Crystal Structure of a Cross-reaction Complex between Fab F9.13.7 and Guinea Fowl Lysozyme

Abstract

The crystal structure of the complex between the cross-reacting antigen Guinea fowl lysozyme and the Fab from monoclonal antibody F9.13.7, raised against hen egg lysozyme, has been determined by x-ray diffraction to 3-Å resolution. The antibody interacts with exposed residues of an α-helix and surrounding loops adjacent to the lysozyme active site cleft. The epitope of lysozyme bound by antibody F9.13.7 overlaps almost completely with that bound by antibody HyHEL10; the same 12 residues of the antigen interact with the two antibodies. The antibodies, however, have different combining sites with no sequence homology at any of their complementarity-determining regions and show a dissimilar pattern of cross-reactivity with heterologous antigens. Side chain mobility of epitope residues contributes to confer steric and electrostatic complementarity to differently shaped combining sites, allowing functional mimicry to occur. The capacity of two antibodies that have different fine specificities to bind the same area of the antigen emphasizes the operational character of the definition of an antigenic determinant. This example demonstrates that degenerate binding of the same structural motif does not require the existence of sequence homology or other chemical similarities between the different binding sites. The crystal structure of the complex between the cross-reacting antigen Guinea fowl lysozyme and the Fab from monoclonal antibody F9.13.7, raised against hen egg lysozyme, has been determined by x-ray diffraction to 3-Å resolution. The antibody interacts with exposed residues of an α-helix and surrounding loops adjacent to the lysozyme active site cleft. The epitope of lysozyme bound by antibody F9.13.7 overlaps almost completely with that bound by antibody HyHEL10; the same 12 residues of the antigen interact with the two antibodies. The antibodies, however, have different combining sites with no sequence homology at any of their complementarity-determining regions and show a dissimilar pattern of cross-reactivity with heterologous antigens. Side chain mobility of epitope residues contributes to confer steric and electrostatic complementarity to differently shaped combining sites, allowing functional mimicry to occur. The capacity of two antibodies that have different fine specificities to bind the same area of the antigen emphasizes the operational character of the definition of an antigenic determinant. This example demonstrates that degenerate binding of the same structural motif does not require the existence of sequence homology or other chemical similarities between the different binding sites. Antibody molecules convey a potentially unlimited molecular diversity and antigen-binding repertoire. Although the genetic elements and mechanisms underlying the generation of this functional and structural diversity have been characterized (43Tonegawa S. Nature. 1983; 302: 575-581Google Scholar), an understanding of the molecular principles that govern antibody specificity remains a major challenge. In this paper, we report the crystal structure of the cross-reaction complex between the antigen Guinea fowl lysozyme (GEL)1 1The abbreviations used are: GELGuinea fowl lysozymemAbmonoclonal antibodyCDRcomplementarity-determining regionr.m.s.root mean squareHELhen lysozymeJQLJapanese quail lysozymePHLpheasant lysozymeLlight chainHheavy chainL1L2, L3, hypervariable regions of the light chainH1, H2, H3hypervariable regions of the heavy chain. 1The abbreviations used are: GELGuinea fowl lysozymemAbmonoclonal antibodyCDRcomplementarity-determining regionr.m.s.root mean squareHELhen lysozymeJQLJapanese quail lysozymePHLpheasant lysozymeLlight chainHheavy chainL1L2, L3, hypervariable regions of the light chainH1, H2, H3hypervariable regions of the heavy chain. and the Fab fragment from the mAb F9.13.7, raised against HEL. Guinea fowl lysozyme monoclonal antibody complementarity-determining region root mean square hen lysozyme Japanese quail lysozyme pheasant lysozyme light chain heavy chain L2, L3, hypervariable regions of the light chain hypervariable regions of the heavy chain. Guinea fowl lysozyme monoclonal antibody complementarity-determining region root mean square hen lysozyme Japanese quail lysozyme pheasant lysozyme light chain heavy chain L2, L3, hypervariable regions of the light chain hypervariable regions of the heavy chain. Avian egg lysozymes have long served as model antigens for investigating the specificity of immune recognition (for review, see 2Benjamin D.C. Berzofsky J.A. East I.J. Gurd F.R.N. Hannum C. Leach S.J. Margoliash E. Michael J.G. Miller A. Prager E.M. Reichlin M. Sercarz E.E. Smith-Gill S.J. Todd P.E. Wilson A.C. Annu. Rev. Immunol. 1984; 2: 67-101Google Scholar). Cross-reaction studies using panels of evolutionarily related avian lysozymes have identified amino acid residues and the general region of the antigen bound by specific mAbs. Using this test, the antibodies could be grouped by their binding specificities (19Harper M. Lema F. Boulot G. Poljak R.J. Mol. Immunol. 1987; 24: 97-108Google Scholar; 26Lavoie T.B. Drohan W.N. Smith-Gill S.J. J. Immunol. 1992; 148: 503-513Google Scholar). Crystallographic analysis of several Fab-lysozyme complexes (1Amit A.G. Mariuzza R.A. Phillips S.E.V. Poljak R.J. Science. 1986; 233: 747-753Google Scholar; 37Sheriff S. Silverton E.W. Padlan E.A. Cohen G.H. Smith-Gill S.J. Finzel B.C. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8075-8079Google Scholar; 11Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Google Scholar; 8Chitarra V. Alzari P.M. Bentley G.A. Bhat T.N. Eisel J.-L. Houdusse A. Lescar J. Souchon H. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7711-7715Google Scholar; 5Braden B.C. Souchon H. Eisele J.-L. Bentley G.A. Bhat T.N. Navaza J. Poljak R.J. J. Mol. Biol. 1994; 243: 767-781Google Scholar) have extended these characterizations of antigenic determinants (or epitopes), and common features have emerged from these studies. 1) The lysozyme-antibody interfaces are large (600-800 Å2) and highly complementary. 2) Both the L and H chains of antibodies make extensive contacts with lysozyme, and the specificity of binding is determined by the CDRs. 3) The contacting residues of lysozyme are discontinuous in sequence but form a contiguous surface. 4) van der Waals' interactions, hydrogen bonds, and, to a much lesser extent, salt bridges mediate the binding of antibodies to lysozyme. 5) Small conformational changes in the antibodies and in lysozyme take place upon complex formation. In addition, these x-ray studies indicated that antibody cross-reactivity results from interactions with epitopes that are shared by heterologous lysozymes or that are closely similar and can be contacted by the antibody with minor conformational rearrangements. In the crystal structure of the cross-reaction complex between Fab F9.13.7 and the heterologous antigen GEL, we have found unexpectedly that the epitope bound by F9.13.7 nearly coincides with that bound by the anti-HEL mAb HyHEL10 (11Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Google Scholar). However, mAbs F9.13.7 and HyHEL10 are structurally different in their L and H chains. We discuss how the two antibodies, with unrelated combining sites, achieve specific, high affinity binding to nearly the same epitope of lysozyme. Preparation of the proteins and crystallization of the complex have been described (27Lescar J. Riottot M.-M. Souchon H. Chitarra V. Bentley G.A. Navaza J. Alzari P.M. Poljak R.J. Proteins. 1993; 15: 209-212Google Scholar). The crystals are monoclinic, space group P21, unit cell dimensions a = 83.7 Å, b = 195.5 Å c = 50.2 Å, β = 108.5°, with two molecules of the complex in the asymmetric unit. Crystal instability upon irradiation precluded merging diffraction data from different crystals. A single data set for structure refinement was collected at 4°C using a MARresearch image plate system and synchrotron radiation from beam line D23 at LURE, Orsay, France (λ = 0.986 Å). A total of 111,931 measurements from 180 images (Δω = 1°) were reduced to 30,232 unique intensities (95% complete to 3-Å resolution) with the MOSFLM package (29Leslie A. Crystallographic Computing. Oxford University Press, Oxford, UK1990: 110-125Google Scholar) and the CCP4 suite (36SERC Daresbury LaboratoryCollaborative Computing Project 4. A Suite of Programs for Protein Crystallography. SERC Daresbury Laboratory, Warrington, United Kingdom1986Google Scholar). The internal merging R-factor is 14% for all data to a nominal resolution of 3 Å (11% for reflections with F > σ(F)). The crystal structure was determined at 4-Å resolution by molecular replacement methods using the program AMoRe (32Navaza J. Acta Crystallogr. 1994; 50: 157-163Google Scholar) as described previously (8Chitarra V. Alzari P.M. Bentley G.A. Bhat T.N. Eisel J.-L. Houdusse A. Lescar J. Souchon H. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7711-7715Google Scholar). The structures of unliganded GEL (28Lescar J. Souchon H. Alzari P.M. Protein Sci. 1994; 3: 788-798Google Scholar), the variable dimer of Fab E225 (3Bentley G.A. Boulot G. Riottot M.-M. Poljak R.J. Nature. 1990; 348: 254-257Google Scholar) and the constant dimer of Fab D1.3 (17Fischmann T.O. Bentley G.A. Bhat T.N. Boulot G. Mariuzza R.A. Phillips S.E.V. Tello D. Poljak R.J. J. Biol. Chem. 1991; 266: 12915-12920Google Scholar) were used as independent search models. Real space averaging between the two independent copies of the complex (SKEW) (6Bricogne G. Acta Crystallogr. 1976; 32: 832-846Google Scholar) significantly improved the quality of the density map, allowing the unambiguous tracing of all the CDRs that had been omitted from the initial models. Refinement was carried out with XPLOR (7Brnger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Google Scholar), and model rebuilding was carried out with the programs FRODO (22Jones T.A. J. Appl. Crystallogr. 1978; 11: 268-272Google Scholar) and QUANTA (Molecular Simulations Inc.). The crystallographic refinement converged to an R-factor of 19% for 23,703 observed intensities (F > 2σ(F)) to a 3-Å resolution. The present model consists of 1130 amino acid residues and does not include solvent molecules. The r.m.s. deviations from ideal bond lengths and angles are, respectively, 0.01 Å and 1.65°. The electron density of the protein backbone is clearly defined for the two independent complexes and unambiguous for all the CDRs in contact with lysozyme (Fig. 1a). The refined coordinates have been deposited with the Protein Data Bank, Brookhaven, NY (4Bernstein F.C. Koetzle T.F. Williams G.J.B. Meyer Jr., E.F. Brice M.D. Rodgers J.R. Kennard O. Simanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Google Scholar), code 1FBI. Total mRNA was purified from 2 × 106 hybridoma cells by the method of acid guanidium thiocyanate extraction (9Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). cDNA was produced from 5 mg of total RNA using Superscript Reverse Transcriptase (Life Technolgies, Inc.) and 25 pmol of H or L chain constant region specific M. D.R. S.J. Science. 1989; Scholar). The cDNA was by chain using the same and a of described by M. D.R. S.J. Science. 1989; and the were chain was by the chain method U. S. was in and by of at two from chain The of the variable regions of the H and L chains have been deposited with and of the amino from the L chain was carried out by a sequence data could be for the H chain to a amino were using J. The Scholar). of the amino acid the of E.A. C. of of of were carried out as described by M. S. Proteins. 1993; 15: A 3 Å, was found to have the electrostatic several the search for the and The of the affinity complexes and were by the crystallographic coordinates of the unliganded lysozyme A. 1992; Scholar; J. Souchon H. Alzari P.M. Protein Sci. 1994; 3: 788-798Google Scholar) the crystal structures of the In all a from was by a molecular carried out at for the binding were using a of the program J. Appl. Crystallogr. 1983; Scholar) with a of Å and der Waals' for protein The coordinates of the complex were from the Protein Data Bank, code The structure of the complex that of other mAb F9.13.7 lysozyme a large contact by the CDRs at the of the variable The two independent copies of the complex in from a different (8Chitarra V. Alzari P.M. Bentley G.A. Bhat T.N. Eisel J.-L. Houdusse A. Lescar J. Souchon H. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7711-7715Google Scholar). backbone however, are closely The r.m.s. between all coordinates is Å Å the constant The two present the same extended with an between the variable and constant of in conformational changes in the antigen chain. of GEL determined at a resolution (28Lescar J. Souchon H. Alzari P.M. Protein Sci. 1994; 3: 788-798Google Scholar) with bound GEL r.m.s. deviations of and Å for chain with the observed between the two copies of lysozyme in the crystal structure of the complex Å). The deviations for a lysozyme at the of the This however, has no in the structure of GEL (28Lescar J. Souchon H. Alzari P.M. Protein Sci. 1994; 3: 788-798Google Scholar), and the observed contacts are extensive and The epitope is of exposed residues from a α-helix and surrounding loops and The area of lysozyme that is in contact with the antibody is residues from the H chain and from the L chain to the binding of lysozyme This is by the upon complex for the H chain and for the L two lysozyme residues interact with the and hypervariable all other lysozyme residues contact residues from the H chain A large of electrostatic interactions are observed at the salt bridges and 12 hydrogen are between antibody and lysozyme residues The of these contacts antibody residues from the and hypervariable in a The antibody F9.13.7 a of the and in the H chain and and in the L chain. with the (Fig. 2) that make a to binding are observed in of the two variable of these and are and and residues that not contact the in the and that are not in contact with lysozyme. these not the of the with that of antibody that has the amino at these (Fig. the of a contacting lysozyme L chain in the against the group of of the variable regions of bound to and of F9.13.7 bound to GEL The hypervariable loops and of and of F9.13.7 contact different regions of the lysozyme surface. Side chain are for the lysozyme at chain of the and loops are as of the region in the and The L chain of F9.13.7 is similar to that of antibodies Proc. Natl. Acad. Sci. U. S. A. 1987; 84: Scholar). The amino acid of F9.13.7 and the Alzari P.M. Poljak R.J. A. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar) at (Fig. and the coordinates can be with a r.m.s. of In the hypervariable loops have the same with two amino acid in the the that their H chain are the determinants of specificity M. S. M. J. Immunol. 1983; Scholar). The of F9.13.7 that of anti-HEL antibody (8Chitarra V. Alzari P.M. Bentley G.A. Bhat T.N. Eisel J.-L. Houdusse A. Lescar J. Souchon H. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7711-7715Google Scholar). The and loops of these two antibodies are closely significantly of the contact to binding regions of lysozyme (Fig. This how the same structural elements can confer binding specificity in different This to the (11Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Google Scholar), to a set of hypervariable is to The long has the same in the two related complex the active site of lysozyme. residues by the diversity are in interactions with residues from CDRs (Fig. the at the of form a that contact with lysozyme residues with the active and the amino an extended that with the The epitope of GEL defined by F9.13.7 overlaps with that of the antibody HyHEL10 (11Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Google Scholar). The same 12 residues of the antigen make of the hydrogen or salt observed in complex The upon Fab-lysozyme are the same for the two complexes for the antibody and for The between the lysozyme epitopes bound by mAbs F9.13.7 and HyHEL10 can be by the by lysozyme upon complex (Fig. binding a nearly the two antibodies have no sequence at any of their CDRs (Fig. 2) and bind the epitope in different (Fig. A can be between the hypervariable loops of F9.13.7 and of of interact with the same region of lysozyme (Fig. In addition, the of F9.13.7 an to that of in a that the active site of lysozyme (Fig. and the of the for and extensive contacts by the region of F9.13.7 are by and in the The of HyHEL10 contributes a single to as does the of F9.13.7 hydrogen and salt bridges are observed in the two In the and are in a similar at the two antibody combining sites, a Å from the of the antigen-binding the same epitope residues are between GEL and are in interactions with the antibody combining sites. A single salt between from the H chain and from lysozyme was found in the the of several exposed residues (11Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Google Scholar). The is different for F9.13.7, the two and that form with residues of the epitope (Fig. interacts with with and with The salt however, is exposed to the solvent and could be by The backbone structures of GEL bound to F9.13.7 and bound to HyHEL10 are similar the sequence between and GEL of Å for that heterologous binding can be with conformational in the lysozyme however, in chain The of the 12 lysozyme residues common to epitopes can be with r.m.s. deviations of Å, but these to Å chain and chain are in the The of chains lysozyme that steric between the two antibody combining sites and for complementarity of the The constant of F9.13.7 for as determined by is × D. Mariuzza R.A. Poljak R.J. Biochem. 1993; Scholar). of F9.13.7 with other avian lysozymes indicated that the affinity for GEL, and quail lysozymes were similar to that for much or no were observed for and lysozymes D. Spinelli S. Souchon H. Riottot M.-M. Mariuzza R.A. Houdusse A. Eisele J.-L. T. Chitarra V. Boulot G. Bhat T.N. Bentley G.A. Alzari P.M. Poljak R.J. 1990; Scholar). The reduced affinity of antibodies F9.13.7 and for has two amino acid at and with has to the that this region could be bound by the two antibodies and that their epitopes could This however, is with the of a complex observed by not In the crystal structures of the Fab (8Chitarra V. Alzari P.M. Bentley G.A. Bhat T.N. Eisel J.-L. Houdusse A. Lescar J. Souchon H. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7711-7715Google Scholar) and the Fab residues from the lysozyme interact with the two antibody combining sites, in with the binding by the two antibodies is (Fig. 3) with a minor conformational of the backbone to steric binding a common set of the F9.13.7 and HyHEL10 antibodies a different pattern of fine specificity heterologous Both antibodies bind and GEL with but differently with and The and complexes have similar high the constant of the and complexes is significantly D.C. Berzofsky J.A. East I.J. Gurd F.R.N. Hannum C. Leach S.J. Margoliash E. Michael J.G. Miller A. Prager E.M. Reichlin M. Sercarz E.E. Smith-Gill S.J. Todd P.E. Wilson A.C. Annu. Rev. Immunol. 1984; 2: 67-101Google Scholar; D. Spinelli S. Souchon H. Riottot M.-M. Mariuzza R.A. Houdusse A. Eisele J.-L. T. Chitarra V. Boulot G. Bhat T.N. Bentley G.A. Alzari P.M. Poljak R.J. 1990; Scholar). epitope binding to different antigenic determinants lysozyme, the two antibodies a similar structural how different to different of a protein epitope Immunol. 1989; Scholar). model carried out for the and complexes that a lysozyme different for the to In the residues of the epitope and for of the total binding in with studies Smith-Gill S.J. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: Scholar), that of or significantly the binding constant for the other residues and of the binding the of amino in salt bridges and these results that the residues at and are in for the in antibody The chain of the antibody combining site in In the is from the solvent by interactions with with and form a the antibody (Fig. In the same is exposed to the solvent in the (Fig. the of active site is differently in the two bound to the in the complex is against the and to the to the of in the The structural of is with the different binding of the antibodies for and lysozyme, for two amino acid changes at the epitope with to and the of the to a by the and loops of HyHEL10 (Fig. The group of the chain is hydrogen of is from the solvent by the of and The F9.13.7 combining site a similar binding the chain is in contact with the of but exposed to the solvent (Fig. The structural could for the of HyHEL10 with has a acid at this Smith-Gill S.J. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: Scholar). The same have a F9.13.7 of a amino acid between and at to the and to be for of have no the affinity of for HyHEL10 Smith-Gill S.J. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: Scholar). The complex common features with other complexes structures have been The molecular in contact upon complex are of in and a similar of contact residues and hydrogen The constant = is of previously complexes = studies of antibody structures have C. Lesk A.M. J. Mol. Biol. 1987; to the existence of a of backbone or for of the hypervariable The results in this this Although the and hypervariable loops of F9.13.7 and have similar (Fig. for of the combining site and are in interactions with different structural This has not for the of antibody combining sites (11Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Google Scholar) but for the molecular mechanisms that antibody a set of chain for hypervariable loops a potentially unlimited functional repertoire. of heterologous binding is by the of the and 12 out of lysozyme residues in contact with the F9.13.7 combining site interact with that of the of amino acid sequence homology in the CDRs of the two antibodies. of unrelated proteins binding a same set of amino acid residues have been D. Spinelli S. Souchon H. Riottot M.-M. Mariuzza R.A. Houdusse A. Eisele J.-L. T. Chitarra V. Boulot G. Bhat T.N. Bentley G.A. Alzari P.M. Poljak R.J. 1990; found that amino acid residues of anti-HEL antibody D1.3 are in interactions with the antigen and an the structure of the complex between the and A. M. Science. 1992; Scholar) that the same set of residues the two a different structure the Tulip W.R. Colman P.M. 1994; 2: found that of the binding site of antibody overlaps with that of the antibody and 5Braden B.C. Souchon H. Eisele J.-L. Bentley G.A. Bhat T.N. Navaza J. Poljak R.J. J. Mol. Biol. 1994; 243: 767-781Google that mAbs and bind a nearly epitope lysozyme this the H chains of the two mAbs from the of the same their regions and L chains are different combining sites and interactions with In of these cross-reacting molecular structural studies of antibodies have that in functional mimicry could be a molecular mimicry (for review, see 1992; and R.A. Poljak R.J. Immunol. 1993; Scholar), the example by a crystallographic of antibodies P.M. A.T. Science. 1992; Scholar). The data that functional mimicry by the two antibodies is in the of any sequence in their combining sites. antigen not the in antibody for S. G. C. D. D. E. C. Williams A. Nature. 1992; Scholar) that heterologous binding by unrelated molecular be a common in This is in with the of immune and to structures that bind to an antigenic determinant. of the and lysozymes an of the to conformational changes are in the observed complementarity of the The of the epitope contributes to steric complementarity to differently shaped combining sites. molecular to be a general underlying in interactions Tulip W.R. Colman P.M. 1994; 2: Scholar). In the of lysozyme, these changes from chain mobility and are not significantly at the backbone This example and other studies of interactions (for review, see D.R. Padlan E.A. Biol. 1992; 2: and Biol. 1993; 3: Scholar) the of conformational changes in immune recognition and have for to We J. for protein and G. A. Bentley and F. A. for in data and We the for to synchrotron