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WO2008044075A1 - Protéine membranaire wza et procédé de sélection d'agents inhibiteurs - Google Patents

Protéine membranaire wza et procédé de sélection d'agents inhibiteurs Download PDF

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WO2008044075A1
WO2008044075A1 PCT/GB2007/050631 GB2007050631W WO2008044075A1 WO 2008044075 A1 WO2008044075 A1 WO 2008044075A1 GB 2007050631 W GB2007050631 W GB 2007050631W WO 2008044075 A1 WO2008044075 A1 WO 2008044075A1
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atom
leu
wza
remark
val
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James Naismith
Kostantinos Beis
Changjiang Dong
Christopher Whitfield
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University of Guelph
University of St Andrews
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University of Guelph
University of St Andrews
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions

Definitions

  • the present invention relates to the protein Wza, an outer membrane auxiliary protein, and its use in a method of selecting for agents which inhibit formation of a bacterial capsular polysaccharide layer.
  • EPSs extracellular polysaccharides
  • the role of EPS is protective; capsules provide essential virulence determinants that allow the bacteria to evade or counteract the host immune response.
  • EPSs play critical roles in forming biofilms and in the colonization of surfaces, such as epithelia and medical implants.
  • EPSs show immense diversity in monomer composition, configuration, linkage sequence and type, and substitution with non-carbohydrate residues. They are linear or branched polymers with reported sizes typically ranging from 10 4 -10 6 daltons. EPSs therefore represent one of the largest and most polar molecules to be transported across a biological membrane and the desolvation of hundreds of carbohydrate rings during passage across the cell envelope seemingly presents an insurmountable kinetic barrier to export.
  • E. coli capsular (K antigen) serotype K30 is the prototype for the group 1 capsule assembly system and a model depicting current understanding of the pathway is shown in Fig. 1a.
  • the K30 polysaccharide is assembled and exported by dedicated proteins encoded by a 12-gene operon.
  • the process requires a member of the OMA (outer membrane auxiliary) protein family for the export (or translocation) of nascent polymer across the outer membrane (see Paulsen et al., (1997) FEMS Microbiol Lett 156:1-8).
  • Assembly of E. coli group 2 capsules follows a substantially different pathway.
  • Group 2 polymers are elongated in the cytoplasm by processive glycosyltransferases and are then exported across the inner membrane by an ATP-binding cassette (ABC) transporter. The details of the two pathways have been reviewed recently (see Whitfield, supra).
  • OMA outer membrane auxiliary
  • the best studied OMA family member is Wza from the E. coli group 1 K30 system.
  • Mature Wza is a 359 amino acid protein that is synthesized as a precursor with a cleavable 20 residue N-terminal signal sequence.
  • Wza is a lipoprotein. Cys 21 is modified by a thioether-linked diacylglyceryl group and its amino group is also acylated. Wza forms oligomers that are stable even in SDS.
  • Wza interacts with an inner membrane tyrosine autokinase protein, Wzc (see Reid et al., (2005) J Bacteriol 187:5470:5481 ). Cryo-negatively stained EM reveals that Wzc forms a tetrameric complex (see Collins et al., (2006) J. Biol Chem. 281 :2144- 2150). Mutations that either eliminate Wzc, or that compromise its phosphorylation, turn off capsular polymer biosynthesis (see Wugeditsch et al., J.
  • the present invention provides a highly purified form of Wza, for example a crystal of Wza, which can be resolved to at least 2.26A.
  • the 2.26A resolution crystal structure provides detailed information on the structure of Wza and its function in EPS transport.
  • the Wza is from E. coli.
  • the crystal of Wza is obtained by crystallisation in 7% PEG 4000, 0.1 M sodium acetate pH4.6 and 0.2M (NH 4 ) 2 S0 4 with 10mg/ml protein.
  • the present invention further provides a method of selecting agents which inhibit the export of EPS from the cytoplasm of a bacteria, said method comprising the steps of:
  • step a) comprises providing a model of Wza resolved to at least 2.6A.
  • the bacteria is a capsular bacteria (ie. normally has a capsule).
  • the model of Wza is based on data obtained by X-ray crystallography studies.
  • the model of Wza has the atomic co-ordinates substantially as set out in Annex 1.
  • the model of Wza has the atomic co-ordinates as set out in Annex 1.
  • the potential inhibitory agent interacts with the PES motif of Wza, in particular the PES motif of E. coli K30 Wza or equivalent residues thereto in other OMAs.
  • the potential inhibitory agent interacts with the PES motif Pfam 02563.
  • the PES domain is annotated in at least 20 proteins (see Pfam 02563) on www.ncbi.nlm.nih.gov) as shown in Fig 12.
  • the potential inhibitory agent interacts with residues 105 to 112 of Domain 1 of E. coli K30 Wza, or equivalent residues thereto in other OMAs.
  • Exemplary other OMAs include BexD, CtrA and KpsD, and the like.
  • the potential inhibitory agent interacts with Tyr 110 of Domain 1 of E. coli K30 Wza, or equivalent residues thereto in other OMAs.
  • Exemplary other OMAs include BexD, CtrA and KpsD, and the like.
  • step (a) comprises conducting structural analysis of at least the PES domain of Wza.
  • this structural analysis includes analysis of the PES domain based on X-Ray crystallography co-ordinates thereof.
  • the structural analysis includes analysis of the data as set out in Annex 1.
  • the model of Wza can be in the form of a computer data or graphic file, and will usually be based upon the X-ray crystal co-ordinates of Wza.
  • the potential inhibitory agent may itself be reviewed in the form of a model, for example a computer data file.
  • the interaction between the enzyme and potential inhibitory agent can be analysed through interaction of the models, and conveniently may be calculated by computer.
  • the structure of the potential inhibitory agent can conveniently likewise be reviewed and analysed in the form of X-ray crystal co-ordinates or approximations thereof.
  • the potential intermolecular interaction between the agent under test and the active site of Wza will be analysed with the aid of a computer.
  • the present invention provides an inhibitory agent for the PES domain of E. coli K30 Wza, or equivalent residues thereto in other OMAs.
  • the inhibitory agent interacts with residues 105 to 112, (for example Tyr 110) of Domain 1 in E. coli K30 Wza, or equivalent residues thereto in other OMAs.
  • the agent may have the effects outlined above.
  • EPS extracellular polysaccharides
  • the structure of mature acylated Wza from E. coli K30 was solved using single-wavelength anomalous diffraction with selenomethionine labeling (Fig 2a).
  • the protein crystallizes in space group P2i2i2i with eight monomers in the asymmetric unit. From the location of the selenium atoms, it was clear the asymmetric unit contained a molecule with eightfold rotational symmetry, consistent with an octamer of molecular weight 320 kDa.
  • the final model contains 2848 amino acid residues, 16 lipid fragments, 16 sulfate ions and 1264 water molecules.
  • the octameric structure is best described as having the shape of a classical "amphora" without the handles (Fig. 2b).
  • Wza has a large internal cavity which is opens at a narrow "neck” and is closed at its base.
  • the long axis of the molecule is approximately 140 A and the diameter at the widest point is 105 A (Figs. 2b, 2c).
  • the eight-fold rotational axis runs parallel to the long axis of molecule.
  • Comparison of the crystal structure to that derived from negatively-stained cryo EM see Beis et al., (2004) J. Biol Chem 279:28227-28232) reveals that the "neck” is missing in the EM structure. This region may be unstructured in the conditions of the EM experiment, or it may interact poorly with the stain. The dimensions of the remainder of the structure match well; in particular the large central cavity is seen.
  • the eight copies of domain 1 combine to form a ring structure (ring 1 ) at the bottom of the Wza, with an eight-fold axis through the centre (Figs. 2b, 2c).
  • Ring 1 presents a concave surface at the base of the structure and the centre is filled by eight loops (residues 105 to 112 of each domain). Measuring from the main chain of these loops, the channel is narrowed to 15 A.
  • Tyr 110 is located at the tip of the loop but is not clearly visible in the experimental map, suggesting the side chain is flexible (Fig. 6a). Once past Tyr 110, the ring has an internal diameter of over 25 A (Figs. 2b, 2c).
  • Domain 2 (residues 68-84 and 175- 252) also has a novel structure, despite having similar overall dimensions to domain 1.
  • This domain has a central five-stranded mixed ⁇ -sheet with three ⁇ -helices on one face.
  • the eight copies ⁇ of Domain 2 also forms an eight-fold symmetric ring structure (ring 2) with an inner diameter of approximately 25 A.
  • Domain 3 (residues 46-64 and 255-344) is a structural duplication of domain 2 (Fig. 4c) and, like domain 2, eight copies form a ring structure (ring 3, Fig. 2b).
  • a longer loop connects two ⁇ -strands and there is an extra ⁇ -strand (residues 49 to 54) compared to domain 2 (Fig.
  • the third ring (which is formed from domain 3) has a significantly larger external diameter of 105 A than that of ring 2.
  • the three rings sit one atop the other with a 10 A spacing between rings 2 and 3. These three rings form the body of the amphora.
  • a ribbon representation of the structure (Fig. 2b) gives the appearance of side holes being present, but these are filled by side chains (Figs. 3a and 3b).
  • Domain 4 comprises the C-terminus of the monomer (residues 345-376) (Fig. 2a) and is an amphipathic helix.
  • the helices create an ⁇ -helical barrel (Fig. 2b) at the "neck" of the molecule.
  • each helix is offset by approximately 35° to the long axis of the molecule and is kinked at Pro 359.
  • the barrel is tapered such that part attached to the third ring has an internal diameter of approximately 30 A. At the open end the internal diameter is 17 A.
  • the N-terminus (residues 22 to 45) is long loop which is wrapped around the top of ring 3 (Figs. 2a, 2b).
  • the central cavity of the protein is open to the solvent (Fig. 2b, Fig. 3a) and has an internal volume of approximately 15000 A 3 . This large volume is comparable to the 84000 A 3 reported for chaperone GroEL GroES (see Xu et al., (1997) Nature 388: 741-750) and 44000 A 3 for the outer membrane drug efflux protein ToIC (see Koronakis et al., (2000) Nature 405: 914-919).
  • the protein surface which encloses the central cavity is polar (Fig. 3a) and lined by residues which exhibit little sequence conservation. Those conserved residues that do exist are principally structural, or are located at subunit interfaces (Fig. 5).
  • While typical cell membranes are composed of a phospholipid bilayer, the outer membrane is distinct and asymmetric.
  • the inner leaflet is composed of glycerophospholipids and the outer leaflet contains the unique glycolipid, lipopolysaccharide.
  • all structures of integral outer membrane proteins have contained a trans-membrane ⁇ -barrel (see Ruiz et al., (2006) Nat Rev Microbiol 4: 57- 66; and Schulz (2002) Biochim Biophys Acta Biomembr 1565: 308-317).
  • Wza is the first example of an ⁇ -helical barrel which crosses the outer membrane of bacteria.
  • transmembrane ⁇ -helices are well known in bacterial inner membrane proteins and eukaryotic membrane proteins, there is no corollary for the transmembrane helical barrel observed in Wza. Wza, therefore expands the repertoire of known trans-membrane protein structures.
  • Some antimicrobial peptides are known to form amphipathic helices (see Bulet et al., (2004) Immunol Rev 198: 169-184) and the C- terminus of Wza may mimic antimicrobial peptide pore formation aggregation in the membrane.
  • the simplest model for export of polysaccharide invokes movement of the carbohydrate from the periplasm to the central cavity of Wza and exit through the helical barrel.
  • the cross-sectional area of the carbohydrate polymer is difficult to estimate reliably, because of the inherent conformational flexibility of the glycosidic linkages.
  • the width of the oligosaccharide is approximately 17 A (Fig. 1 b).
  • the helical barrel with its diameter of 17 A would be sufficient to accommodate the exit of the polymer from the large central cavity (Fig. 2c, Fig. 3a) without structural change.
  • the Wza structure shows no portal between the central cavity and periplasm with the loop at Tyr 110 filling in the base of the structure (Figs. 2d and 6c, 6d). Isolated protein embedded in lipid bilayers shows no ion conductance suggesting the channel is normally closed (C. Whitfield and R.E.W. Hancock, unpublished data). Regulation of the opening of the Wza central cavity would seem desirable in maintaining periplasmic integrity. We propose that the opening of the portal requires a substantial conformational change. Both Wza Yi i o A and Wza Yi i ow derivatives of Wza are fully competent for CPS export (Fig.
  • Wza may also represent a model for the mechanism of export of toxins, enzymes and effector proteins via multimeric "secretin" protein complexes found in type II, III and IV secretion systems in bacteria.
  • a particularly interesting example is the export of single-stranded DNA (a large polar molecule) via type IV systems.
  • EM structures of several secretins show these contain ring-like structures which are capable of accommodating large polar substrates.
  • Carbohydrates make a very large number of hydrogen bonds; one glucose molecule, for example, can make 17 hydrogen bonds.
  • the desolvation of hydroxyl groups is energetically unfavorable. Lactose permease solves this problem by making hydrogen bonds to the two carbohydrate rings inside a central cavity which then undergoes a conformational change releasing the encapsulated sugar on the opposite side of the membrane.
  • a similar model has been proposed for the ATP-dependent lipopolysaccharide lipid A flippase, MsbA, in which the carbohydrate head group is sequestered inside a central cavity whilst the protein undergoes conformational change.
  • Wza homologues from different group 1 polysaccharide biosynthesis systems can complement Wza-deficient E. coli K30 (see Drummelsmith et al., supra and Reid et al., supra). This indicates limited recognition between Wza and the polysaccharide, with specificity controlled elsewhere.
  • Wza defines a new class of integral membrane protein and suggests a mechanism for the transport of the capsular polysaccharide across the bacterial outer membrane. This work lays the structural foundation for the development of novel antimicrobial strategies aimed at blocking the translocation of capsule. Transport processes across membranes are fundamental in biology and the structure Wza suggests a novel mechanism for the translocation of large polar molecules.
  • Figure 1 shows Group 1 capsular polysaccharide export in Gram-negative bacteria schematically.
  • a Model and proposed activities of a hypothetical biosynthetic complex carrying out coordinated synthesis and export of serotype K30 group 1 capsule in E. coli.
  • Individual tetrasaccharide repeat units of the polymer are assembled by a series of enzymes including integral or peripheral membrane proteins on a lipid (undecaprenol diphosphate; und-PP) acceptor, using sugar nucleotide precursors available in the cytoplasm.
  • the und-PP-linked repeat units are "flipped" across the inner membrane by a process involving an integral membrane protein (Wzx). Polymerization occurs at the periplasmic face and is dependent on another integral membrane protein, the putative polymerase, Wzy.
  • Wzx integral membrane protein
  • the monomer of Wza can be decomposed into four domains; these are labeled as D1 to D4 and colored differently.
  • Cys 21 (fatty acid modified N- terminus in mature Wza) is shown as a blue ball and Arg 376 (the last ordered residue) as a red ball.
  • a stereo figure is shown in Fig. 4a.
  • b The Wza octamer is shown in ribbon format. In this view the large central cavity is highlighted by space-filling light orange shape. The octamer is described as an amphora made of four rings (labelled R1 to R4). Ring 1 is formed by eight copies of domain 1 , ring 2 by eight copies of domain 2 and so on.
  • the helical barrel (R4) forms the "neck” of the structure and ring 1 (R1) the "base”.
  • the predicted position of the outer membrane (OM) is marked and is found on the neck of the structure.
  • the C-terminus of the protein is predicted to be exposed on the cell surface and rings 1 , 2 and 3 located inside the periplasm, c,
  • a view of the octamer such that one looks down into the central cavity from outside the cell through the helical barrel.
  • the separation of loops which close the cavity in ring 1 are marked on the diagram, d, Wza is shown as a space filling model colored according to electrostatic charge (blue positive, red negative) and is rotated 180° from the second view, such that one is now looking towards the cavity from periplasm.
  • the concave surface of ring 1 can be seen and is closed by the loop at Tyr 110.
  • Figure 3 shows the central cavity of Wza.
  • a A stereo diagram of the internal cavity. The surface is colored according to polarity, (oxygen red, nitrogen blue and carbon, selenium, sulfur white) and reveals the interior of Wza is polar.
  • the cavity is open at top through the helical barrel, b, Wza is shown as a surface with polar atoms colored (colors are the same as in Fig. 2d.) Wza is oriented as in Fig. 2b.
  • the lipid molecules are shown as black spheres and are located near the top of the structure. There are no gaps in the through the walls o the structure.
  • the helical barrel is clearly non-polar and a band of tryptophan residues is exposed at the base of helical barrel (Fig. 7b).
  • Figure 4 shows a monomer of Wza. a, The four domains are labelled and identified by color. Domain 1 (residues 89-169) is shown in cyan, domain 2 (residues 68-84 and 172- 252) magenta, domain 3 (residues 46-64 and 255-344) orange and domain 4 (residues 345-379) yellow.
  • Domain 1 (residues 89-169) is shown in cyan
  • domain 2 (residues 68-84 and 172- 252) magenta
  • domain 3 (residues 46-64 and 255-344) orange
  • domain 4 (residues 345-379) yellow.
  • the long loop at the N-terminus is colored green, as are loops which connect domains.
  • the N-terminal residue (Cys 21 ) is shown as a blue ball and the C-terminal residue (Arg 376) as a red ball, b,
  • the so called PES (polysaccharide export sequence) motif (colored as blue), PfamO2563, is found between residues 82-195 encompasses domain 1.
  • the sequence motif also includes part of domain 2, in light of the structure we suggest the PES domain is redefined to be residues 89-169 (domain 1 ).
  • Domain 3 (orange) has been superimposed upon domain 2 (magenta). This shows clearly the domain duplication which has occurred.
  • the root mean square deviation of 69 Ca atoms is 1.2k. Domain 3 is slight larger due to a longer loop and an extra ⁇ strand.
  • FIG. 5 shows a sequence alignment of outer membrane auxiliary (OMA). proteins with Wza (residues 57-283) (SEQ ID NO: 1 ). conserveed residues are shown in red, similar residues in blue.
  • BexD (SEQ ID NO: 2) is a capsule export protein from Haemophilus influenzae
  • CtrA (SEQ ID NO: 3) is a capsule export protein from Neisseria meningitidis
  • KpsD SEQ ID NO: 4 is an E. coli group 2 capsule assembly protein.
  • the domains found in the structure of Wza and the conserved PfamO2563 polysaccharide biosynthesis/export motif (PES motif) is marked with + below the residues.
  • PES motif polysaccharide biosynthesis/export motif
  • Figure 6 shows experimental electron density.
  • a The side chain of Tyr 110 was set to low occupancy (0.1 ) throughout the refinement in order to exclude it from phase calculation. Shown in blue chicken wire is the 2Fo-Fc map contoured at 1.2 ⁇ and magenta chicken wire is the Fo-Fc map at 2.6 ⁇ .
  • Six tyrosines are modelled in the second most common confirmation of this residue, two in the most common.
  • the side chain is clearly disordered but there is some weak density in each of the two possible conformations, b, Density can be seen for part of the diacylglycerol group which connects to the S atom of Cys 21.
  • Tyr 110 adopts one set of conformations which completely seal the cavity, d, The same view as in Fig. 6b but with an alternate conformation of Tyr 110 that creates a hole of diameter of 8 A, this is probably too small to allow passage of carbohydrate. Tyr 110 is disordered in the structure, but there is weak density for both conformations.
  • FIG. 7 shows the helical barrel a
  • Wza is shown as a surface with polar atoms colored (colors are as in Fig. 2c). Wza is orientated as the Fig. 2b (first view). The lipid molecules are shown as black spheres and are located near the top of the structure.
  • the helical barrel is clearly non-polar, b, There is a band of tryptophan residues (Trp 350) exposed on the surface of the helical barrel.
  • Such bands of tryptophans are a common feature in transmembrane helices and are thought to act as "lock” keeping the protein embedded in the membrane.
  • Figure 8 shows cell-surface expression of the C-terminal FLAG epitope in
  • Wza FLAG . a The Wza FLAG was clearly detected in sarkosyl-insoluble outer membranes after separation by PAGE and staining with Simply Blue stain, marked * .
  • b The ability of the Wza FLAG derivative to function in K30 CPS assembly was determined by western immunoblots of cell lysates of the wza-null strain E. coli CWG281 using rabbit polyclonal antibodies specific for the K30 polymer.
  • FIG. 9 shows the orientation of Wza in the outer membrane using the PK tag.
  • a Western blot of purified Wza-PK shows the octamer in the unboiled samples and monomer in the boiled samples, indicating the protein behaves like native Wza.
  • the modified protein is competent for capsule formation
  • b E. coli expressing Wza modified by addition of PK tag to the C-terminus (Wza-PK) binds the green fluorescent anti-PK antibody. Under our expression conditions only about 12% of cells seem to bind the antibody strongly.
  • the red anti-alkaline phosphatase antibody locates the periplasm and the DNA is stained blue by DAPI.
  • the scale bar is 15 ⁇ m.
  • c A single cell is shown, the scale bar is 1 ⁇ m.
  • Figure 10 shows mutagenesis of Y110.
  • a K30 capsule expression was assessed by western immunoblotting whole cell lysates using rabbit polyclonal antibodies specific for the K30 polymer.
  • Constructs expressing Wza, Wza Y110A and Wza ⁇ 108"112 were used to transform the wza-null strain E. coli CWG281 (wza ⁇ owaacCI , wza 22m ⁇ n --aadA; GnY, Sp r ) 7 (see Drummelsmith et al. (2000) EMBO J ⁇ 9: 57-66) and expression of the Wza derivatives was induced using 0.006% L-arabinose for 3 hours.
  • the control strain contained pBAD24 vector alone.
  • the mutants are indistinguishable from the native protein, b, Wza monomer expression was determined in western immunoblots probed with polyclonal antibodies specific for Wza after heating at 100 0 C in PAGE sample. All the proteins are expressed, c, The presence of Wza monomers and multimers in the outer membrane was determined by western immunoblots. Outer membranes were prepared as the N-lauryl sarcosinate (sarkosyl)-insoluble fraction, as described previously (see Drummelsmith et al., supra). Samples were added to PAGE buffer and incubated at 100 0 C or room temperature (RT). These data show that for the Wza ⁇ 108"112 mutant only a very small amount of protein is at the outer membrane.
  • Figure 11 shows protein mass spectrometry.
  • a The peak at 40330 ⁇ 10Da corresponds to the predicted weight of 40340 Da. This is derived from mature protein (39550) plus the three predicted palmitoyl groups (717) and a glycerol moiety (73), the classical lipoprotein signature,
  • b The selenomethionine protein peak at 40810 is increased from native by 470, which corresponds to 10 selenium atoms per monomer ( 79 Se- 29 S).
  • Figure 12 shows PES domain.
  • coli LE392 cells were transformed with plasmid pWQ126 (pBAD24 expressing Wza - see Drummelsmith et al., (2000) EMBO J ⁇ 9: 57-66).
  • the cells were grown in M9 minimal medium containing 2 % (w/v) casamino acids until the OD ⁇ oonm reached to 0.8.
  • the cells were then harvested and washed twice using M9 medium, and then transferred into the same amount of the M9 medium containing a mixture of all L-amino acids (except methionine and cysteine) each at a final concentration of 50 mg/L, together with 50 mg/L selenomethonine.
  • the selenomethionine-labeled Wza was over- expressed by induction using 0.008 % (w/v) L-arabinose and incubation overnight at 30 0 C. Purification of the labeled protein was the same as for the native protein. However, the crystallization condition was optimized to 7 % PEG4000, 0.1 M sodium acetate pH 4.6 and 0.2 M (NH 4 ) 2 SO 4 with 10 mg/ml protein containing 80 mM NaCI, 0.008 % (w/v) DDM and 20 mM Tris pH 8.0. Mass spectrometry confirmed Wza has full incorporation of selenium and does have the classical diacylgycerol and N-acylation lipoprotein modification (see Fig. 10).
  • the crystals of native Wza and, especially the selenomethionine derivative, are highly variable and even within the same crystal some regions give better data than others.
  • the 2.26 A data set was collected on ID29 of the ESRF on a single selenomethionine crystal protected with 25 % (w/v) glycerol in the mother liquid and cooled to 100 K. Data were recorded on an ASDC 3 x 3 tiled array CCD detection. Six data sets were collected at three wavelengths using different crystals and reduced with MOSFLM / SCALA. In the end, only one peak wavelength data set allowed location of 78 (out of 80) selenium ions by SHELX using data to a resolution up to 3.2 A. The correct solution had a frequency of 1 in 600 trials.
  • a Anomalous completeness corresponds to the fraction of possible acentric reflections for which an anomalous difference has been measured.
  • b Rmerge ⁇ hki ⁇
  • the Wza oligomer is formed by a network of H- bonds, salt bridges and hydrophobic contacts.
  • the subunit association between two Wza monomers is as follows: the N-terminal loop (residues 22-35) is wrapped around ring 3 via two salt bridges from Arg 33 and Asp 44, and Asp 47 and Lys 34. Further stabilization is provided by a H-bond (Tyr 341 and GIn 27) and a van der Waal's contact (Arg 349 and GIn 27).
  • the first ring is stabilized by inter-subunit hydrogen bonds between Asp 99 and Tyr 130, Thr 116 and Arg 111 , Arg 111 and Arg 111 on an adjacent monomer. Van der Waals interactions between Ne 190 and Lys 172, MSE 230 and Thr 176, MSE 230 and Phe 248, and two hydrogen bonds (Asp196 and Tyr174, Asn 199 and Thr 176) stabilize the second ring.
  • the third ring is stabilized by salt-bridges (Asp 272 and Lys 256, GIu 280 and Lys 256), hydrogen bonds (lie 288 and GIn 266, Arg 273 and Asp 253), and a van der Waal's contact between Ala 323 and Tyr 341. There is a potential hydrogen bond between His 365 and GIu 369 in adjacent transmembrane ⁇ -helices.
  • the underlined restriction sites (EcoRI and Pst ⁇ , respectively) were used for cloning the amplified DNA fragment into pBAD24.
  • E. coli LE392 cells containing either pWQ126 (native Wza) or pWQ390 (Wza FLAG ) were grown at 37oC to an O.D. 6 oo ⁇ m of 0.6. Recombinant protein expression was induced by incubation for an additional 2h in 0.01 % (w/v) L-arabinose.
  • Bacterial cells (2 O.D. 6 oo ⁇ m units) were collected by centrifugation, resuspended in 1 ml 5% formaldehyde and incubated at room temperature for 1 hour. The fixed cells were washed twice with 1 ml PBS and resuspended in 100 ⁇ l PBS.
  • the cell suspensions were applied to poly-L-lysine-treated microscope slides and immunofluorescence microscopy was performed as described previously (see Clarke et al., (2004) J. Biol Chem 279: 35709-35714) using anti-FLAG M2 Mab (Sigma) and Rhodamine-Red-X-conjugated goat anti-mouse Ab (Jackson Immunoresearch) as primary and secondary antibodies, respectively.
  • K30 capsule expression was assessed by western immunoblotting whole cell lysates (see Hitchcock et al., (1983) J. Bacteriol 154: 269-277) using rabbit polyclonal antibodies specific for the K30 polymer.
  • cell-free lysates were prepared by sonication and cell envelopes wre probed with polyclonal antibodies specific for Wza (see Nesper et al., (2003) J. Biol Chem 278: 49763-72).
  • Outer membranes were purified based on their insolubility in N-lauryl sarcosinate (sarkosyl) (see FiNp et al., (1973) J. Bacteriology n5: 717-722).
  • the stability of multimeric forms of the Wza derivatives in SDS was used to distinguish between monomers and multimers.
  • REMARK REMARK 3 REFINEMENT REFINEMENT.
  • REMARK 3 PROGRAM REFMAC 5.2.0019
  • REMARK 3 AUTHORS MURSHUDOV,VAGIN,DODSON REMARK 3 REMARK 3 REFINEMENT TARGET : MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT.
  • REMARK 3 CROSS-VALIDATION METHOD THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.19008 REMARK 3 R VALUE (WORKING SET) : 0.18818 REMARK 3 FREE R VALUE : 0.22608 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 5.0 REMARK 3 FREE R VALUE TEST SET COUNT : 10587 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN.
  • REMARK 3 ALL ATOMS 23866 REMARK 3 REMARK 3 B VALUES.
  • REMARK 3 FROM WILSON PLOT (A**2) NULL REMARK 3 MEAN B VALUE (OVERALL, A**2) : 36.102 REMARK 3 OVERALL ANISOTROPIC B VALUE.
  • REMARK 3 B11 A* k 2) -0.46 REMARK 3 B22 (A* * 2) 0.09 REMARK 3 B33 (A * k 2) 0.37 REMARK 3 B12 (A* * 2) 0.00 REMARK 3 B13 (A* * 2) 0.00 REMARK 3 B23 (A * "2) 0.00 REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR.

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Abstract

La présente invention concerne un cristal de Wza, une protéine membranaire externe, résolue à 2,26 Å. L'invention concerne en outre un procédé de sélection d'agents qui inhibent l'exportation de Polysaccharides Extracellulaires (EPS) du cytoplasme d'une bactérie, ledit procédé comportant les étapes consistant: a) à se procurer un modèle de Wza résolu à 2,26 Å ; b) à identifier un agent inhibiteur potentiel de Wza ; c) à analyser l'interaction de l'agent inhibiteur potentiel avec Wza ; et d) à sélectionner un agent capable d'interagir avec Wza pour inhiber l'exportation de EPS du cytoplasme d'une bactérie.
PCT/GB2007/050631 2006-10-13 2007-10-12 Protéine membranaire wza et procédé de sélection d'agents inhibiteurs Ceased WO2008044075A1 (fr)

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GB0620304A GB0620304D0 (en) 2006-10-13 2006-10-13 Protein
GB0620304.6 2006-10-13

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WO2008044075A1 true WO2008044075A1 (fr) 2008-04-17

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9963518B2 (en) 2011-08-22 2018-05-08 Oxford University Innovation Limited Cyclic oligosaccharides for use in the treatment and prevention of bacterial infection

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BEIS ET AL: "Two-step purification of outer membrane proteins", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, BUTTERWORTH & CO., GUILDFORD, GB, vol. 39, no. 1-3, 15 August 2006 (2006-08-15), pages 10 - 14, XP005564787, ISSN: 0141-8130 *
BEIS KONSTANTINOS ET AL: "Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 27, 2 July 2004 (2004-07-02), pages 28227 - 28232, XP002470740, ISSN: 0021-9258 *
DONG CHANGJIANG ET AL: "Wza the translocon for E-coli capsular polysaccharides defines a new class of membrane protein", NATURE (LONDON), vol. 444, no. 7116, November 2006 (2006-11-01), pages 226 - 229, XP002470742, ISSN: 0028-0836 *
REIS KONSTANTINOS ET AL: "Crystallization and preliminary X-ray diffraction analysis of Wza outer-membrane lipoprotein from Escherichia coli serotype O9a:K30.", ACTA CRYSTALLOGRAPHICA SECTION D BIOLOGICAL CRYSTALLOGRAPHY, vol. 60, no. 3, March 2004 (2004-03-01), pages 558 - 560, XP002470741, ISSN: 0907-4449 *
WHITFIELD CHRIS: "Biosynthesis and assembly of capsular polysaccharides in Escherichia coli", ANNUAL REVIEW OF BIOCHEMISTRY, vol. 75, 2006, pages 39 - 68, XP002470743, ISSN: 0066-4154 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9963518B2 (en) 2011-08-22 2018-05-08 Oxford University Innovation Limited Cyclic oligosaccharides for use in the treatment and prevention of bacterial infection

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