WO2001098349A2

WO2001098349A2 - Recombinant avidin monomer and its use in biotin binding

Info

Publication number: WO2001098349A2
Application number: PCT/GB2001/002702
Authority: WO
Inventors: Markku Kulomaa; Olli H. Laitinen; Edward A. Bayer; Meir Wilchek
Original assignee: Ark Therapeutics Ltd
Current assignee: Ark Therapeutics Ltd
Priority date: 2000-06-19
Filing date: 2001-06-19
Publication date: 2001-12-27
Anticipated expiration: 2002-12-19
Also published as: AU2001274262A1; WO2001098349A3; GB0015016D0

Abstract

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance.

Description

RECOMBINANT AVIDIN AND ITS USE IN BIOTIN BINDING Field of the Invention

This invention relates to a recombinant avidin and its use in biotin binding. Background of the Invention Many proteins are composed of more than one subunit. The principles and rationale for the spatial arrangement of subunits (or monomers) into dimers, trimers, and tetramers are still not well understood. The role of multimeric associations in binding and protein activity is still a mystery, particularly for proteins in which each subunit contains the necessary information for binding or activity. An example of this phenomenon is the aldolase homotetramer, in which each subunit exhibits identical catalytic activity.

Avidin is a homotetramer (molecular weight c.63,000), each monomer of which binds a single biotin molecule with the highest known affinity in nature between a protein and a ligand. The three-dimensional structure of avidin has been solved, and the interactions between the various residues at the intersubunit interfaces have been determined.

The avidin tetramer is essentially a dimer of dimers consisting of three types of monomer-monomer interactions, as discussed in detail by Livnah et al, PNAS USA 90:5076-80 (1993). The 1-4 intermonomer interface forms a structurally cohesive dimer. The two monomers are so tightly integrated that this particular dimer can essentially be regarded structurally as a single entity.

By contrast, the 1-2 and 1-3 interfaces each comprise only a selected number of critical interactions (buried surface areas of 557 A² and 147 A² per monomer, respectively). Thus, the dimer-dimer interaction is dictated by the combined 1-2 and 1-3 interfaces. The 1-3 interface consists of a contribution of only three amino acids from each monomer, namely Met-96, Val-115, and lle-117. This hydrophobic interaction has a contact surface area of 147 A², which is a mere 20% of the dimer-dimer interaction.

The 1-4 interface is characterised by extensive polar and hydrophobic interactions. Some of the polar interactions consist of an intricate network of hydrogen- bonding interactions, sometimes involving conserved water molecules. The ten hydrogen-bonding interactions observed for the side chains of two of these residues, Asn- 54 and Asn-69 (each from the opposite monomer), are particularly extensive. Because of the 2-fold symmetry axis, normal to the plane of the 1-4 interface, the site is duplicated on opposite poles of the overall interacting interface. The two residues are positioned within the same site but on opposite monomers. Laitinen et al, FEBS Lett. 461:52-58 (1999), describes dimeric, biotin-binding forms of both chicken avidin and bacterial streptavidin. In both proteins, a binding-site tryptophan was converted to a lysine.

- . This.tryptophan/Trp-UO in. avidin and Trp-120 in streptavidin) plays a dual role in both proteins. On one hand, it serves a biological role as one of the major biotin- binding residues. On the other hand, it plays a major structural role in characterising one of the three intermonomer interactions (the 1-2 interface). The consequence of the tryptophan-lysine exchange was 2-fold; (a) the affinity constant of the biotin binding was reduced, and the binding could be reversed, and (b) the mutated protein formed a stable dimer in solution. Because dimer formation requires the combined counteraction of two interfaces, the inference was such that a drastic mutation also affected the weaker 1-3 interface, leaving the extensive 1-4 interface intact. Summary of the Invention

The present invention is based on the discovery of monomeric avidin (or streptavidin), i.e. a monomeric form of avidin that does not form a tetramer in the absence of biotin, but which retains biotin-binding activity. More specifically, the present invention is based on the construction of a set of avidin mutants in which selected 1-3 and 1-4 interface residues were modified to alanine. Successive mutation of these residues caused a progressive weakening of the quaternary structure. In the absence of biotin, two of the mutants proved to be stable, soluble monomers. Surprisingly, the presence of biotin regenerated stable avidin tetramers in these two mutants. This is apparently the first instance in which a small ligand that binds to a single subunit causes subunit assembly into a stable tetramer. This suggests that, although a monomer can bind a ligand, stability factors can favour formation of the native oligomer. This may be important for designing novel binding proteins and for demonstrating the role(s) of the quaternary structure in maintaining stability of multisubunit proteins.

This invention relates to new forms of avidin that are useful for a variety of functions, especially those already associated with avidin. One of these is described in WO-A-99/42577, the content of which is incorporated herein by reference. The lower binding affinity between biotin and the new forms of avidin means that reactions that may previously have been irreversible are now reversible, depending on the circumstances, allowing enhanced therapy. A biotin-bound drug can be released/replaced. Thus, one aspect of the invention comprises monomeric avidin conjugated to a therapeutic agent or other substance of interest, or the use of the monomeric avidin togetherwith a biotin-substance conjugate. A further aspect lies in a protein comprising a membrane-spanning domain and an extracellular domain comprising monomeric avidin.

Detailed Description of the Invention

It is preferred that the minimal number of changes in the interface residues among the subunits of the avidin tetramer shoud be made, which lead to disruption of the avidin tetramer into monomers, without destroying the strong binding specificity for biotin. This can be achieved by converting selected interface residues, e.g. to alanine, via site-directed mutagenesis. All three hydrophobic residues in the 1-3 interface may be mutagenised. In addition, two critical 1-4 interface residues, Asn-54 and Asn-69, may be modified. Alteration of the latter two residues to alanine would preclude the formation of the extensive hydrogen-bonding network involving these residues, thus abolishing the conserved water molecule and perturbing the entire 1-4 interface. The combination of these mutations may thus result in a substantial weakening of the tetrameric assembly of avidin. Indeed, as indicated below, in the absence of biotin, both Avm-[3,4] mutants were found to exist in the monomeric state in solution. When biotin was introduced, however, a stable avidin tetramer was assembled, presumably because of the additional interaction in the 1-2 interface between Trp-110 (from the adjacent monomer) and biotin in its binding pocket. This result demonstrates the importance of the 1-2 interaction for the stability of the avidin molecule. It is known that biotin enhances the stability of native avidin, and it is shown below that biotin not only stabilises the native protein but that it can also play a role in the assembly of tetrameric avidin from the monomer.

The experiments reported below also shed light on the importance of the major 1 -2 interface and Trp-110 for tetramer formation. It appears that, in order to preserve high affinity biotin binding, the binding-site residues should be retained, particularly the aromatic ones. In contrast, a certain amount of latitude is permissible in modifying the other residues, including the loop and j3-strand residues. The results also demonstrate that it is possible to manipulate the inter-facial residues quite freely to alanine, thereby reducing both hydrophobic interactions (e.g. in the 1-3 interface) and hydrogen bonding (in the 1-4 interface).

The efficient isolation of all mutants on 2-iminobiotin-agarose may indicate that the monomeric mutants formed tetramers upon interaction with the immobilised ligand. Under acidic conditions, the mutated proteins were converted to the monomers, which could be reassociated to the tetramer upon interaction with free or immobilised ligands. This indicates that constant interaction with the ligand is a prerequisite for tet- ramerisation-of the interface, mutants. These results also imply that biotin is not a necessary element for proper folding of the monomeric avidin. Together with the results reported by Laitinen et al, supra, concerning the dimeric avidin and streptavidin mutants, this also indicates that the avidin j3-barrel perse is a stable tertiary structure and capable of folding correctly without the involvement of neighbouring monomers. According to Ellison era/, Protein Sri. V.S.A.92:1754-8 (1995), the mechanism that protects the native avidin-biotin complex against proteinase K treatment appears to be the closure and consequent rigidity of the loop between j3-strands 3 and 4. Compared with native avidin, however, all the mutants reported below were rapidly digested in the absence of biotin by this enzyme but were all stable in the presence of biotin. Weakening of the quaternary structure of the tetramer seems to correlate well with susceptibility to protease digestion, as well as reduced resistance against denaturation in SDS-PAGE. Upon regeneration of the Avm-[3,4a] and Avm[3,4b] tetramers by adding biotin, the extreme stability characteristics (similar to those exhibited by the native protein) were restored. The results reported below may have broader implications regarding why avidin and streptavidin appear as tetramers in the native state and how proteins form oligomers in general. It seems that unusually high ligand-binding affinity and the exceptional stability of avidin and streptavidin are two sides of the same coin. Apparently, the two properties have coevolved, and it is difficult to separate them. The biotin-induced monomer-tetramer transition is the first example of a monomeric ligand that causes association of protein components. This phenomenon may be amenable to biotechnological application. The capacity to produce a monovalent form of avidin, which can then undergo oligomerisation upon addition of biotin, iminobiotin, or other avidin-binding ligands, opens new avenues for the production of different intracellular fusion proteins that can be switched on by oligomerisation. The possibility of subsequent coupling of such a fusion protein with a natural counterpart or substrate may allow control of protein-protein interactions and of cellular processes involving oligomerisation. The mutated avidins may also be applicable for reversible assembly and dismantling of working proteins in living cells. A product of the type described in WO-A-99/42577, incorporating monomeric avidin, and a biotinylated drug, can be used in therapy. The relatively weak binding can be utilised in reversible binding, e.g. for application of a drug intended to treat cancer or-heart-disease..

By way of example, in order to obtain a product of the invention, one or more of the three hydrophobic amino acids that line the 1-3 interface and selected polar residues that play a major role in the 1-4 interface are changed, e.g. to alanine. This has the effect of reducing the hydrophobic nature of the first set and eliminating hydrogen-bonding potential of the second. The target residues were selected on the basis of the 3D structure of avidin (Livnah et al, supra), taking into account their suspected contribution to the respective intermonomer interface. The mutants that have been prepared are listed in Table I.

Table

Construction of Recombinant Bacmids and Baculoviruses

Chicken egg white avidin cDNA was mutated by the megaprimer method (1) using pGEMAV (2) as a template. When combined mutants were created, the former constructs were used as a template. After a second PCR amplification, the products were digested with Bglll and Hindlll, extracted from agarose, and cloned into the BamHIIHindlll-treated pFASTBACI plasmid to construct recombinant vectors. The vectors were transformed into JMTO9 cells, and the nucleotide sequences were confirmed by dideoxynucle_oti.de. sequencing using an automated DNA sequencing instrument. The preparations of recombinant viruses were completed according to the manufacturer's instructions of the Bac-ToBacmf baculovirus expression system (Life Technologies, Inc., Gaithersburg, MD). The primary virus stocks were amplified for large scale production of mutants, and the titres of virus stocks were determined by a plaque assay procedure (3). Production and Purification of Mutant Avidins

Mutant avidins were produced in baculovirus-infected insect cells (4). Purification on 2-iminobiotin-agarose column was performed from cell extracts grown in biotin-free medium (5). Biotin Binding Assays The determination of binding constants for avidin and its mutants to iminobiotin was performed using an lASyS optical biosensor (6). Reversibility of biotin binding was determined by competitive binding to biotinylated biosensor surfaces and by competitive biotin-binding enzyme-linked immunosorbent assay (4).

To study the reversibility of biotin binding, an assay based on optical biosensor technology was designed. The results showed that only native avidin and Avm-4a were completely irreversible. The three mutants exhibited levels of reversibility between 30 and 45%. In this group of mutants, reversible biotin binding was highest for Avm-[3,4a].

Measurement of the actual binding constants of native avidin and the mutants for 2-iminobiotin, using the lASyS optical biosensor, indicated that interaction of mutants Avm-3 and Avm-4a with 2-iminobiotin surfaces was similar to that of native avidin (Table II). Calculation of the Kd values from k~ and kd and direct determination of Kd from the binding curves gave comparable values. Because of the monomer- tetramer transition following biotin binding, the lASyS biosensor could not be used to evaluate the actual binding constants for mutants Avm-[3,4a] and Avm-[3,4b]; in qualitative terms, however, these mutants also showed strong binding to 2-iminobiotin. Table II

^a Dissociation constant calculated from K_ass and K-,^ values derived from association analysis using the plot K__π against protein concentration. Dissociation constant determined directly from binding curves.

Structure Analysis

The molecular mass of a mutant was calculated from the known amino acid composition using the GCG package program Peptidesort (Genetic Computer Group, Madison, Wl). SDS-PAGE, immunoblot analyses, and assays for protease sensitivity were performed according to Laitinen et al, supra. For stability analysis, protein samples were combined with sample buffer and incubated at selected temperatures for 20 mm before being subjected to SDS-PAGE (7). Quaternary states of untreated or biotin-treated avidin and mutant samples were defined by FPLC (4).

All mutants were purified from cell lysates. Isolation of the mutants on iminobiotin-agarose resulted in pure products of high yields (data not shown), comparable with those reported earlier (5,6). In general, the 2-iminobiotin purification scheme used was very efficient in terms of protein purity, because no contaminating proteins could be observed in Coomassie-stained SDS-PAGE gels.

The stability of the mutants was analysed by SDS-PAGE (4). When compared with native avidin, all the mutants showed decreased stability. Avm-3 and Avm-4a exhibited partial tetrameric structure in SDS-PAGE at ambient temperatures in the absence of biotin. In contrast, Avm-[3,4a] and Avm-[3,4b] migrated as monomers, even at room temperature in the absence of biotin. In the presence of biotin, all mutants formed tetramers that displayed stability characteristics similar to those of native avidin.

In protease assays, all mutants showed reduced resistance to proteinase K degradation compared with native avidin. In the absence of biotin, all mutants were rapidly degraded. Upon addition of biotin, however, complete resistance (similar to native avidin) was restored to all mutants except Avm-[3,4a], which exhibited only 75% resistance to degradation. The quaternary status of the mutants was analysed by gel filtration FPLC. The theoretical molecular mass for all mutant monomers without the oligosaccharide side chain is between 14,127 and 14,300 daltons. According to the FPLC data, the observed

_mass.forA.vm^3,4a], inihe absence of biotin, was 13,990 daltons. After saturation with biotin, the mass was calculated to be 58,900 daltons. The corresponding masses for

Avm-[3,4b] were 14,280 and 55,960 daltons. These values suggest that, in the absence of biotin, Avm-[3,4a] and Avm-[3,4b] were monomers, but that they formed tetramers upon biotin binding. In contrast, Avm-3 and Avm-4a formed tetramers even in the absence of biotin. References

1. Sarkar and Summer, (1990), Biotechniques 8, 404-407.

2. Airenne et al, (1994), Gene 144, 75—80.

3. O'Reilly eta/, (1994), Boculovirus Expression Vectors. A Laboratory Manual, pp. 130-132, Oxford University Press, New York. 4. Laitinen et al, (1999), supra.

5. Airenne et a/, (1997), Protein Expression Purif 9, 100-108.

6. Marttila et al, (1998), FEBS Lett. 441 , 313-317.

7. Bayer et al, (1996), Electraphoresis 17, 1319-1324.

Claims

1. Monomeric avidin.

2. Avidin according to claim 1 , wherein one or more of the hydrophobic residues in the 1-3 interface is changed.

3. Avidin according to claim 1 or claim 2, wherein Asn-54 and/or Asn-69 is changed.

4. Avidin according to claim 2 or claim 3, wherein each changed residue is Ala.

5. The use of avidin according to any of claims 1 to 4, in the presence of biotin, wherein either or each of the avidin and the biotin is conjugated to a substance of interest.

6. A protein comprising as membrane-spanning domain and an extracellular domain comprising avidin according to any of claims 1 to 4.