ILE A 160 -11. 687 13. 412 -1. 849 1 .00 23 .36 o
ATOM 414 CB ILE A 160 -10. 332 10. 648 -3. 102 1 .00 13 .38 c
ATOM 415 CGI ILE A 160 -10. 222 9. 171 -2. 716 1 .00 16 .69 c
ATOM 416 CG2 ILE A 160 -9. 259 11. 496 -2. 410 1 .00 13 .88 c
ATOM 417 CD1 ILE A 160 -10. 434 8. 876 -1. 218 1 .00 16 .03 c
ATOM 418 N THR A 161 -11. 846 13. 214 -4. 075 1 .00 17 .80 N
ATOM 419 CA THR A 161 -11. 841 14. 665 -4. 226 1 .00 26 .03 C
ATOM 420 C THR A 161 -13. 076 15. 277 -3: 555 1 .00 28 .38 C
ATOM 421 o THR A 161 -12. 988 16. 322 -2. 909 1 .00 33 .41 o
ATOM 422 CB THR A 161 -11. 672 15. 104 -5. 691 1 .00 21 .89 c
ATOM 423 OGl THR A 161 -12. 713 14. 543 -6. 493 1 .00 24 .51 o
ATOM 424 CG2 THR A 161 -10. 328 14. 619 -6. 221 1 .00 25 .70 c
ATOM 425 N GLY A 162 -14. 212 14. 597 -3. 672 1 .00 24 .97 N
ATOM 426 CA GLY A 162 -15. 431 15. 029 -3. 008 1 .00 28 .64 C
ATOM 427 C GLY A 162 -15. 301 15. 035 -1. 494 1 .00 31 .19 C
ATOM 428 o GLY A 162 -15. 708 15. 990 -0. 838 1 .00 38 .20 o
ATOM 429 o SER A 163 -14. 021 15. 650 2. 021 1 .00 35 .87 o
ATOM 430 N SER A 163 -14. 733 13. 969 -0. 936 1 .00 16 .42 N
ATOM 431 CA SER A 163 -14. 534 13. 869 0. 505 1 .00 21 .88 C
ATOM 432 C SER A 163 -13. 663 14. 999 1. 046 1 .00 30 .50 C
ATOM 433 CB SER A 163 -13. 932 12. 508 0. 883 1 .00 26 .94 C
ATOM 434 OG SER A 163 -14. 845 11. 449 0. 610 1 .00 30 .82 o
ATOM 435 o LEU A 16 -12. 035 18. 477 1. 709 1 .00 46 .76 o
ATOM 436 N LEU A 164 -12. 521 15. 233 0. 413 1 .00 37 .49 N
ATOM 437 CA LEU A 164 -11. 617 16. 275 0. 882 1 .00 35 .37 C
ATOM 438 C LEU A 164 -12. 286 17. 637 0. 848 1 .00 38 .66 C
ATOM 439 CB LEU A 164 -10. 332 16. 309 0. 054 1 .00 34 .11 C
ATOM 440 CG LEU A 164 -9. 470 15. 049 0. 102 1 .00 29 .48 C
ATOM ' 441 CDl LEU A 164 -8. 294 15. 189 -0. 846 1 .00 27 .19 c
ATOM 442 CD2 LEU A 164 -9. 003 14. 765 1. 523 1 .00 36 .17 c
ATOM 443 o ARG A 165 -14, 913 2,0. 614 1. 315 1 .00 37 .62 o
ATOM ' 444 N ARG A 165 -13. 136 17. 866 -0. 146 1 .00 32 .12 N
ATOM 445 CA ARG A 165 -13. 803 19. 158 -0. 243 1 .00 35 .85 C TOM 446 C ARG A 165 -14. 620 19. 455 1. 012 1 .00 37 .46 C
ATOM 447 CB ARG A 165 -14. 677 19. 235 -1. 496 1 .00 37 .45 C TOM 448 CG ARG A 165 -13. 920 19. 657 -2. 742 1 .00 49 .01 C
ATOM 449 CD ARG A 165 -14. 859 19. 877 -3. 914 1 .00 56 .20 c
ATOM 450 NE ARG A 165 -14. 138 20. 278 -5. 117 1 .00 66 .94 N ATOM 451 CZ ARG A 165 -14.724 20.611 -6.263 1.00 72.78 C
ATOM 452 NHl ARG A 165 -16. 046 20 .592 -6 .365 1 .00 77 .79 N
ATOM 453 NH2 ARG A 165 -13. 988 20 .966 -7 .310 1 .00 68 .67 N
ATOM 454 0 LYS A 166 -15. 313 19 .000 5 .284 1 .00 48 .21 o
ATOM 455 N LYS A 166 -14. 972 18 .402 1 .744 1 .00 31 .64 N
ATOM 456 CA LYS A 166 -15. 756 18 .537 2 .967 1 .00 41 .60 C
ATOM 457 C LYS A 166 -14. 848 18 .818 4 .160 1 .00 44 .94 C
ATOM 458 CB LYS A 166 -16. 563 17 .261 3 .221 1 .00 53 .00 C
ATOM 459 CG LYS A 166 -17. 282 16 .716 1 .992 1 .00 59 .19 C
ATOM 460 CD LYS A 166 -18. 309 17 .701 1 .456 1 .00 66 .52 C
ATOM 461 CE LYS A 166 -19. 000 17 .167 0 .207 1 .00 68 .96 C
ATOM 462 NZ LYS A 166 -18. 100 17 .152 -0 .980 1 .00' 68 .84 N
ATOM 463 o ILE A 167 -11. 670 20 .875 3 .588 1 .00 34 .32 o
ATOM 464 N ILE A 167 -13. 546 18 .846 3 .905 1 .00 37 .61 N TOM 465 CA ILE A 167 -12. 554 19 .101 4 .944 1 .00 41 .22 C
ATOM 466 C ILE A 167 -11. 941 20 .485 4 .725 1 .00 39 .53 C
ATOM 467 CB ILE A 167 -11. 463 18 .000 4 .956 1 .00 34 .31 C
ATOM 468 CGI ILE A 167 -11. 992 16 .723 5 .615 1 .00 37 .25 C
ATOM 469 CG2 ILE A 167 -10. 232 18 .461 5 .705 1 .00 34 .56 C
ATOM 470 CD1 ILE A 167 -13. 205 16 .113 4 .941 1 .00 40 .56 C
ATOM 471 o LYS A 168 -9. 040 21 .726 5 .192 1 .00 23 .56 o
ATOM 472 N LYS A 168 -11. 749 21 .239 5 .803 1 .00 29 .78 N
ATOM 473 CA LYS A 168 -11. 224 22 .593 5 .672 1 .00 32 .80 C
ATOM 474 C LYS A 168 -9. 876 22 .597 4 .966 1 .00 28 .01 C
ATOM 475 CB LYS A 168 -11. 105 23 .278 7 .033 1 .00 44 .68 C
ATOM 476 CG LYS A 168 -10. 46? 24 .663 6 .959 1 .00 56 .50 C
ATOM 477 CD LYS A 168 -10. 549 25 .404 8 .290 1 .00 66 .28 C
ATOM 478 CE LYS A 168 -9. 903 24 .607 9 .413 1 .00 70 .41 c
ATOM 479 NZ LYS A 168 -9. 902 25 .350 10 .705 1 .00 74 .89 N
ATOM 480 o GLY A 169 -9. 166 22 .047 1 .888 1 .00 36 .18 o
ATOM 481 GLY A 169 -9. 678 23 .582 4 .101 1 .00 28 .92 N
ATOM 482 CA GLY A 169 -8. 415 23 .742 3 .413 1 .00 24 .51 C
ATOM 483 C GLY A 169 -8. 307 22 .881 2 .175 1 .00 36 .87 C
ATOM 484 o SER A 170 -4. 604 22 .144 -0 .185 1 .00 15 .56 o
ATOM 485 N SER A 170 -7. 240 23 .100 1 .424 1 .00 35 .23 N
ATOM 486 CA SER A 170 -6. 954 22 .280 0 .266 1 .00 14 .37 C
ATOM 487 C SER A 170 -5. 563 21 .761 0 .499 1 .00 13 .89 C
ATOM 488 CB SER A 170 -7. 009 23 .136 -0 .995 1 .00 29 .91 C
ATOM 489 OG SER A 170 -8. 248 23 .822 -1 .067 1 .00 29 .67 o
ATOM 490 o TYR A 171 -4. 299 18 .802 0 .191 1 .00 12 .38 o
ATOM 491 N TYR A 171 -5. 448 20 .899 1 .503 1 .00 10 .65 N
ATOM 492 CA TYR A 171 -4. 147 20 .430 1 .948 1 .00 12 .10 C
ATOM 493 C TYR A 171 -3. 585 19 .343 1 .043 1 .00 13 .26 ,C
ATOM 494 CB TYR A 171 -4. 244 19 .918 3 .395 1 .00 12 .58 C
ATOM 495 CG TYR A 171 -4. 709 20 .984 4 .379 1 .00 12 .08 C
ATOM ^ 496 CD1 TYR A 171 -4. 470 22 .327 4 .135 1 .00 16 .11 ■ C
ATOM 497 CD2 TYR A 171 -5. 383 20 .648 5 .548 1 .00 16 .20 C
ATOM 498 CE1 TYR A 171 -4. 890 23 .314 5 .021 1 .00 19 .09 C
ATOM 499 CE2 TYR A 171 -5. 796 21 .633 6 .451 1 .00 13 .54 c
ATOM 500 CZ TYR A 171 -5. 555 22 .962 6 .168 1 .00 13 .81 c
ATOM L501 OH TYR A 171 -5. 975 23 .937 7 .039 1 .00 15 .97 o
ATOM 502 N ASP A 172 -2. 307 19 .022 t .253 1 .00 9 .82 N
ATOM 503 CA ASP A 172 -1. 629 17 .974 0 .501 1 .00 8 .60 c
ATOM 504 C ASP A 172 -1. 730 16 .652 1 .238 1 .00 11 .80 c
ATOM 505 o ASP A 172 -1. 438 16 .586 2 .415 1 .00 9 .41 o
ATOM 506 CB ASP A 172 - -0. 140 18 .301 0 .310 1 .00 12 .77 c
ATOM 507 CG~ ASP A 172 0. 092 19 .637 -0 .358 1 .00 15 .75 c ATOM 508 ODl ASP A 172 -0.,833 20.152 -1.018 1.00 14.66 o
ATOM 509 OD2 ASP A 172 1. 211 20. 169 -0 .224 1 .00 21 .92 o
ATOM 510 N TYR A 173 -2. 115 15. 595 0 .532 1 .00 7 .86 N
ATOM 511 CA YR A 173 -2. 257 14. 277 1 .147 1 .00 4 .38 • C
ATOM 512 C TYR A 173 -1. 463 13. 250 0 .385 1 .00 9 .93 c
ATOM 513 0 TYR A 173 -1. 754 12. 984 -0 .784 1 .00 7 .53 o
ATOM 514 CB TYR A 173 -3. 734 13. 858 1 .131 1 .00 6 .85 c
ATOM 515 CG TYR A 173 -4. 576 14. 772 1 .971 1 .00 9 .90 c
ATOM . 516 CD1 TYR A 173 -4. 763 14. 506 3 .327 1 .00 10 .83 c
ATOM 517 CD2 TYR A 173 -5. 124 15. 928 1 .442 1 .00 10 .95 c
ATOM 518 CE1 TYR A 173 -5. 503 15. 339 4 .127 1 .00 8 .72 c
ATOM 519 CE2 TYR A 173 -5. 873 16. 786 2 .241 1 .00 13 .27 c
ATOM 520 CZ TYR A 173 -6. 060 16. 478 3 .583 1 .00 10 .36 c
ATOM .521 OH TYR A 173 -6. 788 17. 302 4 .411 1 .00 13 .80 o
ATOM 522 N TRP A 174 -0. 468 12. 651 1 .032 1 .0,0 9 .13 N
ATOM 523 CA- TRP A 174 0. 251 11. 550 0 .395 1 .00 7 .55 c
ATOM 524 C TRP A 174 -0. 700 10. 448 -0 .045 1 .00 12 .47 c
ATOM 525 o TRP A 174 -1. 647 10. 111 .0 .664 1 .00 12 .72 o
ATOM 526 CB TRP A 174 1. 249 10. 908 1 .362 1 .00 8 .33 c
ATOM 527 CG TRP A 174 2. 459 11. 705 1 .687 1 .00 9 .67 c
ATOM 528 CD1 TRP A 174 2. 886 12. 040 2 .932 1 .00 10 .97 c
ATOM 529 CD2 TRP A 174 3. 430 12. 242 0 .771 1 .00 12 .48 c
ATOM 530 NE1 TRP A 174 4. 043 12. 763 2 .858 1 .00 8 .55 N
ATOM 531 CE2 TRP A 174 . 4. 403 12. 908 1 .547 1 .00 11 .45 c
ATOM 532 CE3 TRP A 174 3. 555 12. 252 -0 .618 1 .00 14 .70 c
ATOM 533 CZ2 TRP A 174 5. 498 13. 560 0 .981 1 .00 12 .34 R c
ATOM 534 CZ3 TRP A 174 4. 652 12. 897 -1 .185 1 .00 14 .92 c
ATOM 535 CH2 TRP A 174 5. 604 13. 547 -0 .387 1 .00 14 .74 c
ATOM 536 N VAL A 175 -0. 438 9. 882 -1 .216 1 .00 7 .83 N
ATOM 537 CA VAL A 175 -1. 106 8. 642 -1 .633 1 .00 4 .55 C
ATOM 538 C VAL A 175 -0. 066 7. 589 -1 .954 1 .00 7 .36 C
ATOM 539 o VAL A 175 1. 128 7. 871 -1 .983 1 .00 9 .57 o
ATOM 540 CB VAL A 175 -2. 018 8. 859 -2 .857 1 .00 10 .53 C
ATOM 541 CGI VAL A 175 -3. 078 9. 909 -2 .545 1 .00 9 .84 C
ATOM 542 CG2 VAL A 175 -1. 196 9. 258 -4 .078 1 .00 13 .91 C
ATOM 543 N GLY A 176 -0. 507 6. 362 -2 .216 1 .00 9 .00 N
ATOM 544 CA GLY A 176 0. 429 5. 254 -2 .354 1 .00 12 • 78 C
ATOM 545 C GLY A 176 1. 124 5. 135 -3 .707 1 .00 13 .77 C
ATOM 546 o GLY A 176 1. 279 4. 028 -4 .224 1 .00 12 .69 o
ATOM 547 N LEU A 177 1. 552 6. 262 -4 .269 1 .00 13 .70 N
ATOM 548 CA LEU A 177 2. 213 6. 296 -5 .575 1 .00 10 .19 C
ATOM 549 C LEU A 177 3. 659 6. 694 -5 .429 1 .00 7 .71 C
ATOM 550 o LEU A 177 3. 969 7. 674 -4 .752 1 .00 11 .44 o
ATOM 551 CB LEU A 177 1. 555 7. 344 -6 .492 1 .00 12 .51 C
ATOM 552 CG LEU A 177 0. 493 6. 966 -7 .517 1 .00 21 .14 C
ATOM 553 CD1 LEU A 177 0. 965 5. 828 -8 .418 1 .00 19 .80 C
ATOM 554 CD2 LEU A 177 0. 151 8. 199 -8 .341 1 .00 19 .56 C TOM 555 N SER A 178 4. 555 5. 960 -6 .074 1 .00 10 .34 N
ATOM 556 CA SER A 178 5. 969 6. 345 -6 .071 1 .00 11 .73 C
ATOM 557 C SER A 178 6. 586 6. 113 -7 .435 1 .00 17 .46 C
ATOM 558 o SER A 178 6. 132 5. 252 -8 .208 1 .00 14 .79 o
ATOM 559 CB SER A 178 6. 767 5. 584 -4 .995 1 .00 13 .49 C
ATOM 560 OG SER A 178 6. 732 4. 186 -5 .234 1 .00 18 .65 o
ATOM 561 N GLN A 179 7. 624 6. 886 -7 .723 1 .00 17 .30 N
ATOM 562 CA GLN A 179 8. 241 6. 898 -9 .041 1 .00 23 .18 C
ATOM. 563 C GLN A 179 9. 619 6. 284 -8 .926 1 .00 27 .01 c
ATOM 564 0 GLN A 179 10. 425 6. 724 -8 .109 1 .00 25 .55 o ATOM 565 CB GLN A 179 8.345 8.338 -9.548 1.00 32.38 C
ATOM 566 CG GLN A 179 8. 813 8. 498 -10. 992 1 .00 39 .96 C
ATOM 567 CD GLN A 179 8. 912 9. 959 -11. 411 1 .00 42 .43 C
ATOM 568 OEl GLN A 179 9. 389 10. 805 -10. 653 1 .00 48 .08 o
ATOM 569 NE2 GLN A 179 8. 447 10. 264 -12. 616 . . 1 .00 44 .05 N
ATOM 570 o ASP A 180 12. 037 6. 334 -11. 254 1 .00 56 ,80 o
ATOM 571 N ASP A 180 9. 883 5. 267 -9. 740 1 .00 55 .47 N
ATOM 572 CA ASP A 180 11. 200 4. 635 -9. 779 1 .00 66 .41 C
ATOM 573 C ASP A 180 12. 264 5. 596 -10. 297 1 .00 59 .10 C
ATOM 574 CB ASP A 180 11. 172 3. 372 -10. 644 1 .00 78 .58 C
ATOM 575 CG ASP A 180 11. 127 2. 102 -9. 818 1 .00 89 .77 C
ATOM 576 ODl ASP A 180 11. 686 2. 099 -8. 700 1 .00 94 .87 o
ATOM 577 OD2 ASP A 180 10. 534 1. 107 -10. 285 1 .00 91 .61 o
ATOM 578 0 GLY A 181 15. 955 6. 926 -11. 879 1 .00 59 .66 o
ATOM 579 N GLY A 181 13. 431 5. 573 -9. 664 1 .00 51 .15 N
ATOM 580 CA GLY A 181 14. 513 6. 468 -10. 030 1 .00 55 .76 C
ATOM 581 C GLY A 181 15. 188 6. 126 -11. 346 1 .00 57 .32 C
ATOM 582 o HIS A 182 15. 045 4. 790 -15. 457 1 .00 63 .57 o
ATOM . 583 N HIS A 182 14. 905 4. 941 -11. 877 1 .00 58 .47 N
ATOM 584 CA HIS A 182 15. 539 4. 504 -13. 120 1 .00 61 .43 C
ATOM 585 C HIS A 182 14. 602 4. 569 -14. 329 1 .00 63 .70 c
ATOM 586 CB HIS A 182 1-6. 118 3. 093 -12. 961 1 .00 57 .96 c
ATOM 587 CG HIS A 182 17. 208 3. 004 -11. 939 1 .00 60 .53 c
ATOM 588 ND1 HIS A 182 18. 472 3. 513 -12. 152 1 .00 61 .28 N
ATOM 589 CD2 HIS A 182 17. 222 2. 475 -10. 692 1 .00 60 .88 C
ATOM 590 CE1 HIS A 182 19. 217 3. 299 -11. 082 1 .00 62 .97 C
ATOM 591 NE2 HIS A 182 18. 482 2. 671 -10. 181 1 .00 61 .21 N
ATOM 592 o SER A 183 11. 099 6. 069 -16. 324 1 .00 58 .04 o
ATOM 593 N SER' A 183 13. 309 4. 384 -14. 088 1 .00 62 .42 N
ATOM 594 CA SER A 183 12. 335 4. 362 -15. 172 1 .00 63 .28 C
ATOM 595 C SER A 183 11. 544 5. 662 -15. 249 1 .00 61 .65 C
ATOM 596 CB ' SER A^ 183 11. 372 3. 187 -14. 997 1 .00 67 .11 c
ATOM 597 OG SER A^ 183 10. 560 3. 364 -13. 849 1 .00 71 .73 o
ATOM 598 o GLY A 184 8. 146 7. 853 -14. 006 1 .00 42 .36 o
ATOM 599 N . GLY A 184 11. 375 6. 311 -14. 102 1 .00 62 .92 N
ATOM 600 CA GLY A 184 10. 513 7. 474 -14. 005 1 .00 56 .60 C
ATOM 601 C GLY A 184 9. 063 7. 030 -14. 005 1 .00 50 .08 C
ATOM 602 o ARG A 185 7. 427 4. 583 -11. 698 1. '00 27 .14 o
ATOM 603 N ARG A 185 8. 861 5. 715 -13. 999 1 .00 48 .04 N
ATOM 604 CA ARG A 185 7. 520 5. 148 -14. 046 1 .00 39 .54 C
ATOM 605 C ARG A 185 6. 849 5. 057 -12. 677 1 .00 26 .51 C
ATOM 606 CB ARG A 185 7. 521 3. 782 -14. 733 1 .00 42 .63 c
ATOM 607 CG ARG A 185 7. 745 3. 857' -16. 235 1 .00 53 .33 C
ATOM 608 CD ARG A 185 7. 641 2. 486 -16. 885 1 .00 63 .82 c
ATOM 609 NE ARG A 185 6. 299 1. 919 -16. 778 1 .00 69 .81 N
ATOM 610 CZ ARG- A 185 5. 919 1. 059 -15. 837 1 .00 70 .70 C
ATOM 611 NHl ARG A 185 6. 780 0. 658 -14. 909 1 .00 66 .34 N
ATOM 612 NH2 ARG A 185 4. 676 0. 598 -15. 825 1 .00 73 .35 N
ATOM 613 N TRP A 186 5. 606 5. 507 -12. 645 ■ 1 .00 17 .51 N
ATOM 614 CA TRP A 186 4. 808 5. 525 -11. 436 1 .00 13 .10 C
ATOM 615 C TRP A 186 4. 166 4. 174 -11. 183 1 .00 12 .84 C
ATOM 616 o TRP A 186 3. 640 3. 543 -12. 111 1 .00 14 .52 o
ATOM 617 CB TRP A 186 3. 716 6. 581 -11. 590 1 .00 12 .09 C
ATOM 618 CG TRP A 186 ■ 4. 239 7. 973 -11. 564 1 .00 11 .92 C
ATOM 619 CD1 TRP A 186 4. 477 8. 790 -12. 638 1 .00 16 .22 C
ATOM 620 CD2 TRP A 186 4. 583 8. 731 -10. 398 1 .00 11 .12 C
ATOM 621 NE1 TRP A 186 4. 945 10. 012 -12. 203 1 .00 18 .77 N ATOM 622 CE2 TRP A 186 5.024 9.996 -10.835 1.00 17.24 C
ATOM 623 CE3 TRP A 186 4 .562 8. 461 -9. 029 1 .00 13. 20 C
ATOM 624 CZ2 TRP A 186 5 .434 10. 991 -9. 946 1 .00 19. 24 C
ATOM 625 CZ3 TRP A 186 4 .962 9. 446 -8. 150 1 .00 14. 58 C
ATOM 626 CH2 TRP A 186 5 .397 10. 694 -8. 609 1 .00 20. 85 C
ATOM 627 0 LEU A 187 3 .304 3. 460 -7. 310 1 .00 12. 57 o
ATOM 628 N LEU A 187 4 .212 3. 734 -9. 928 1 .00 10. 97 N
ATOM 629 CA LEU A 187 3 .554 2. 513 -9. 502 1 .00 14. 56 C
ATOM 630 " C LEU A 187 2 .827 2. 729 -8. 183 1 .00 13. 10 C
ATOM 631 CB LEU A 187 4 .589 1. 413 -9. 298 1 .00 12. 21 C
ATOM 632 CG LEU A 187 5 .246 0. 860 -10. 551 1 .00 13. 40 c
ATOM 633 CD1 LEU A 187 6 .291 -0. 186 -10. 170 1 .00 19. 16 c
ATOM 634 CD2 LEU A 187 4 .191 0. 271 -11. 486 1 .00 18. 37 c
ATOM 635 N TRP A 188 1 .676 2. 091 -8. 034 1 .00 10. 20 N
ATOM 636 CA TRP A 188 1 .045 1. 987 -6. 729 1 .00 10. 19 C
ATOM 637 C TRP A 188 1 .860 1. 006 -5. 909 1 .00 13. 49 C
ATOM 638 0 TRP A 188 2 .696 0. 265 -6. 455 1 .00 13. 55 o
ATOM 639 CB TRP A 188 -0 .403 1. 502 -6. 846 1 .00 11. 33 C
ATOM 640 CG TRP A 188 -1 .274 2. 447 -7. 590 1 .00 11. 38 C
ATOM 641 CD1 TRP A 188 -1 .792 2. 272 -8. 845 1 .00 15. 05 c
ATOM 642 CD2 TRP A 188 -1 .725 3. 732 -7. 146 1 .00 . 9. 40 c
ATOM '643 NE1 TRP A 188 -2 .541 3. 360 -9. 203 1 .00 12. 40 N
ATOM 644 CE2 TRP A 188 -2 .515 4. 276 -8. 181 1 .00 9. 64 c
ATOM 645 CE3 TRP A 188 -1 .537 4. 478 -5. 977 1 .00 12. 51 C
ATOM 646 CZ2 TRP A 188 -3 .117 5. 534 -8. 084 1 .00 10. 31 c
ATOM 647 CZ3 TRP A 188 -2 .141 5. 714 -5. 879 1 .00 13. 50 c
ATOM 648 CH2 TRP A 188 -2 .919 6. 234 -6. 925 1 .00 12. 45 c
ATOM 649 0 GLN A 189 2 .699 -2. 133 -3. 083 1 .00 14. 83 o
ATOM 650 N GLN A 189 1 .626 0. 996 -4. 601 1 .00 11. 21 N
ATOM 651 C GLN A 189 2 .104 -1. 323 -3. 794 1 .00 15. 39 c
ATOM 652 CA . AGLN A 189 2 .408 0. 168 -3. 685 0 .50 10. 19 c
ATOM 653 CB , AGLN A 189 2 .263 0. 655 -2. 236 0 .50 11. 92 c
ATOM 654 CG . AGLN A 189 3 .134 1. 853 -1. 910 0 .50 10. 40 c
ATOM 655 CD , AGLN A 189 3 .099 2. 239 -0. 442 0 .50 15. 35 c
ATOM 656 OEIAGLN A 189 2 .070 2. 674 0. 078 0 .50 23. 64 o
ATOM 657 NE2AGLN A 189 4 .231 2. 087 0. 233 0 .50 11. 05 N
ATOM 658 CA BGLN A 1-89 2 .438 0. 168 -3'. 723 0 .50 10. 52 C
ATOM 659 CB BGLN A 189 2 .412 0. 724 -2. 300 0 .50 10. 79 C
ATOM 660 CG BGLN A 189 2 .912 2. 153 -2. 268 0 .50 12. 60 c
ATOM 661 CD BGLN A 189 4 .217 2. 314 -3. 037 0 .50 16. 52 c
ATOM 662 OE1BGLN A 189 5 .266 1. 868 -2. 582 0 .50 15. 44 0
ATOM 663 NE2BGLN A 189 4 .154 2. 936 -4. 217 0 .50 10. 14 N
ATOM 664 0 ASP A 190 1 .340 -4. 629 -6. 763 1 .00 16. 00 o
ATOM 665 N. ASP A 190 1 .186 -1. 691 -4. 686 1 .00 13. 16 N
ATOM 666 CA ASP A 190 0 .925 -3. 110 -4. 935 1 .00 15. 34 c
ATOM 667 C ASP A 190 1 .552 -3. 521 -6. 273 1 .00 17. 31 c
ATOM 668 CB ASP A 190 -0 .576 -3. 421 -4. 898 1 .00 13. 68 c
ATOM 669 CG ASP A 190 -1 .363 -2. 641 -5. 945 1 .00 17. 07 c
ATOM 670 ODl ASP A 190 -2 .609 -2. 606 -5. 861 1 .00 16. 47 o
ATOM 671 OD2 ASP A 190· -0 .732 -2. 056 -6. 851 1 .00 13. 45 o
ATOM 672 o GLY A 191 2 .628 -2. 909 -10. 487 1 .00 16. 59 o
ATOM 673 N GLY A 191 2 .324 -2. 613 -6. 863 1 .00 15. 88 N
ATOM 674 CA GLY A 191 3 .003 -2. 880 -8. 121 1 .00 13. 07 . C
ATOM 675 C GLY A 191 2 .167 -2. 652 -9. 377 1 .00 12. 71 c
ATOM 676 o SER A 192 0 .833 0. 391 -10. 157 1 .00 13. 90 o
ATOM 677 N SER A 192 0 .936 -2. 182 -9. 223 1 .00 15. 23 N TOM 678 CA SER A 192 0 .116 -1. 878 -10. 397 1 .00 13. 66 C ATOM 679 C SER A 192 0.392 -0.469 -10..913 1.00 13.45 C
ATOM 680 CB SER A 192 -1. .368 -2. ,033 -10. 090 1 .00 13 .22 C
ATOM 681 OG SER A 192 -1. 812 -1. 058 -9. 163 1 .00 14 .42 o
ATOM 682 o SER A 193 -1. 973 1. .426 -13. 040 1 .00 18 .63 o
ATOM 683 N SER A 193 0. 118 -0. 238 -12. 200 1 .00 13 .96 N
ATOM 684 CA SER A 193 0. 378 1. 064 -12. 814 1 .00 15 .39 C
ATOM 685 C SER A 193 -0. 878 1. 924 -12. 797 1 .00 17 .29 C
ATOM 686 CB SER A 193 0. 823 0. 893 -14. 273 1 .00 21 .16 C
ATOM 687 • OG SER A 193 . 2. 061 0, 204 -14. 363 1 .00 23 .38 o
ATOM 688 o PRO A 194 -1. 461 4. 178 -14. 997 1 .00 13 .21 o
ATOM 689 N PRO A 194 -0. 722 3. 224 -12. 534 1 .00 11 .74 N
ATOM 690 CA PRO A 194 -1. 895 4. 095 -12. 647 1 .00 12 .20 C
ATOM 691 C PRO A 194 -2. 291 4. 286 -14. 102 1 .00 13 .77 C
ATOM 692 CB PRO A 194 -1. 409 5. 42 -12. 070 1 .00 13 .05 C
ATOM 693 CG PRO A 194 0. 073 5. 402 -12. 250 1 .00 20 .78 C
ATOM 694 CD PRO A 194 0. 479 3. 957 -12. 096 1 .00 15 .03 C
ATOM 695 o SER A 195 -3. 131 7. 119 -15. 196 1 .00 12 .17 o
ATOM 696 N SER A 195 -3. 560 4. 585 -14. 323 1 .00 11 .79 N
ATOM 697 CA SER A 195 -4. 029 4. 936 -15. 653 1 .00 11 .97 C
ATOM 698 C SER A 195 -3. 561 6. 338 -16. 042 1 .00 13 .41 C
ATOM 699 CB SER A 195 -5. 553 4. 872 -15. 705 1 .00 13 .99 C
ATOM 700 OG SER A 195 -6. 121 6. 010 -15. 073 1 .00 14 .54 o
ATOM 701 o PRO A 196 -3. 707 10. 264 -17. 314 1 .00 14 .43 o
ATOM 702 N PRO A 196 -3. 646 6. 661 -17. 337 1 .00 10 .66 N
ATOM 703 CA PRO A 196 -3. 244 7. 982 -17. 812 1 .00 10 .30 C
ATOM 704 C PRO A 196 -A- 059 9. 108 -17. 160 1 .00 11 .64 C
ATOM 705 CB PRO A 196 -3. 528 7. 903 -19. 315 1 .00 11 .83 C
ATOM 706 CG PRO A 196 -3. 316 6. 461 -19. 648 1 .00 14 .80 C
ATOM 707 CD PRO A 196 -3. 838 5. 705 -18. 444 1 .00 9 .99 C
ATOM 708 o GLY A 197 -5. 871 11. 014 -13. 683 1 .00 13 .21 o
ATOM 709 N GLY A 197 -5. 152 8. 782 -16. 480 1 .00 9 .62 N
ATOM 710 CA GLY A 197 -5. 915 9. 792 -15. 761 1 .00 9 .87 C
ATOM 711 C GLY A 197 -5. 416 10. 073 -14. 348 1 .00 12 .55 C
ATOM 712 N LEU A 198 -4. 481 9. 253 -13. 886 1 .00 9 .10 N
ATOM 713 CA LEU A 198 -4. 025 9. 324 -12. 507 1 .00 12.52 C
ATOM 714 C LEU A 198 -2. 508 9. 374 -12. 432 i .00 13 .12 C
ATOM 715 o LEU A 198 -1. 896 8. 758 -11. 553 1 .00 13 .99 o
ATOM 716 CB LEU A 198 -4. 566 8. 126 -11. 723 1 .00 15 .03 C
ATOM 717 CG LEU A 198 -6. 100 8. 136 -11. 676 1 .00 15 .97 C
ATOM 718 CD1 LEU A 198 -6. 680 6. 764 -11. 365 1 .00 17 .75 C
ATOM 719 CD2 LEU A 198 -6. 591 9. 188 -10. 682 1 .00 21 .63 c
ATOM 720 N LEU A 199 -1. 906 10. 119 -13. 351 1 .00 12 .23 N
ATOM 721 CA LEU A 199 -0. 467 10. 340 -13. 332 1 .00 12 .95 C
ATOM 722 C LEU A 199 -o . 181 11. 668 -12.654 1 .00 15 .29 C
ATOM 723 o LEU A 199 -0. 780 12. 695 -12. 998 1 .00 17 .91 o
ATOM 724 CB LEU A 199 0. 104 10. 371 -14. 742 1 .00 13 .68 C
ATOM 725 CG LEU A 199 -0. 057 9. 078 -15. 540 1 .00 20 .98 C
ATOM 726 CD1 LEU A 199 0. 490 . 9. 249 -16. 963 1 .00 18 .89 c
ATOM ' 727 CD2 LEU A 199 0. 629 7. 942 -14. 826 1 .00 25 .70 c
ATOM 728 N PRO A 200 0. 731 11. 663 -11. 682 1 .00 13 .06 N
ATOM 729 CA PRO A 200 1. 031 12. 951 -11. 055 1 .00 15 .98 c
ATOM 730 C PRO A 200 1. 562 13. 948 -12. 076 1 .00 17 .03 c
ATOM 731 o PRO A 200 2. 294 13. 567 -12. 990 1 .00 21 .50 o
ATOM 732 CB PRO A 200 2. 119 12. 591 -10. 049 .1 .00 14 .40 c
ATOM 733 CG PRO A 200 1. 802 11. 164 -9. 661 1 .00 12 .58 c
ATOM 734 CD PRO A 200 1. 399 10. 545 -10. 991 1 .00 10 .22 c
ATOM 735 N ALA A 201 1. 187 15. 213 -11. 924 1 .00 15 .07 N ATOM 736 CA ALA A 201 1.684 16.266 -12.808 1.00 20.52 C
ATOM 737 C ALA A 201 3. 186 16 .449 -12 .615 1 .00 28 .10 C
ATOM 738 0 ALA A 201 3. 708 16 .242 -11 .524 1 ; oo 32 .55 o
ATOM 739 CB ALA A 201 0. 945 17 .569 -12 .544 1 .00 25 .64 C
ATOM 740 0 GLU A 202 5. 182 19 .253 -12 .910 1 .00 75 .44 "O TOM 741 N GLU A 202 3. 876 16 .832 -13 .683 1 .00 52 .31 N
ATOM 742 CA GLU A 202 5. 328 16 .971 -13 .647 1 .00 67 .08 C
ATOM 743 C. GLU A 202 5. 767 18 .174 -12 .816 1 .00 71 .59' C
ATOM 744 CB GLU A 202 5. 878 17 .078 -15 .066 1 .00 74 .28 C
ATOM 745 , CG GLU A 202 5. 414 15 .963 -15 .983 1 .00 81 .03 C
ATOM 746 CD GLU A 202 5. 873 16 .157 -17 .415 1 .00 86 .16 C
ATOM 747 OEl GLU A 202' 6. 410 17 .242 -17 .725 1 .00 86 .52 0
ATOM 748 OE2 GLU A 202 5. 697 15 .226 -18 .230 1 .00 87 .99 o TOM 749 o GLN A 209 11. 573 11 .436 -3 .871 1 .00 41 .46 o
ATOM 750 N GLN A 209 14. 170 10 .593 -4 .234 1 .00 45 .27 N TOM 751 CA GLN A 209 13. 073 9 .768 -4 ,729 1 .00 44 .07 C
ATOM 752 C GLN A 209 11. 771 10 .561 -4 .720 1 .00 39 .40 C
ATOM 753 CB GLN A 209 12. 930 8 .499 -3 .888 1 .00 42 .37 C
ATOM 754 CG GLN A 209 11. 827 7 .560 -4 .356 1 .00 46 .80 C
ATOM 755 CD GLN A 209 ί 11. 589 6 .416 -3 .385 1 .00 51 .76 C
ATOM 756 OEl GLN A 209 12. 485 6 .029 -2 .635 1 .00 56 .89 o
ATOM 757 NE2 GLN. A 209 10; 373 5 .878 -3 .387 1 .00 49 .04 N TOM 758 N VAL A 210 10. 880 10 .244 -5 .653 1 .00 30 .09 N
ATOM 759 CA VAL A 210 9. 663 11 .026 -5 .824 1 .00 24 .23 C
ATOM 760 C VAL A 210 8. 381 10 .225 -5 .550 1 .00 13 .21 C
ATOM 761 o VAL A 210 8. 267 9 .047 -5 .910 1 .00 16 .25 o
ATOM 762 CB VAL A 210 9. 619 11 .675 -7 .219 1 .00 24 .95 C
ATOM 763 CGI VAL A 210 8. 363 12 .495 -7 .380 1 .00 18 .54 C
ATOM 764 CG2 VAL A 210 10. 852 12 .550 -7 .420 1 .00 27 .65 C
ATOM 765 N CYS A 211 7. 435 10 .877 -4 .881 .1 .00 12 .40 N
ATOM 766 CA CYS A 211 6. 180 10 .253 -4 .489 1 .00 12 .44 C
ATOM 767 C CYS A 211. 5. 002 11 .133 -4 .838 1 .00 10 .67 C
ATOM 768 o CYS A 211 5. 147 12 .328 -5 .071 1 .00 14 .49 o
ATOM 769 CB CYS A 211 6. 162 10 .003 -2 .981 1 .00 17 .15 C
ATOM 770 SG CYS A 211 7. 429 8 .848 -2 .424 1 .00 21 .93 s
ATOM 771 N GLY A 212 3. 818 .10 .538 -4 .868 1 .00 13 .11 N
ATOM 772 CA GLY A 212 2. 646 11 .275 -5 .297 1 .00 11 .08 C
ATOM 773 C GLY A 212 1. 740 11 .691 -4 .146 1 .00 11 .14 C
ATOM 774 o GLY A 212 1. 648 11 .015 -3 .119 1 .00 8 .88 o
ATOM 775 N TYR A 213 1. 039 12 .802 -4 .350 1 .00 9 .39 N
ATOM 776 CA TYR A 213 • 0. 049 13 .262 -3 .392 1 .00 8 .06 " C
ATOM 777 C TYR A 213 -1. 151 13 .872 -4 .089 1 .00 11 .37 c
ATOM 778 o TYR A 213 -1. 071 14 .252 -5 .248 1 .00 8 .32 o
ATOM 779 CB TYR A 213 0. 655 14 .279 -2 .422 1 .00 6 .45 c
ATOM 780 CG TYR A 213 1. 124 15 .612 -2 .985 1 .00 9 .67 c
ATOM 781 CD1 TYR A 213 0. 380 16 .773 -2 .782. 1 .00 12 .72 c
ATOM 782 CD2 TYR A 213 2. 335 15 .722 -3 .661 1 .00 12 .98 c
ATOM 783 CE1 TYR A 213 0. 826 18 .013 -3 .253 1 .00 16 .09 c
ATOM 784 CE2 TYR A 213 2. 786 16 .957 -4 .148 1 .00 11 .06 c TOM 785 CZ TYR A 213 2. 026 18 .098 -3 .940 1 .00 15 .46 c
ATOM 786 OH TYR A 213 2. 486 19 .328 -4 .406 1 .00 15 .04 o
ATOM 787 N VAL A 214 -2. 277 13 .931 -3 .383 1 .00 9 .88 N
ATOM 788 C VAL A 214 -3. 628 15 .956 -3 .252 1 .00 11 .28 c
ATOM 789 o VAL A 214 -3. 390 16 .116 -2 .059 1 .00 9 .38 o
ATOM 790 CA , AVAL A 214 -3. 463 14 .590 -3 .906 0 .50 12 .78 c
ATOM 791 CB , AVAL A 214 -4. 726 13 .756 -3 .641 0 .50 14 .75 c
ATOM 792 CGIAVAL A 214 -5. 949 14 .434 -4 .237 0 .50 9 .83 c ATOM 793 CG2AVAL A 214 -4.569 12.361 -4.212 0.50 22.03 C
ATOM 794 CA BVAL A 214 -3 .461 14 .595 -3. 909 0.50 12.73 C
ATOM 795 CB BVAL A 214 -4 .749 13 .760 -3. 698 0.50 14.82 C
ATOM 796 CG1BVAL A 214 -4 .740 12 .527 -4. 575 0.50 20.83 C
ATOM 797 CG2BVAL A 214 -4 .924 13 .370 -2. 237 0.50 6.30 C
ATOM 798 N LYS A 215 -4 .027 16 .945 -4. 042 1.00 11.27 N
ATOM .799 CA LYS A 215 -4 .321 18 .279 -3. 524 1.00 9.37 C
ATOM 800 C LYS A 215 -5 .523 18 .805 -4. 283 1.00 17.11 C
ATOM 801 0 LYS A 215 -5 .449 19 .010 -5. 493 1.00 16.01 O
ATOM 802 CB LYS A 215 -3 .145 19 .222 -3. 735 1.00 7.00 C
ATOM 803 CG LYS A 215 -3 .327 20 .570 -3. 045 1.00 12.38 C
ATO 804 CD LYS A 215 -2 .221 21 .555 -3. 459 1.00 14.65 C
ATOM 805 CE LYS A 215 -2 .257 22 .830 -2. 614 1.00 15.94 C
ATOM 806 NZ LYS A 215 -2 .048 22 .540 -1. 157 1.00 15.04 N
ATOM 807 N SER A 216 -6 .631 19 .004 -3. 576 1.00 13.63 N
ATOM 808 CA SER A 216 -7 .876 19 .378 -4. 216 1.00 13.70 C
ATOM 809 C SER A 216 -8 .253 18 .337 -5. 281 1.00 15.54 C
ATOM 810- o SER A 216 -8 .539 17 .189 -4. 957 1.00 18.53 O
ATOM 811 CB SER A 216 -7 .767 20 .776 -4. 820 1.00 18.84 C
ATOM 812 OG SER A 216 -7 .466 21 .712 -3. 791 1.00 26.59 · O
ATOM 813 N ASN A 217 -8 .249 18 .728 -6. 551 1.00 15.50 N TOM 814 . CA ASN A 217 -8 .696 17 .814 -7. 601 1.00 18.09 C
ATOM 815 C ASN A 217 -7 .555 17 .165 -8. 383 1.00 19.88 C
ATOM 816 o ASN A 217 -7 .789 16 .466 -9. 375 1.00 19.84 O
ATOM 817 CB ASN A 217 -9 .613 18 .549 -8. 576 1.00 23.23 C
ATOM 818 CG ASN A 217 -10 .865 19 .071 -7. 913 1.00 32.11 C
ATOM 819 ODl ASN A 217 -11 .549 18 .348 -7. 192 1.00 26.94 O
ATOM 820 ND2 ASN A 217 -11 .165 20 .345 -8. 142 1.00 41.14 N
ATOM 821 N SE A 218 -6 .326 17 .398 -7. 936 1.00 17.89 N
ATOM 822 CA SER A 218 -5 .153 17 .048 -8. 729 1.00 15.47 C
ATOM 823 C SER A 218 -4 .210 16 .072 -8. 028 1.00 19.12 C
ATOM 824 o SER A 218 -4 .043 16 .133 -6. 815 1.00 12.77 O
ATOM 825 CB SER A 218 -4 .381 18 .316 -9. 063 1.00 23.32 C *
ATOM 826. OG SER A 218 -5 .202 19 .216 -9. 795 1.00 24.89 O
ATOM 827 N LEU A 219 -3 .591 15 .186 -8. 805 1.00 17.17 N
ATOM 828 CA LEU A 219 -2 .465 14 .388 -8. 325 1.00 14.3 C
ATOM 829 C LEU A 219 -1 .166 15 .085 -8. 708 1.00 14.82 C
ATOM 830 0 LEU A 219 -0 .964 15 .430 -9. 877 1.00 15.09 O
ATOM 831 CB LEU A 219 -2 .490 12 .987 -8. 927 1.00 15.42 C
ATOM 832 CG LEU A 219 -3 .477 12 .009 -8. 306 1.00 20.30 C
ATOM 833 CD1 LEU A 219' -3 .657 10 .775 -9. 190 1.00 24.67 C
ATOM 834 CD2 LEU A 219 -2 .958 11 .612 -6. 946 1.00 19.47 C
ATOM 835 N LEU A 220 -0 .308 15 .311 -7. 713 1.00 12.81 N
ATOM 836 CA LEU A 220 0 .966 16 .003 -7. 904 1.00 7.17 C
ATOM 837 C LEU A 220 2 .138 15 .168 -7. 427 1.00 12.93 C
ATOM 838 0 LEU A 220 1 .959 .14 .129 -6. 800 1.00 13.24 O
ATOM 839 CB LEU A 220 0 .953 17 .335 -7. 129 1.00 10.43 C
ATOM 840 CG LEU A 220 -0 .228 18 .243 -7. 498 1.00 17.80 C
ATOM 841 CD1 LEU A 220 -0 .198 18 .653 -8. 9 1.00 20.78 C
ATOM 842 CD2 . LEU A 220 -0 .258 19 .475 -6. 602 1.00 16.37 C
ATOM 843 SER A 221 3 .340 15 .670 -7. 696 1.00 12.45 N
ATOM 844 CA SER A 221 4 .588 14 .987 -7'. 403 1.00- 16.08 C
ATOM 845 e SER A 221 5 .438 15 .770 -6. 398 1.00 13.43 C
ATOM 846 o SER A 221 5 .494 17 .001 -6. 446 1.00 16.45 O
ATOM 847 CB SER A 221 5 .378 14 .848 -8. 704 1.00 17.83 C
ATOM 848 OG SER A 221 6 .620 14 .225 -8. 468 ' 1.00 33.51 O
ATOM 849 N SE A 222 6 .117 15 .055 -5. 503 1.00 11.49 N ATOM 850 CA SER A 222 6.970 15.705 -4.506 1.00 14.98 C
ATOM 851 C SER A 222 8. 129 14. 809 -4. 087 1 .00 19 .42 C
ATOM 852 o SER A 222 8. 021 13. 583 -4. 109 1 .00 14 .37 o
ATOM 853 CB SER A 222 6. 150 16. 067 -3. 264 1 .00 23 .85 c
ATOM 854 OG SER A 222 6. 977 16. 601 -2. 239 1 .00 23 .68 o
ATOM 855 N ASN A 223 9. 238 15. 422 -3. 690 1 .00 14 .95 N
ATOM 856 CA ASN A 223 10. 254 14. 698 -2. 942 1 .00 15 .93 c
ATOM 857 C ASN A 223 9. 561 13. 968 -1. 792 1 .00 19 .34 c
ATOM 858 0 ASN A 223 8. 729 14. 555 -1. 092 1 .00 13 .62 o
ATOM 859 CB ASN A 223 11. 286 15. 689 -2. 402 1 .00 16 .98 c
ATOM 860 CG ASN A 223 12. 470 15. oio- -1. 771 1 .00 23 .79 c
ATOM 861 ODl ASN A 223 12. 326 14. 234 -0. 827 1 .00 22 .81 o
ATOM 862 ND2 ASN A 223 13. 659 15. 293 -2. 293 1 .00 34 .02 N TOM 863 N CYS A 224 9. 887 12. 690 -1. 608 1 .00 16 .77 N
ATOM 864 CA CYS A 224 9. 232 11. 856 -0. 598 1 .00 15 .48 C
ATOM 865 C CYS A 224 9. 537 12. 274 0. 839 1 .00 16 .89 C
ATOM 866 o CYS A 224 8. 786 11. 940 1. 774 1 .00 15 .97 o
ATOM 867 CB CYS A 224 9. 634 10. 396 -0. 787 1 .00 16 .28 C
ATOM . 868 SG CYS A 224 9. 253 9. 750 -2. 421 1 .00 24 .65 S
ATOM 869 N ASP A 225 10. 648 12. 992 1. 002 1 .00 11 .31 N
ATOM 870 CA ASP A 225 11. 131 13. 432 2. 310 1 .00 13 .57 C
ATOM 871 C ASP A 225 10. 643 14. 845 2. 587 1 .00 21 .06 ' C
ATOM 872 o ASP A 225 11. 427 15. 764 2. 844 1 .00 21 .71 o
ATOM 873 CB ASP A 225 12. 658 13. 374 2. 356 1 .00 18 .91 C
ATOM 874 CG ASP A 225 13. 221 13. 753 3. 724 1 .00 31 .91 C
ATOM 875 ODl ASP A 225 12. 584 13. 437 4. 756 1 .00 27 .89 o
ATOM 8.76 OD2 ASP A 225 14. 309 14. 366 3. 762 1 .00 32 .11 o
ATOM 877 N THR A 226 9. 329 15. 006 2. 527 1 .00 12 .38
ATOM 878 CA THR A 226 8. 678 16. 249 2. 894 1 .00 13 .82 c
ATOM 879 C THR A 226 7. 459 15. 915 3. 742 1 .00 10 .32 c
ATOM 880 o THR A 226 6. 934 14. 808 3. 662 1 .00 13 .56 o
ATOM 881 CB THR A 226 8. 242 17. 041 1. 639 1 .00 19 .45 c
ATOM 882 OGl THR A 226 7. 404 16. 215 0. 817 1 .00 17 .99 o
ATOM 883 CG2 THR A 226 9. 458 17. 447 0. 826 1 .00 17 .65 c
ATOM 884 N TRP A 227 6. 989 16. 876 4. 530 1 .00 10 .43 N
ATOM 885 CA TRP A 227 5. 852 16. 653 5. 433 1 .00 7 .24 C
ATOM 886 £ TRP A 227 4. 527. 17. 028 4. 790 1 .00 13 .17 C
ATOM 887 o TRP A 227 4. 332 18. 174 4. 378 1 .00 12 .26 o
ATOM 888 CB TRP A 227 6. 027 17. 489 6. 715 .1 .00 10 .28 C
ATOM 889 CG TRP A 227 6. 881 16. 864 7. 768 1 .00 11 .26 c
ATOM 890 CD1 TRP A 227 7. 361 15. 589 7. 796 1 .00 16 .27 · c
ATOM 891 CD2 TRP A 227 7. 368 17· 502 8. 957 1 .00 12 .42 c
ATOM 892 NE1 TRP A 227 8. 108 15. 390 8. 939 1 .00 14 .60 N
ATOM 893 CE2 TRP A 227 8. 122 16. 551 9. 665 1 .00 12 .79 C
ATOM 894 , CE3 TRP A 227 7. 211 18. 781 9. 503 1 .00 18 .28 C
ATOM 895 CZ2 TRP A 227 8. 735 16. 843 10. 882 1 .00 20 .62 C
ATOM 896 CZ3 TRP A 227 7. 823 19. 066 10. 709 1 .00 17 .89 C
ATOM 897 CH2 TRP A 227 8. 571 18. 102 11. 384 1 .00 19 .48 c
ATOM 898 N LYS A 228 3. 622 16. 053 4. 687 1 .00 10 .59 N
ATOM 899 CA LYS A 228 2. 284 16. 288 4. 156 1 .00 11 .16 C
ATOM 900 C LYS A 228 1. 320 15. 476 4. 976 1 .00 10 .20 C
ATOM1 901 o LYS A 228 1. 738 14. 607 5. 735 1 .00 10 .79 o
ATOM 902 CB LYS A 228 2. 206 15. 856 2. 684 1 .00 8 .62 C
ATOM 903 CG LYS A 228 3. 166 16. 628 1. 787 1 .00 6 .83 C
ATOM 904 CD LYS A 228 3. 015 16. 213 0. 330 1 .00 10 .96 C
ATOM 905 CE LYS A 228 3. 537 17. 310 -0. 595 1 .00 14 .78 C
ATOM 906 NZ LYS A 228 4. 981 17. 583 -0. 390 1 .00 25 .40 N ATOM 907 N TYR A 229 0.027 15.733 4.842 1.00 7.91 N
ATOM 908 CA TYR A 229 -0 .936 14. 832 5. 466 1 .00 6. 20 C
ATOM 909 C TYR A 229 -0 .931 13. 539 4. 643 1 .00 6. 45 C
ATOM 910 o TYR A 229 -0 .186 13. 423 3. 672 1 .00 8. 93 o
ATOM 911 CB TYR A 229 -2 .324 15. 443 5. 440 1 .00 6. 10 c
ATOM 912 CG TYR A 229 -2 .413 16. 755 6. 187 1 .00 8. 17 c
ATOM 913 CD1 TYR A 229 -2 .141 17. 972 5. 550 1 .00 8. 69 c
ATOM 914 CD2 TYR A 229 -2 .762 16. 777 7. 530 1 .00 10. 49 c
ATOM 915 CE1 TYR A 229 -2 .216 19. 187 6. 252 1 .00 8. 72 c
ATOM 916 CE2 TYR A 229 -2 .837 17. 980 8. 23 1 .00 10. 31 c
ATOM 917 CZ TYR A 229 -2 .577 19. 175 7. 580 1 .00 11. 40 c TOM 918 OH TYR A 229 -2 .653 20. 368 8. 286 1 .00 10. 79 o
ATOM 919 N PHE A 230 -1 .755 12. 575 5. 036^ 1 .00 8. 96 N TOM 920 CA PH.E A 230 -1 .858 11. 327 4. 283 1 .00 6. 45 c
ATOM · 921 C PHE A 230 -3 .252 10. 717 4. 374 1 .00 7. 56 c
ATOM 922 o. PHE A 230 -4 .053 11. 084 5. 226 1 .00 11. 11 o
ATOM 923 CB PHE A 230 -0 .790 10. 328 4. 735 1 .00 6. 92 c
ATOM . 924 CG PHE A 230 -0 .852 9. 980 6. 182 1 .00. 10. 36 c
ATOM .925 CD1 PHE A 230 -1 .655 8. 949 6. 629 1 .00 12. 40 c
ATOM 926 CD2 PHE A 230 -0 .084 10. 673 7. 095 1 .00 11. 47 c
ATOM . 927 CE1 PHE A 230 -1 .695 8. 618 7. 976 1 .00 13. 36 c
ATOM 928 CE2 PHE A 230 -0 .118 10. 356 8. 455 1 .00 13. 16 c
ATOM 929 CZ PHE A 230 -0 .929 9. 336 8. 890 1 .00 12. 18 c
ATOM 930 N ILE A 231 -3 .562 9. 821 3. 442 1 .00 9. 12 N
ATOM 931 CA ILE A 231 -4 .835 9. 116 3. 475 1 .00 13. 25 c
ATOM 932 C ILE A 231 -4 .527 7. 630 * 3. 408 1 .00 9. 05 c
ATOM 933 o ILE A 231 -3 .719 7. 211 2. 576 1 .00 10. 55 o
ATOM 934 CB ILE A 231 -5 .739 9. 503 2. 280 1 .00 11. 73 c
ATOM 935 CGI ILE A 231 -6 .120 10. 980 2. 377 1 .00 10. 02 c
ATOM 936 CG2 ILE A 231 -6 .996 8. 682 2. 298 1 .00 12. 84 c
ATOM 937 CD1 ILE A 231 -6 .646 11. 571 1. 054 1 .00 7. 16 c
ATOM 938 N CYS A 232 -5 .139 6. 865 4. 311 1 .00 10. 83 • N
ATOM 939 CA CYS A 232 -4 .988 5. 417 4. 346' 1 .00 10. 93 c
ATOM 940 C CYS' A 232 -6 .256 4. 782 3. 832 1 .00 10. 32 c
ATOM 941 o CYS A 232 -7 .337 5. 369 3. 907 1 .00 13. 83 o
ATOM 942 CB CYS A 232 -4 .772 4. 903 5. 773 1 .00 15. 44 c
ATOM 943 SG CYS A 232 -3 .392 5. 618 6. 681 1 .00 17. 10 S
ATOM 944 N GLU A 233 -6 .116 3. 553 3. 346 1 .00 11. 33 N
ATOM 945 CA GLU A 233 -7 .233 2. 806 2. 792 1 .00 10. 39 C
ATOM 946 C GLU A 233 -7 .124 1. 346 3. 196 1 .00 12. 76 C TOM 947 o GLU A 233 -6 .047 0. 758 3. 126 1 .00 13. 93 o
ATOM 948 CB GLU A 233 -7 .222 2. 896 1. 265 1 .00 12. 53 C
ATOM 949 CG GLU A 233 -8 .240 1. 985 0. 585 1 .00 12. 85 C
ATOM 950 CD GLU A 233 -8 .237 2. 089 -0. 926 1 .00 16. 23 c
ATOM 951 OEl GLU A 233 -9 .161 1. 520 -1. 555 1 .00 14. 83 o-
ATOM 952 OE2 GLU A 233 -7 .317 2. 722 -1. 493 1 .00 12. 96 o
ATOM 953 N LYS A 234 -8 .241 0. 749 3. 589 1 .00 15. 49 N
ATOM 954 CA LYS A 234 -8 .277 -0. 697 3. 753 1 .00 17. 07 c
ATOM 955 C LYS A 234 -9 .625 -1. 251 3. 335 1 .00 19. 92 c
ATOM 956 o LYS A 234 -10 .598 -0. 500 3. 206 1 .00 18. 94 o
ATOM 957 CB LYS A 234 -7 .961 -1. 098 5. 196 1 .00 20. 47 c
ATOM 95'8 CG LYS A 234 -8 .'958 -0. 620 6. 227 1 .00 15. 77 c
ATOM 959 CD LYS A 234 -8 .485 -1. 096 7. 615 1 .00 23. 98 c
ATOM 960 CE LYS A 234 -9 .187 -0. 393 8. 752 1 .00 35. 09 c
ATOM 961 NZ LYS A 234 -8 .770 -0. 952 10. 086 1 .00 28. 41 N
ATOM 962 N TYR A 235. -9 .666 -2. 565 3. 126 1 .00 21. 53 N
ATOM 963 CA TYR A 235 -10 .891 -3. 246 2. 741 1 .00 23. 84 C ATOM 964 C TYR A 235 -11.922 -3.131 3.852 1.00 23.11
ATOM 965 0 TYR A 235 -11. 623 -3. 388 5 .023 1.00 23.43
ATOM 966 CB TYR A 235 -10. 631 -4. 733 2 .478 1.00 23.99
ATOM 967 -CG TYR A 235 -9. 817 -5. 073 1 .240 1.00 22.87
ATOM 968 CDl TYR A 235 -10. 298 -4. 804 -0 .036 1.00 25.03
ATOM 969 CD2 TYR A 235 -8. 582 -5. 708 1 .355 1.00 25.61
ATOM 970 CE1 TYR A 235 -9. 559 -5. 132 -1 .160 1.00 21.63
ATOM 971 CE2 TYR A 235 -7. 836 -6. 040 0 .239 1.00 26.01
ATOM 972 CZ TYR A 235 -8. 333 -5. 757 -.1 .021 1.00 27.88
ATOM 973 OH TYR A 235 -7. 596 -6. 095 -2 .146 1.00 25.83
ATOM 974 o ALA A 236 -14. 421 -5. 137 3 .866 1.00 25.08
ATOM 975 N ALA A 236 -13. 139 -2. 746 3 .485 1.00 25.41
ATOM 976 CA ALA A 236 -14. 257 -2. 797 4 .418 1.00 24.94
ATOM 977 C ALA A 236 -14. 608 -4. 258 4 .714 1.00 30.51
ATOM 978 CB ALA A 236 -15. 461 -2. 064 3 .848 1.0,0 32.29
ATOM 979 o LEU A 237 -17. 380 -5. 636 4 .882 1.00 39.84
ATOM 980 N . LEU A 237 -15. 118 -4. 509 5 .912 1.00 31.55
ATOM 981 CA LEU A 237 -15. 477 -5. '863 6 .322 1.00 40.28
ATOM 982' C LEU A 237 -16. 660 -6. 397 5 .523 1.00 40.58
ATOM 983 CB LEU A 237 -15. 801 -5. 899 7 .815 1.00 39.27
ATOM 984 CG LEU A 237 -14. 688 -5. 447 8 .756 1.00 46.66
ATOM 985 CDl LEU A 237 -15. 155 -5. 525 10 .199 1.00 50.26
ATOM 986 CD2 LEU A 237 -13. 436 -6. 288 8 .551 1.00 49.68
TER 987 LEU A .237
END

Claims

1. A polypeptide complex conjugated to a therapeutic agent or a detectable label, wherein the polypeptide comple comprises
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A.
2. The polypeptide complex of claim 1, wherein the first polypeptide is a spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titin, myosin, tubulin, catenin, keratin, cytokeratin, nestin, lamin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
3. The polypeptide complex of claim 1 or claim 2, wherein the first polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 41 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of
SEQ ID NOs 41 to 89; and/or
c) an actin binding fragment of a) or b).
4. The polypeptide complex according to any one of claims 1 to 3, wherein the second polypeptide comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 26 to 40; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 26 to 40; and/or
c) a fragment of a) or b) which binds an actin binding domain.
5. The polypeptide complex according to any one of claims 1 to 4, wherein the Clec9A comprises
a) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8;
b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 1 to 8.
6. The polypeptide complex according to any one of claims 1 to 5, wherein when bound to Clec9A, the complex further comprises RNF41 bound to the Clec9A.
7. The polypeptide complex of claim 6, wherein the RNF41 comprises
5 a) an amino acid sequence as provided in any one of SEQ ID NOs 21 to 25;
b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 21 to 25; and/or
c) a fragment of a) or b) which binds Clec9A.
10 8. The polypeptide complex according to any one of claims 1 to 7, wherein the therapeutic agent is an antigen.
9. The polypeptide complex of claim 8, wherein the antigen is a cancer antigen, a self-antigen, an allergen, and/or an antigen from a pathogenic and/or infectious
15 organism.
10. The polypeptide complex according to any one of claims 1 to 7, wherein the therapeutic agent is a cytotoxic agent.
20 11. The polypeptide complex according to any one of claims 1 to 7, wherein the therapeutic agent is a drug and or pharmacological agent.
12. The polypeptide complex according to any one of claims ί to 11, wherein the first and second polypeptides are separate polypeptide chains, or form part of a single
25 polypeptide chain.
13. The polypeptide according to any one of claims 1 to 12, wherein the actin is filamentous actin or a filamentous fragment thereof.
30 14. A compound that binds a polypeptide complex which comprises:
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and 35 wherein the polypeptide complex is capable of binding Clec9A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A.
15. The compound of claim 14 which is a polypeptide.
16. The compound of claim 15 which is an antibody or antigen-binding fragment thereof.
17. The compound of claim 16, wherein the antibody is a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
18. The compound according to any one of claims 14 to 17 which is conjugated to a therapeutic agent or a detectable label.
19. The compound of claim 18, wherein the therapeutic agent is one or more of a cytotoxic agent, a drug, or a pharmacological agent.
20. The compound according to any one of claims 14 to 19, wherein the actin is filamentous actin or a filamentous fragment thereof.
21. A composition comprising a polypeptide complex according to any one of claims 1 to 13, or a compound according to any one of claims 14 to 20, and a pharmaceutically acceptable carrier.
22. The composition of claim 21 which further comprises an adjuvant.
23. The composition of claim 21 or claim 22, wherein the polypeptide complex or compound is encapsulated in, or exposed on the surface of, a liposome.
24. A method of modulating an immune response in a subject, the method comprising administering to the subject at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of/binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and
v) a composition according to any one of claims 21 to 23.
25. The method of claim 24, wherein
i) an immune response to an antigen is induced and/or enhanced, and/or ii) an immune response to a self-antigen or allergen is reduced.
26. A method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13, ,
iii) a compound according to any one of claims 14 to 20, and
iv) a composition according to any one of claims 21 to 23, and
administering said cells to the subject.
27. The method of claim 26, wherein the cells have been isolated from the subject.
28. The method according to any one of claims 24 to 27 which comprises modulating a humoral and/or T-cell mediated response.
29. The method of claim 28 which comprises modulating naive CD8+ T-cell activation and/or naive CD4+ T-cell activation.
30. A method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject at least one of i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and
y b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and
iv) a composition according to any one of claims 21 to 23.
31. The method of claim 30 which comprises administering a polypeptide complex of claim 10 or claim 11 and/or a compound of claim 19 or claim 20.
32. A method of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering to the subject at least one of i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and
iv) a composition according to any one of claims 21 to 23.
33. A method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and iv) a composition according to any one of claims 21 to 23.
34. A method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9 A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and
iv) a composition according to any one of claims 21 to 23.
35. The method according to any one of claims 32 to 34, wherein the subject is suffering from a. disease selected from: graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
36. Use of at least one of
i) a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and
iv) a composition according to any one of claims 21 to 23,
for the manufacture of a medicament for at least one of
a) modulating an immune response in a subject,
b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject, c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) modulating the antigen recognition, processing and or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
37. A
i) polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
ii) a polypeptide complex according to any one of claims 1 to 13,
iii) a compound according to any one of claims 14 to 20, and
iv) a composition according to any one of claims 21 to 23,
for use in at least one of
a) modulating an immune response in a subject,
b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) modulating the antigen recognition, processing and or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and r
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a 5 portion thereof, or surrounding cells, in a subject.
38. A method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
10 i) contacting a cell with a compound that binds a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and
b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
15 wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
. ii) determining whether the polypeptide complex is present or absent, wherein the presence of the complex polypeptide provides a diagnosis, prognosis
20 and/or status of the disease.
39. The method of 38, wherein the compound is an antibody or antigen-binding fragment thereof.
25 40. The method of claim 38 or claim 39, wherein the compound is detectably labelled.
41. The method according to any one of claims 38 to 40 which is performed in vivo on a subject.
30
42. The method according to any one of claims 38 to 40 which is performed in vitro on a sample obtained from a subject.
43. The method according to any one of claims 38 to 42, wherein the disease is 35 selected from: graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
44. A method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
i) exposing a cell to the therapy, and
ii) detecting a cell with a disrupted cell membrane, a dying cell or a dead cell, or a portion thereof, using a method according to any one of claims 38 to 43,
wherein the presence of a cell with a disrupted cell membrane, a dying cell or a dead cell indicates that the therapy is effective.
45. The method of claim 44, wherein the cell in step i) is in vivo.
46. The method of claim 44 or claim 45, wherein the therapy is administered to a subject.
47. The method according to any one of claims 44 to 46, wherein the subject has cancer or an infection.
48. The method according to any one of claims 44 to 47, wherein step ii) is performed on a sample obtained from a subject.
49. The method according to any one of claims 44 to 48, wherein the therapy is drug therapy or radiotherapy.
.
50. A method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
i) contacting a cell with a compound that binds a polypeptide complex comprising
a) a first polypeptide comprising an actin binding domain, and b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9 A, and ii) determining whether binding of the compound to the polypeptide complex is present or absent,
wherein the compound binding to the polypeptide indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
51. A method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a polypeptide complex according to any one of claims 1 to 10, 12 or 13, or a compound of claim 17, and
ii) isolating cells bound to the polypeptide complex or compound.
52. The method of claim 51, wherein the cells obtained from step ii) are administered to a subject.
53. The method of claim 52, wherein the cells are administered to treat and/or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
54. A method of detecting dendritic cells, or a subset or precursors thereof, in asample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a polypeptide complex according to any one of claims 1 to 10 or 12, or a compound of claim 18, and
ii) detecting cells bound to the compound.
55. A method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
i) administering to the subject a polypeptide complex according to any one of claims 1 to 10, 12 or 13, or a compound of claim 18, and,
ii) detecting cells bound to the compound.
56. The method according to any one of claims 51 to 55, wherein the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CDl lc, HLADR, Clec9A, XCRI and BDCA3.
57. A method of detecting a cell with a disrupted,cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
i) contacting a cell with a compound that binds a polypeptide complex comprising
5 a) a first polypeptide comprising an actin binding domain, and
b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in 10 isolation, and wherein the compound is not Clec9A, and
ii) determining whether binding of the compound to the polypeptide is present or absent,
wherein the compound binding to the polypeptide complex indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying or is dead.
15
58. A compound which
i) binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l,
ii) binds Clec9A and which through stearic hindrance reduces the binding of the 20 one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
a) a first polypeptide comprising an actin binding domain, and
b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, or
25 iii) competes with the one or both tryptophan residues of Clec9A for binding to the polypeptide complex.
59. The compound of claim 58, wherein the compound is a polypeptide.
30 60. The compound of claim 59, wherein the polypeptide is an antibody or antigen- binding fragment thereof.
61. The compound according to any one of claims 58 to 60, wherein the compound does not affect the binding of RNF41 to Clec9A.
62. A Composition comprising a compound according to any one of claims 58 to 61 , and a pharmaceutically acceptable carrier.
63. A method of reducing an immune response in a subject, the method comprising 5 administering to the subject a compound according to any one of claims 58 to 61 and/or a composition of claim 62.
64. A method of reducing an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to a
10 compound according to any one of claims 58 to 61 and/or a composition of claim 62, and administering said cells to the subject.
65. A method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject a compound
15 according to any one of claims 58 to 61 and/or a composition of claim 62.
66. The method of claim 65, wherein the disease is an autoimmune disease or inflammation.
20 67. A method of reducing the uptake and or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering a compound according to any one of claims 58 to 61 and/or a composition of claim 62.
25 68. A method of reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound according to any one of claims 58 to 61 and/or a composition of claim 62.
30
69. A method of reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound according to any one of claims 58 to 61 and/or a composition of claim 62.
70. Use of a compound according to any one of claims 58 to 61 and/or a composition of claim 62 for the manufacture of a medicament for at least one of
i) reducing an immune response in a subject,
ii) reducing an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
iii) treating and/or preventing in a subject a disease involving dendritic cells or precursors thereof,
iv) reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof* in a subject,
v) reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and vi) reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
71. A compound according to any one of claims 58 to 61 and/or a composition of claim 62 for use in at least one of
i) reducing an immune response in a subject,
ii) reducing an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
iii) treating and/or preventing in a subject a disease involving dendritic cells or precursors thereof,
iv) reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
v) reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and vi) reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
72. An isolated and/or exogenous polynucleotide encoding
i) a polypeptide complex according to any one of claims 1 to 11, wherein the first polypeptide and the second polypeptide form part of a single polypeptide chain, or ii) a compound according to any one of claims 13 to 19 or 58 to 61, wherein the 5 compound is a polypeptide.
73. A vector comprising a polynucleotide of claim 72.
74. The vector of claim 73 which is an expression vector.
10
75. A host cell comprising a polynucleotide of claim 72, and/or a vector of claim 73 or claim 74.
76. The host cell of claim 75 which is a bacterial, yeast, insect, animal or plant cell.
" 15
77. An enriched population of dendritic cells and/or precursors thereof, obtained by a method according to any one of claims 51 to 53.
78. An expanded dendritic cell population, and/or precursors thereof, obtained by 20 culturing an enriched population of dendritic cells and/or precursors thereof according to claim 77.
79. A composition comprising a polynucleotide of claim 72, a vector of claim 73 or claim 74, a host cell of claim 75 or claim 76, and/or a cell population of claim 77 or
25 claim 78, and a pharmaceutically acceptable carrier.
80. A crystal of the C-type lectin-like domain of human Clec9A.
81. A set of atomic coordinates, or subset thereof, provided in Appendix I.
30
82. A computer-readable medium having recorded thereon data representing the atomic coordinates, or subset thereof, provided in Appendix I and/or a model produced using the atomic coordinates.
35 83. A computer-assisted method of identifying a compound that binds Clec9A, the method comprising i) docking the structure of a candidate compound to a structure defined by the atomic coordinates, or subset thereof, provided in Appendix I, and
ii) identifying a candidate compound which may bind Clec9A.
5 84. The method of claim 83 which further comprises synthesising or obtaining an identified candidate compound and determining if the compound binds Clec9A.
85. A computer-assisted method for identifying a compound which binds a polypeptide complex which comprises
10 a) a first polypeptide comprising an actin binding domain, and
b) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, the method comprises the steps of:
15 i) comparing a structure defined by the atomic coordinates, or subset thereof, provided in Appendix I, to a computer database of chemical structures, and
ii) selecting from the database a chemical structure which is complementary or similar to the structure defined by the atomic coordinates, or subset thereof, provided in Appendix I.
20
86. The method of claim 85 which further comprises synthesising or obtaining a selected candidate compound and determining if the compound binds the polypeptide complex.
25 87. The set of atomic coordinates of claim 81, the computer-readable medium of claim 82, or the method of according to any one of claims 83 to 86, wherein the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
30 88. A method of identifying a compound which binds a polypeptide complex comprising
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
35 wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, the method comprising a) contacting the complex polypeptide with a candidate compound,
b) determining whether the compound binds the polypeptide complex, and c) optionally selecting a compound which binds the polypeptide complex.
89. A method of identifying a compound which binds a polypeptide complex comprising '
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, arid wherein the polypeptide complex is capable of binding Clec9A, the method comprising a) exposing the polypeptide complex to a binding partner which binds the polypeptide complex, and a candidate compound,
b) assessing the ability of the candidate compound to compete with the binding partner for binding to the polypeptide complex, and
c) optionally selecting a compound which competes with the binding partner for binding to the polypeptide complex.
90. The method of claim 89, wherein the binding partner is Clec9A or a soluble fragment thereof.
91. A method of identifying a compound which binds one or both of the tryptophan " residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO.l, the method comprising
a) contacting Clec9A or a fragment thereof comprising one or both of the tryptophan residues with a candidate compound,
b) determining whether the compound binds one or both of the tryptophan residues,
c) determining if the compound reduces or inhibits the binding of Clec9A or a fragment thereof to a polypeptide complex comprising - i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and
d) optionally selecting a compound which binds one or both of the tryptophan residues and which reduces or inhibits the binding of Clec9A or a fragment thereof to the polypeptide complex.
92. A method of identifying a compound which binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l, the method comprising
a) exposing Clec9A or a fragment thereof comprising one or both of the tryptophan residues to a binding partner which binds one or both of the tryptophan residues, and a candidate compound,
b) assessing the ability of the candidate compound to compete with the binding partner for binding to one or both of the tryptophan residues,
c) determining if the compound reduces or inhibits the binding of Clec9A or a fragment thereof binding a polypeptide complex comprising
i) a first polypeptide comprising an actin binding domain, and
ii) a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain,
wherein the actin or fragment thereof is bound to the actin binding domain, and
d) optionally selecting a compound which competes with the binding partner for binding to one or both of the tryptophan residues and which reduces or inhibits the binding of Clec9A or a fragment thereof to the polypeptide complex.
93. A compound identified by the method of according to any one of claims 83 to 92.
94. A kit comprising one or more of a polypeptide complex according to any one of claims 1 to 13, a compound according to any one of claims 14 to 20 or 58 to 61, a polynucleotide of claim 72, a vector of claim 73 or claim 74, a host cell of claim 75 or claim 76, a cell population of claim 77 or claim 78, and a composition according to any one of claims 21 to 23, 62 or 74.
95. Isolated actin or a fragment thereof conjugated to a therapeutic agent or a detectable label, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain.
96. The conjugated actin or fragment thereof of claim 95 which is filamentous actin or a filamentous fragment thereof.
97. The conjugated actin or fragment thereof of claim 95 or claim 96, wherein the therapeutic agent is one or more of an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
98. A composition comprising conjugated actin or a fragment thereof according to any one of claims 95 to 97, and a pharmaceutically acceptable carrier.
99. A method of modulating an immune response in a subject, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98.
100. The method of claim 99, wherein
i) an immune response to an antigen is induced and/or enhanced, and/or ii) an immune response to a self-antigen or allergen is reduced.
101. A method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is riot bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98, and
administering said cells to the subject.
102. The method of claim 101, wherein the cells have been isolated from the subject.
103. A method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject at least one of i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98.
104. The method of claim 103 which comprises administering the conjugated actin or fragment thereof of claim 97.
105. A method of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering to the subject at least one of i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98.
106. A method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98.
107. A method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is riot Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98.
108. The method according to any one of claims 105 to 107, wherein the subject is suffering from a disease selected from: graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
109. The method according to any one of claims 99 to 108, wherein the actin or fragment thereof is filamentous actin or a filamentous fragment thereof.
110. Use of at least one of
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98,
for the manufacture of a medicament for at least one of
a) modulating an immune response in a subject,
b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject, d) modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead^cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
111. A
i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
ii) a compound which binds actin or a fragment thereof of i), wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
iii) actin or a fragment thereof according to any one of claims 95 to 97, and iv) a composition of claim 98,
for use in at least one of
a) modulating an immune response in a subject,
b) modulating an immune response to an antigen in a subject by exposing dendritic cells or precursors thereof in vitro to the compound, and administering said cells to the subject,
c) treating and/or preventing a disease involving dendritic cells or precursors thereof in a subject,
d) modulating the uptake and or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject,
e) modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, and
f) modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject.
112. A method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
i) contacting a cell with a compound that binds actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain, and wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain,
ii) determining whether the actin or fragment thereof is present or absent, wherein the presence of the actin or fragment thereof provides a diagnosis, prognosis and/or status of the disease.
113. The method of claim 112, wherein the compound is detectably labelled.
114. The method of claim 112 or claim 113 which is performed in vivo on a subject, or which is performed in vitro on a sample obtained from a subject.
115. A method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
i) exposing a cell to the therapy, and
ii) detecting a cell with a disrupted cell membrane, a dying cell or a dead cell, or a portion thereof, using a method according to any one of claims 112 to 1 14, wherein the presence of a cell with a disrupted cell membrane, a dying cell or a dead cell indicates that the therapy is effective.
116. A method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
i) contacting a cell with a compound that binds actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain, and wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain, and
ii) determining whether binding of the compound to the actin or fragment thereof is present or absent, wherein the compound binding to the actin or a fragment thereof indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
117. A method of enriching dendritic cells, or a subset or precursors thereof, from a 5 sample comprising ,
i) contacting a sample comprising dendritic cells or precursors thereof with a conjugated actin or fragment thereof of claim 95 or claim 96, and
ii) isolating cells bound to the conjugated actin or fragment.
10 118. A method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
i) contacting a sample comprising dendritic cells or precursors thereof with a conjugated actin or fragment thereof of claim 95 or claim 96, and
ϋ) detecting cells bound to the conjugated actin or fragment.
15
119. A method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
i) administering to the subject a conjugated actin or fragment thereof of claim 95 or claim 96, and,
0 ii) detecting cells bound to' the conjugated actin or fragment.
120. A method of detecting a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
i) contacting a cell with a compound that binds actin or a fragment thereof, 25 wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain, and wherein the compound is not Clec9A or a polypeptide comprising an actin binding domain, and
ii) determining whether binding of the compound to the actin or fragment 30 thereof is present or absent,
wherein the compound binding to the actin or fragment thereof indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying or is dead.
121. An enriched population of dendritic cells and/or precursors thereof, obtained by
35 a method of claim 117.
122. An expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof according to claim 121.
123. A composition comprising a cell population of claim 121 or claim 122, and a pharmaceutically acceptable carrier.
124. A method of identifying a compound which binds actin or a fragment thereof, the method comprising
a) exposing actin or a fragment thereof to Clec9A or a soluble fragment thereof and a candidate compound, wherein the actin or a fragment thereof is not bound to a polypeptide comprising an actin binding domain,
b) assessing the ability of the candidate compound to compete with the CIec9A or a soluble fragment thereof for binding to actin or a fragment thereof, and
c) optionally selecting a compound which competes with the Clec9A or a soluble fragment thereof for binding to the actin or a fragment thereof.
125. A compound identified by the method of claim 124.
126. A kit comprising One or more of a conjugated actin or fragment thereof according to any one of claims 95 to 97, a cell population of claim 121 or claim 122, and a composition of claim 98 or claim 123.
PCT/AU2012/001233 2011-10-14 2012-10-12 Molecules which bind clec9a Ceased WO2013053008A2 (en)

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