Structure of anti-peptide antibody complexes

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Structure of anti-peptide I.A. Dept. of Molecular

Wilson

Biology,

(*I, J.B. Ghiara

The use of peptides to generate an immune response has provided a tremendous set of tools for probing a variety of biological phenomena. The ability to specifically target antibodies to particular peptide sequences has permitted the tracking of protein molecules and viruses within the cell. An important general methodology for the purification of proteins is the epitope addition method (Field et al., 1988) where a peptide tag is added, usually to the terminus of an expressed protein, and anti-peptide antibodies against the tag are then used to bind the protein. In addition to its general use in the study of biological systems, the ability to express monoclonal anti-peptide antibodies has contributed greatly to the increase in our knowledge of the structural basis of immune recognition. Recent work has concentrated on peptides of high biological interest, including several peptides from viral pathogens. The current use of peptides to generate neutralizing antibodies to viruses and other microbial agents is now fulfilling the initial promise from the early experiments with anti-peptide antibodies (Lerner, 1982, 1984). This short review deals primarily with an analysis of the X-ray structures of antibody-peptide complexes and the contribution of these studies to our understanding of antibody-antigen interactions. Peptide

antibody and R.L.

The Scripps Research Institute, La Jolla, CA 92037 (USA)

Introduction

conformation

The question of whether peptides adopt a regular secondary structure when bound to antibodies was answered with the structure determination of the antimyohemerythrin C-helix antibody BI312, in complex with a 1Pmer peptide (Stanfield et a/., 1990). The peptide was ordered for the first seven residues EVVPHKK (fig. le), which corresponded well to the binding site determined by epitope mapping (Fieser ef al., 1987). The peptide formed a type II p-turn (*) To whom correspondence should be addressed.

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complexes

Stanfield 10666 N. Torrey Pines Road,

around residues VPHK. The second antibody bound peptide structure to be determined was that of a peptide corresponding to influenza virus haemagglutinin (Rini et al., 1992). Twenty antibodies were raised against a 36-mer peptide, but surprisingly almost all recognized only a five-to-six-mer sequence, DVPDYA (Wilson et al., 1984). The structure of the peptide bound to one of these antibodies, Fab 17/9, contains an extended chain terminating in a type I p-turn (fig. Id). Several other peptide-antibody structures have now been determined in which the peptides also show some preference for turn structures when bound to antibodies. However, this is not always true, as other conformations for the peptide have now been seen. The anti-HIV-l gp120 antibodypeptide complex contains a primarily extended peptide structure that stretches along a 25 ,&groove between the light and heavy chains of Fab 50.1 (fig. lb) (Rini et al., 1993). Peptides from cholera toxin (Shoham, 1993) and from the tip of the V3 loop of HIV-l gp120 (Ghiara et al., 1994) also contain p-turn structures but are more complicated and contain other conformational features (fig. lc,a). For exampie, the peptide in the Fab 59.1 complex contains a type II turn followed by a type III-I double turn. Another major question is whether the peptides adopt similar structures free in solution, as bound peptides, and in the environment of the native protein from which they were derived. For the C-helix peptide of myohemerythrin, the antibody-bound peptide clearly differs from its counterpart in the native protein (Stanfield et al., 1990) and from its NMR solution structure (Dyson et al., 1988), but corresponds to the structure of the same region in apomyohemerythrin when bound to BI312 (Stanfield and Wilson, unpublished results). On the other hand, the haemagglutinin peptide in many ways has a similar conformation to its protein counterpart, the only major backbone deviation being the psi angle of the central proline residue (Rini ef al., 1992). Again the solution structure was different from either the

a.

b.

d.

e. Fig. 1. Stereoview of the conformation of five peptides when bound to different anti-peptide antibodies. The peptides are oriented as they would appear when looking directly dowh into the antibody combining site. The peptides are from the complexes with (a) Fab 59.1, (b) Fab 50.1, (c) Fab TE33, (d) Fab 17/9 and (e) Fab B1312. The peptides all contain some element of regular secondary structure whife bound to Fab. The coordinates for 50.1 (IGGI), 17/9 (IHIM), and B1312 (21GF) are available from the Brookhaven Protein Data Bank, Fab 59.1 coordinates were provided by JBG, and TE33 coordinates were kindly provided by M. Shoham.

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bound peptide or the protein (Dyson et al., 1985). The HIV1 peptide structure in the 50.1 complex was largely as predicted (LaRosa el al., 1990; Rini et al., 1993) and its partial p-turn agreed with NMR results (Chandrasekhar ef al., 1991). For the 59.1 complex, the actual structure deviated significantly from the prediction, as a double turn was observed rather than the predicted single type-II p-turn. This peptide sequence corresponds to the major neutralizing determinant of HIV1 (Ghiara et al., 1994) and so it may be of great value to know its biologically active conformation when considering its potential use in drug or vaccine design. Hence, it is clear that with careful selection, anti-peptide monoclonal antibodies can recognize peptide conformations that are similar to their structures in intact proteins. Many uses of antipeptide antibodies, however, do not need to have such strict structural fidelity. But, for any experiments which depend on protein conformation (White and Wilson, 1987), careful selection of antibodies is required in the initial production stage. Antibody-peptide

interactions

The size of the antigenic determinant bound by anti-peptide antibodies is remarkably constant and reflects, to some extent, the limitation of recognizing a linear peptide sequence. The epitopes vary from 7-12 residues with an average of around 7-8 residues. The sizes of buried interacting surfaces of the antibodies and antigens are also remarkably similar for different antibody-antigen complexes. For eight Fab complex structures determined with linear eptides, the average buried surface area is 480 8,4 for the peptide (ranging from 422-620 R) and 550 R (ranging from 483-725 R) for the antibody (see reviews by Wilson and Stanfield, 1993 ; and Stanfield and Wilson, 1993). The corresponding numbers for antibody- rotein interactions correspond to around 700-900 8 for buried surface areas of both protein and Fab (see reviews by Davies et al., 1990; Colman, 1988) which indicates a maximum area for the Fab surface that can interact with regular-shaped globular proteins. Hence, peptides manage to access only around 50-70 % of the possible Fab binding site surface and given the small numbers of peptide residues bound, can only achieve this extent of interaction by substantially burying themselves in the Fab combining site. In general, there appears to be little correlation of the area of antibody surface buried by ligand with the relative affinity of the interaction.Very small ligands such as progesterone (Arevalo et al., 1993a,b) or fluorescein (Herron et al., 1989 bury comparatively little surface (- 280 8?) o‘g the antibody (reviewed in Wilson and Stanfield, 1993), yet can still bind with nanomolar or better affinities. The shapes of the antibody combining sites for anti-peptide antibodies are more like the clefts or

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grooves seen for haptenic ligands. Indeed, the BI312 and 1719 antibodies have classic clefts or pockets, whereas the 50.1 Fab has a long groove for the peptide ligand to bind. However, there are exceptions. Fab 59.1 binds its peptide in a multiple turn conformation and has a relatively flat binding pocket when bound to the peptide (Ghiara et al., 1994). Antiprotein antibodies tend also to have flatter and more undulatory surfaces that correspond more to the shapes of globular proteins (see review, Wilson and Stanfield, 1993). Conformational

changes

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induced

fit

Early studies of protein-antibody structures suggested that conformational changes were not likely to play a major role in antibody-ant.igen recognition (Amit eCal., 1986). However, small changes were seen for the antigen in other lysozyme- and neuraminidase-Fab complexes and were on the order of l-2 i% for backbone and around 2 8, for side chains (see review Davies et al., 1990 ; Colman, 1988 ; Tulip ef al., 1992). These studies could not determine whether the antibody changed conformation as the structures of both the free and bound Fabs were required to provide the definitive evidence for whether any structural changes occur in antibodies upon ligand complexation. The structures of anti-peptide antibodies have been instrumental in showing the range of conformational changes that occur in antibodies on ligand binding. Crystallization of the free and bound forms of peptide antibodies (Wilson et al., 1991; Stura et al., 1993) has proved to be more facile than expected and the subsequent structure determinations lead to several conclusions about the extent of the conformational flexibility or plasticity of antibodies. For anti-peptide antibodies, antigen-mediated changes range from sidechain and small segmental shifts (l-2 & of CDR loops (B1312, Stanfield et al., 1990), to large rearrangements of either the H3 loop (17/9, Rini et al., 1993 ; Schulze-Gahmen et al., 1994) or the V,/V, interface (50. I, Stanfield et al., 1993; BV04, Herron et al., 1991) or combinations thereof (50.1 and BV04). The major rearrangement in the H3 loop of the 17/9 antibody provided firm evidence for induced fit as a mechanism for antibody-antigen recognition (Rini et al., 1992) and complemented the other examples, such as D1.3 (Bhat et al., 1990), BV04 (Herron et al., 1991) and B1312 (Stanfield et al., 1990). The opportunity to observe multiple free and bound forms of the same antibody in different environments was clearly important in the discriminations of induced fit versus lock-and-key models. The 17/9 antibody has been solved as several complexes under different conditions and with different peptides (Rini et ul., 1992; Schulze-Gahmen et al., 1993). Comparisons of these structures have shown

a.

b.

e.

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that the bound Fab is very similar in all cases. The two independent structures that have been determined for the unliganded 17/9 Fab show that the same conformation exists for each of these free forms, both of which are distinctly different from the bound form (Schulze-Gahmen et al., 1993). A related antibody to 17/9, Fab 26/9 also shows the same bound peptide conformation and illustrates how even limited amino acid changes in the binding site can accommodate and preserve the peptide conformation (Churchill et al., submitted).

Vaccine and drug development The advent of anti-peptide antibodies heralded a new opportunity for producing synthetic vaccines. The initial success of this approach was limited, but recent findings indicate that peptides may indeed be valuable for producing broadly neutralizing antibodies to fight microbial pathogens. A discussion of neutralizing anti-peptide antibodies is confined here to those whose structures have been determined. The anti-influenza peptide antibody 17/9 has been shown to bind to the low pH or fusion active form of the influenza virus. The haemagglutinin changes conformation at a pH that corresponds to its environment when in the endosome (pH 4.8-5.3), and which perturbs its trimeric structure (Skehel et al., 1982). The haemagglutinin antibodies have been very useful in monitoring such biologically important conformational changes (White and Wilson, 1987) and suggest that antibodies against the normally inaccessible, but biologically active forms of viral surface antigens, could be effective neutralizing agents. However, the restriction of activation to the endosomal compartment for this influenza example has proved problematic for the use of neutralizing antibodies. On the other hand, the anti-HIV1 antibodies generated against peptides of the V3 loop of the surface glycoprotein gp120 can neutralize the virus at neutral pH, by preventing the fusion of viral and cellular membranes (Weiss, 1992). Such antibodies have been shown to be effective in neutralizing HIV1 and protecting chimpanzees from infection (Emini et al., 1992). This protection is usually isolate-

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specific, but broadly neutralizing antibodies that recognize conserved structural epitopes within the predominantly hypervariable V3 region would be more useful as general anti-HIV1 vaccines. Such antibodies for HIV1 have now been made (WhiteScharf et al., 1993). Anti-peptide antibodies that have been carefully selected for their ability to neutralize the virus, or those generated against conformationally constrained peptides to ensure recognition of the relevant epitope in the intact antigen, are likely to be more effective as viral neutralizing agents. Furthermore, in the absence of a structure for an intact viral antigen, such as in the case of HIV1 gp120, a good strategy appears to be the design of constrained analogues that mimic the structure of the major neutralizing determinant of the virus. We have found that the complex structure of such determinants can be pieced together from the solution of overlapping epitopes that bind to different neutralizing monoclonal antibodies (Ghiara et al., 1994, submitted; Rini el al., 1993). This novel use of anti-peptide antibodies has provided a means to determine the structure of the V3 loop of HIVl. Structural studies on these antibody-peptide complexes suggest how the virus might interact with potential secondary receptors and hence provide structural insight into the biological role of the V3 loop. The peptide structure derived from such studies can then be used to design new synthetic vaccines or small molecule drugs to inhibit receptor binding. The approach of using antibodies, biological activity selected for their, in complex with peptide determinants may indeed prove to be a general method of mapping important epitopes on the surfaces of pathogens, especially when it is not possible to derive those structures by conventional methods. The authors thank M. Shoham for kindly providing the unpublished TE33 coordinates and A. Gordon for valuable assistance. This work was supported by NIH grants GM-38794 (Bl312). GM-49497 (B1312), AI-23498 (17/g), and GM-46192 (50.1, 59.1) to I.A.W. and the Scripps Research Institute Graduate Program (J.B.G.). R.L.S. is a Scholar of the American Foundation for AIDS Research.

Fig. 2. The structures of five Fab-peptide complexes. These Fab-peptide complexes are in the same orientation as the peptides in fig. 1 (a) Fab-59.1 with HIV1 gp120 peptide; (b) Fab 50.1 with HIV1 gp120 peptide; (c) Fab TE33 with cholera toxin peptide; (d) Fab 17/9 with flu haemagglutinin peptide; (e) Fab B1312 with myohaemerythrin peptide. The majority of interactions between the peptides and Fab are with the heavy chain of the Fab.

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References Amit, A.G., Mariuzza, R.A., Phillips, S.E.V. & Poljak, R.J. (I986), Three-dimensional structure of an antigen-antibody complex at 2.8 .&resolution. Science, 233,141-l%. Arevalo, J .H., Stura, E.A., Taussig, M. J. & Wilson, 1.A. (1993a), Three-dimensional structure of an antisteroid Fab’ and progesterone-Fab’ complex. J. Mol. Biol., 231, 103-118. Arevalo, J.H., Taussig, M.J. & Wilson, 1.A. (1993b), Molecular basis of cross-reactivity and the limits of antibodyantigen complementarity. Nature (Land.), 365, 859-863. Bhat, T.N., Bentley, G.A., Fischmann, T.O., Boulot, G. & Poljak, R.J. (1990). Small rearrangements in structures of Fv and Fab fragments of antibody Dl.3 on antigen binding. Nature (Land.), 347, 483-485. Chandrasekhar, K., Profy, A.T. & Dyson, H.J. (1991). Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-I envelope glycoprotein gpl20. Biochemistry, 30, 9187-9194. Colman, P.M. (1988), Structure of antibody-antigen complexes: implications for immune recognition. A&. Immunol.,

43, 99-132.

Davies, D.R., Padlan, E.A. & Sheriff, S. (1990), Antibodyantigen complexes. Ann. Rev. Biochem., 59.439-473. Dyson, H. J., Cross, K. J., Houghten, R.A., Wilson, I.A., Wright, P.E. & Lerner, R.A. (1985), The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn. Nature (Lond.), 318, 480-483. Dyson, H.J., Rance, M., Houghten, R.A., Wright, P.E. & Lerner, R.A. (1988), Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix. J. Mol. Biol., 201, 201-217. Emini, E.A., Schleif, W.A., Nunberg, J.H., Conley, A.J., Eda, Y., Tokiyoshi, S., Putney, S.D., Matsushita, S., Cobb, K.E., Jett, C.M., Eichberg, J.W. & Murthy, K.K. (1992), Prevention of HIV-l infection in chimpanzees by gpl20 V3 domain-specific monoclonal antibody. Nature (Lond.), 355, 728-730. Field, J., Nikawa, J.-I., Broek, D., MacDonald, B., Rodgers, L., Wilson, I.A., Lerner, R.A. & Wigler, M. (1988), Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevkiae by use of an epitope addition method. Mol. Cell. Biol., 8, 2159-2165. Fieser, T.M., Tainer, J.A., Geysen, H.M., Houghten, R.A. & Lerner, R.A. (1987), Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti-peptide antibodies with a protein alphahelix. Proc. Natl. Acad. Sci. (Wash.), 84, 8568-8512. Ghiara, J.B., Stura, E.A., Stanfield, R.L., Profy, A.T. & Wilson, I.A. (1994), Science (in press). Herron, J.N., He, X.M., Mason, M.L., Voss, E.W.J. & Edmundson, A.B. (1989), Three-dimensional structure of a fluorescein-Fab complex crystallized in 2-methyl-2-4-pentanediol. Proteins, 5, 271-280. Herron, J .N., He, X.M., Ballard, D. W ., Blier, P.R., Pace, P.E., Bothwell, A.L., Voss, E.W. Jr. & Edmundson, A.B. (1991), An autoantibody to single-stranded DNA: comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex. Proteins, 11, 159-175. LaRosa, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A., Profy, A.T., Lewis, J.A., Langlois, A.J.,

Dreesman, G.R., Boswell, R.N., Shadduck, P., Holley, L.H., Karplus, M., Bolognesi, D.P., Matthews, T.J., Emini, E.A. & Putney, S.D. (1990). Conserved sequence and structural elements in HIV-l principal neutralizine determinant. Science. 249. 932-935. Lerner, R.A. (19%2), Tapping the immunological repertoire produce antibodies of predetermined specificity. Nature (Lond.), 299, 592-596. Lerner, R.A. (1984). Antibodies of predetermined specificity in biology and medicine. Adv. Immunol., 36, l-44. Rini, J.M., Schulze-Gahmen, U. & Wilson, I.A. (1992), Structural evidence for induced fit as a mechanism for antibody-antigen recognition. Science, 255, 959-965. Rini, J.M., Stanfield, R.L., Stura, E.A., Salinas, P.A., Profy, A.T. & Wilson, I.A. (1993), Crystal structure of an HIV-l neutralizing antibody 50.1 in complex with its V3 loop peptide antigen. Proc. Nat/. Acad. Sci. (Wash.), 90, 6325-6329. Schulze-Gahmen, U., Rini, J.M. & Wilson, I.A. (1993), Detailed analysis of the free and bound conformations of an antibody. J. Mol. Biol., 234, 1098-l 118. Shoham, M. (1993), Crystal structure of an anticholera toxin peptide complex at 2.3 A. J. Mol. Biol., 232, 1169-l 175. Skehel, J.J., Bayley, P.M., Brown, E.B., Martin, S.R., Waterfield. M.D.. White. J.M.. Wilson. I.A. & Wiley, D.d. (1982), Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. Natl. Acad. Sci. (Wash.), 79, 968-972. Stanfield, R.L., Fieser, T.M., Lerner, R.A. & Wilson, I.A. (1990), Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 8, Science, 248, 712-719. Stanfield, R.L., Takimoto-Kamimura, M., Rini, J.M., Profy, A.T. & Wilson, I.A. (I993), Major antigeninduced domain rearrangements in an antibody. Structure,

I, 83-93.

Stanfield, R.L. & Wilson, I.A. (1993), X-ray crystallographic studies of antibody-peptide complexes. Immunomethods, 3, 21 l-221. Stura, E.A., Gieser, G.G. &Wilson, I.A. (1993), Crystallization of antibodies and antibody-antigen complexes. Immunomethods, 3, 164- 179. Tulip, W.R., Varghese, J.N., Laver, W.G., Webster, R.G. &Colman, P.M. (1992), Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex. J. Mol. Biol., 227, 122-148. Weiss, R.A. (1992), Human immunodeficiency virus receptors. Virology, 3, 79-84. White, J.M. & Wilson, I.A. (1987), Anti-peptide antibodies detect steps in a protein conformational change : low-pH activation of the influenza virus hemagglutinin. J. Cell Biol., 105, 2887-2896. White-Scharf, M.E., Potts, B.J., Smith, L.M., SokoIowski, K.A., Rusche, J.R. & Silver, S. (1993), Broadly neutralizing monoclonal antibodies to the V3 region of HIV-1 can be elicited by peptide immunization. Virology, 192, 197-206. Wilson, I.A., Niman, H.L., Houghten, R.A., Cherenson, A.R.. Connollv. M.L. & Lerner. R.A. 11984). The struciure of an’ ‘antigenic determinant in‘ a p&tein. Ce(l, 37, 767-778. Wilson, I.A., Rini, J.M., Fremont, D.H., Fieser, G.G. & Stura, E.A. (1991), X-ray crystallographic analysis of free and antigen-complexed Fab fragments to investigate structural basis of immune recognition. Meth. Enzymol., 203, 153-176. Wilson, I.A. & Stanfield, R.L. (1993), Antibody-antigen interactions. Curr. Opin. Strut. Biol., 3, 113-118.

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DISCUSSION P.M.

Alzari :

This Forum reflects some of the present concerns involving the structural implications of immune recognition by antibodies. The availability of a now considerable database of antibody structures, complemented and potentiated by the widespread use of molecular biology techniques in functional studies, has prompted a number of studies on structurefunction relationship. Thus, the pioneering work of Kabat, Chothia, and others that were based on a limited number of amino acid sequences and threedimensional structures, is now being substantiated by similar studies on larger databases. Two examples in this Forum are the analysis of sequence and structure patterns related to V, subgroups by F. Saul, and the observation of Abergel er al. that only a small percentage of the residues in regions displaying hypervariable sequences (CDR) contributes to the construction of an antigen-binding site. Smith-Gill describes studies of anti-lysozyme antibodies that highlight the different nature of structural and functional epitopes, in agreement with the ideas put forward by van Regenmortel and others about the operative character of the epitope definition. Jerne’s proposal that some antiidiotypic antibodies carry a structure (internal image) that mimics the original antigen is discussed by Amzel et al. in light of recent structural evidence. They correctly conclude that, although true molecular mimicry by antibodies may be a rare phenomenon in general, peptide antigens are the system in which mimicry could be most easily accomplished (as their remarkable work on the complex between angiotensin II and an antiidiotypic antibody strongly suggests). The effects of amino acid substitutions on antigen recognition are discussed by Colman, who remarks the importance of considering the structural context in which these changes take place. He then speculates on the effective size of the antibody repertoire, noting that tolerance of non-conservative substitutions at the antigen-antibody interface indicates that different antibodies can convey a same specificity (a notion strongly supported by his work on mutant neuraminidase-antibody complexes), whereas conformational adaptability of the antibody structure might indicate that a single antibody can serve to specifically bind a number of different antigens (something that remains to be demonstrated). A number of crystallographic studies has provided a more complete picture of antigen-antibody interactions in various systems. Bentley et al. describe two examples of cross-reactive complexes in antilysozyme antibodies. Although binding of the same

antibody to homologous ligands appears to be largely the consequence of a conserved epitope, distinct strategies are used by Fab D1.3 to recognize heterologous molecular surfaces. Novel features (the use of aromatic Trp and His residues in forming hydrogen bonds with sugar hydroxyls) were observed by Cygler in a carbohydrate-antibody complex, distinguishing it from other sugar binding proteins. However, further studies of Fab-carbohydrate complexes are required in order to determine to what extent these observations are likely to prove general. Antibody-peptide complexes are reviewed by Wilson, who notes that several antibody-bound peptides show some preference for turn structures and that the average buried surface area is about 500 A2 for peptide. These studies have also been important to evidence the plasticity of the antigen-binding site of antipeptide antibodies, for which antigen-mediated changes range from local side-chain movements to large rearrangements of H3 loops and Vu/V, interfaces. Edmundson et al. also describes ligand-induced conformational changes on the structures of the Meg light chain dimer and of Fab NC6.8, whose unliganded and antigen-bound forms were crystallized in different space groups. A significant variation of the elbow angle between the native and complexed Fab NC6.8 molecules prompted the authors to speculate on a possible conformational mechanism for the activation of the complement cascade, although it is difficult to assess the influence of distinct packing environments on the observed structural changes. The contribution of hydration to the energetics of molecular assembly has been recognized for a long time. In particular, there is some consensus on the role of water molecules in perfecting surface complementarity and in mediating intermolecular hydrogen bonds (see, for example, Mariuzza et al., Bentley et al. and Cygler in this Forum). However, the view of the hydrophobic effect as the major driving force in protein association is a more controversial issue. In an interesting paper, Mariuzza et al. describe calorimetric and crystallographic studies of the FvDl.3-lysozyme reaction. Their experiments revealed a large negative enthalpy change, favourable to binding, and a smaller unfavourable entropy change. According to Mariuzza et a/., these results support the notion that the dominant forces stabilizing this complex arise from hydrogen bond formation and van der Waals interactions rather than the hydrophobic effect. Furthermore, they invoke the number of ordered water molecules observed in the unliganded and complexed crystal structures as a possible explanation for the insufficiency of hydrophobic interactions to account for complex stabilization.

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However, the usual meaning of hydrophobicity (or hydrophobic interactions) refers to the total free energy of transfer of non-polar solutes into aqueous solutions. In other words, at least part of the enthalpit contribution to protein association may actually arise from the hydrophobic effect. The dynamic behaviour of water on protein binding remains another unresolved question, and further studies probably reserve unexpected surprises. Two recent examples include studies of proteins under osmotic stress, which revealed an impressive number of hydration waters involved in ligand-binding reactions, and high resolution NMR studies of protein hydration, indicating residence times of solvation waters in the subnanosecond range even when the hydration sites contain well-ordered water in the Xray structures. Undoubtedly, a better understanding of the role of water in protein recognition is one of the major challenges for future research. G.A. Bentley : Comments

on the contribution

by Amzel et al.

The paper by Amzel et al. argues that, while evidence for a structural internal image in their system of anti-angiotensin II antibodies is indirect, it offers the most likely explanation for their observations on the structure of the complex formed between the mAb3 131 and angiotensin II, and for its relationship to the mAb1 110. The murine mAb1 110, obtained from the immune response to angiotensin II, was used to obtain polyclonal antiidiotypic antibodies in rabbit. The latter were then used in turn to produce antiantiidiotypic antibodies in the same strain of mouse that provided the mAb1 110. The mAb3 13 1 thus obtained also binds to angiotensin II with high affinity. As an alternative to the internal image hypothesis, the authors put forward the following possibility. Because the rabbit polyclonal Ab2 binds to the murine mAb1 110 with high affinity, it selects a mAb3 within the same strain of mouse which is very similar to the mAb 1 by recognizing an idiotope which is either completely or largely distinct from the antigen binding site. Indeed, the nucleotide sequence data presented in their paper (Garcia et al., 1992b) reveals a very close resemblance between the two antibodies in both the variable light and heavy chain coding regions, showing that but for the D region, the gene segments coding for 110 and 13 1 were derived from the same germ-line source or at least from the same gene families. The D coding segment and the junction regions of mAb3 13 1, however, provide an alternative genetic solution to form what is likely to be a very similar structure for the H3 loop present in mAb1 110; the authors accordingly reject idiotope selection as an explanation in favour of an internal

IN IMMUNOLOGY structural image of angiotensin II being carried by the Ab2. Yet I think that the use of the same (or similar) V,, J,, V, and J, gene segments still renders idiotope selection as a very plausible explanation; given the diversity of the antibody repertoire, the very close similarity between 110 and 131 may be more than a coincidence. A note of caution to the internal image hypothesis for the mAb3 131-angiotensin 11 complex could be added here. Angiotensin II is deeply buried in the complex with all residues of the peptide making direct interatomic contacts with the Fab moiety. Thus, if a CDR on the Ab2 were to carry a structural internal image of the peptide, it too would have to be equally buried in the antigen binding site without incurring steric hindrance between the other CDR of the Ab2 and mAb1 110. For example, if an L3 loop similar to that of REI were to mimic angiotensin II, as suggested (Garcia et al., 1992a), there might be difficulty in avoiding steric hindrance, since the residues at each end of the CDR are very likely to be buried, as shown in table I of the paper by Abergel et al. in this Forum. The image-carrying CDR might have to be of longer than average CDR length for this hypothesis to remain a possibility. References Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T. & Amzel, L.M. (1992a), Science, 257, 502-507. Garcia, K.C., Desiderio, S.V., Ronco, P.M., Verroust, P.J. & Amzel, L.M. (1992b), Science, 257, 528-53 1.

E.A. Padlan,

C. Abergel and J.P. Tipper:

The binding of an antibody to its antigen is exquisitely specific and various aspects of this specificity are discussed by the contributors to this Forum. Yet many antibodies have been demonstrated to be multispecific or polyreactive, i.e., they are capable of binding to several different antigens, although with low affinity (see, e.g., Burastero et al., 1988). What is the structural basis for this polyreactivity? Crystallographic studies of a number of antibodies complexed to their specific antigens confirm the notion that the specificity of antibody-antigen interactions is due to the complementarity of the interacting surfaces, not only in terms of shape but also in terms of physical and chemical properties. This notion of complementarity has led to the suggestion that the combining site of an antiidiotypic antibody may mimic the structure of the original antigen (Jerne, 1974) and that the antiidiotypic antibody itself may be useful as a vaccine (Nisonoff and Lamoyi, 1981) (see, however, the contributions of Amzel et al. and of Bentley et af. to this Forum). Numerous studies have demonstrated that any depar-

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ture from complementarity can have a significant effeet on the binding (see, e.g., the contributions of P.M. Colman and of S.J. Smith-Gill). Is the fit between antibody combining site and antigenic determinant less tight in the case of polyreactive antibodies? Indeed, the presence of an unfilled cavity in the interface would be energetically costly and would result in lower affinity. Solvent molecules may smooth out an imprecise fit between the interacting surfaces and even enhance binding (see, e.g., the contributions of M. Cygler and of Mariuzza et a/.). However, the immobilization of solvent molecules will result in a decrease in the entropy of the system and subtract from the total binding energy. It has been demonstrated that the antibody combining site is not a rigid structure; rather it is deformable, and it has been suggested that this plasticity may enhance antigen binding in the manner of an “induced fit” (see, e.g., the contributions of A.B. Edmundson et al. and of I.A. Wilson). Are the combining sites of polyreactive antibodies unusually plastic so that they can assume many different conformations and thereby complement many different antigenic structures? Here, again, entropic considerations are important, since the immobilization of structural elements upon complex formation will result in a decrease in entropy. Could it be that the combining sites of polyreactive antibodies are enriched in structural elements that make them more “sticky”? For example, is there a greater incidence in their CDR of aromatic side chains (which can contribute more to the binding energy than the smaller, apolar, aliphatic side chains (Padlan, 1990)), or of charged residues (which are less subject to directional constraints and which can contribute significantly to the binding energy through the formation of strong ionic interactions (GonzalezQuintial et al., 1990))? It may be that polyreactivity is simply a manifestation of the utilization of different areas of the hypervariable surface in binding. In fact, it is found that only a quarter to a third of the CDR surface is in contact with antigen and that, in the main, only the most variable residues are used in specific interactions (see the contribution of C. Abergel et a/.). Is there a greater utilization of the less variable positions in polyreactive antibodies? In this regard, it is interesting that polyreactivity is usually associated with natural or pre-immune antibodies which are encoded by unmutated or essentially unmutated germline genes (see, e.g., Baccala et al., 1989). It is obvious that we can still only speculate on the structural basis for these “not-very-specific” antibody-antigen interactions. However, the understanding of the concept of polyreactivity is important and must be pursued. This importance is

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emphasized by the fact that the existence of polyreactive natural antibodies affords a distinct survival advantage, since it provides an organism the ability to cope, albeit initially only weakly, with an antigen that it had not previously encountered. A better defence against subsequent challenge by the same antigen is then achieved through affinity maturation (see, e.g., the contribution of S. Spinelli and P.M. Alzari). References Baccala, R., Quang, T.V., Gilbert, M., Ternynck, T. & Avrameas, S. (1989), Proc. Natl. Acad. Sci. (Wash.), 86, 4624. Burastero, S.E., Casali, P., Wilder, R.L. & Notkins, A.B. (1988), J. Exp. Med., 168, 1979. Gonzalez-Quintial, R., Baccala, R., Alzari, P.M., Nahmias, C., Mazza, G., Fougereau, M. & Avrameas, S. (1990), Eur. J. Immunol., 20, 2383. Jerne, N.K. (1974), Ann. Immunol. (Inst. Pasteur), 12X, 373. Nisonoff, A. & Larnoyi, E. (1981), Clin. fmmunol. Pathol., 21, 397. Padlan, E.A. (1990), Proteins. Struct. Funct. Genet., 7, 112.

A.B. Edmundson, L.W. Guddat, L. Shan, Z.-C. Fan and B.L. Hanson: It is gratifying to feel that each contribution to this Forum can be considered as a starting point for a review article. We are especially impressed by the strides made in the understanding of the interactions of antibodies with protein antigens. In addition to the shape complementarity, hydrogen bonding and ion pairing between two proteins, water molecules are found to be included rather than completely excluded from the binding process (Bentley et al. ; Mariuzza et al.; this Forum). We and others have noted that ordered water molecules tend to be concentrated at the exposed surfaces of the CDR in unliganded Fab, particularly along the junction of the light and heavy chains. It seems inevitable that many of these solvent molecules are displaced during complexation with protein antigens, but some are retained to bridge the space between juxtaposed residues. Such phenomena are not limited to block-end types of interactions between proteins. For example, water molecules are directly involved in the binding of peptides (Wilson et al.), carbohydrates (Cygler) and a small guanidine-based sweetener (Edmundson et a/.). With relatively large areas buried in the binding of proteins it is not always obvious which residues are-most critical (Abergel et al. ; Amzel et al. ; Bentley ef al. ; Colman; Mariuzza et al. ; Saul ; SmithGill). Non-conservative exchanges are sometimes tolerated in the interface without loss of specificity (Colman). In other cases, like the histidine-glutamine

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antigenic variation in lysozymes, the substitution may be completely incompatible with the binding capability of an antibody (Bentley et al. ; Mariuzza et al.). Glutamine and histidine have very similar hydrogen bonding capabilities at physiological pH values. Without considering steric restrictions, we were initially surprised at the discrimination shown by the D1.3 antibody for the two lysozymes. In a test system with the Meg light chain dimer, however, histidine behaved very differently from glutamine when doubly protonated (Edmundson et al.). The direction of entry of the peptide N-acetyl-His-Pro into the binding cavity could be reversed merely by changing the pH from 7.2 (no charge on the imidazole ring) to < 6.2 (positive charge). Even when the tests are not conducted under on-off conditions, it is desirable to consider the possible effects of the binding environment or the pK of histidine side chains. In more protected active sites like cavities, slots or fissures, the analyses are usually less complicated. High affinity is often associated with nearly maximal utilization of potential binding participants, as evidenced by the complexation of antibodies with fluorescein (Herron et al., 1989) and 2-phenyloxazolone (Spinelli and Alzari). In the latter example, formation of a suitable active site requires a short CDR3 of the heavy chain and the presence of a strategically placed glycine residue in position 3 of the D segment. Again there is a Hisamidated side chain (Asn or Gin) interchange among the somatic mutations (at position L33), this time in the antibody instead of the antigen. Rather than eliminating binding, the substitutions lead to ten-fold increases in affinity. Because of the volume differences, we do not consider Asn and Gln as equivalent contact residues. In agreement with this opinion, NQ22.18.7 and NQ10.12.4.6 have 250-fold differences in affinity which are probably related to a substitution of Asn for Gin in position L33. The second pertinent mutation in the active site, Phe for Tyr in L35, was explained in terms of a disruption of a possible hydrogen bond of Tyr with Gln L88, which is in contact with the ligand. As expected, most random mutations did not disturb the architecture of the active site or the binding patterns. It appears to us that Ox-l antibodies have CDR sequences that are near the top of the affinity maturation stages necessary to maintain a relatively deep pocket and therefore are not subject to major changes. The late dominance of V,4.51-TEPC25 antibodies apparently represents a comprehensive design change which enhances the on-rate kinetic constants. These observations are in contrast to the antifluorescyl antibodies. David Kranz, James Herron and Edward Voss found that members of an idiotype family varied in affinity over five orders of magnitude. The on-rates differed by a factor of 7, but the off-rates varied by three orders of magnitude.

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Thus, affinity maturation in the anti-fluorescyl series progressed by prolongation of the off-rates. We have determined the three-dimensional structure of the molecule (4-4-20) with the highest affinity (10” M-‘) in the family. A comparison of the 4-4-20 contact residues with the sequences of the other antibodies suggests that the substitution of arginine for histidine raises the affinity by two orders of magnitude. It is more than a coincidence that this mutation site corresponds to the L33 hot spot in the anti-phenyloxazolone series. In the discussion of cross-reactivity, Bentley et al. mention that “natural” autoantibodies can bind to diverse self antigens with different sequences. By serendipity, the human Pot IgM selected for study by Robert Raison in Sydney, Australia, turns out to be such a molecule. When we determined the structure of the Pot Fv (released from the IgM by pepsin), we found that the active site region presents an undulating surface with little or no access to the interface of the VL and VH domains. Even more unusual, there are few CDR residues on the surface. Gordon Tribbick and Allen Edmundson were unable to bind tethered peptides to the IgM by Mario Geysen’s methods, an occurrence noted in fewer than 5 % of all antibodies tested in Geysen’s laboratory. Recently, Stuart Tangye, Paul Ramsland and Robert Raison demonstrated that this natural antibody is polyspecific, with activities toward IgG components (probably light chains), spectrin and other selfantigens. Structural studies on the binding of carbohydrates by antibodies are progressing quite nicely (Cygler). In the first 50 years of modern immunological research, there were probably more papers on the complexation of these antigens than any others, notably in the laboratories of Michael Heidelberger, Elvin Kabat and their colleagues. The stacking of aromatic side chains like Trp and His to hydrophobic patches on the monosaccharides and the extensive hydrogen bonding to the hydroxyl groups predicted by the pioneers are beautifully illustrated in the crystal structures of the complexes. As in recurring themes with other types of antigens, water molecules play important roles in bridging hydrogen bonds between sugar and protein constituents and also provide improved shape complementarity. We have screened thousands of peptides for binding to IgG, IgM and light chain dimers by Geysen’s technology and have determined the crystal structures of more than 35 peptides with the Meg dimer. There are few, if any areas of conflict between these results and the conclusions of the more recent (and elegant) peptide binding studies reported by Wilson et al. The last vestiges of opposition to induced fit mechanisms seem to have disappeared and the field can now concentrate on other issues, such as those outlined by Graham Bentley in his introductory remarks.

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F.A. Saul:

Abergel et al. analyse the three-dimensional structures of antigen-antibody complexes of D1.3, HyHEL-5, HyHEL-10, and NC41, correlating hypervariability of CDR with antigen binding. The antigenbinding segments occur primarily in the central portion of the CDR surface; as a result, the combining sites represent only a fraction of the total CDR surface. This observation, along with other contributions to this Forum, suggests that perhaps we should redefine the precise positions of the CDR by taking into account structural information as well as the large sequence database now available. Amzel et al. describe requirements for antiidiotypic antibodies to mimic antigen; short peptide antigens are best suited. Indeed, experimental evidence provided by anti-angiotensin II Ab3 antibody strongly suggests that Ab2 carries an internal image of the antigen. As noted by the authors, structural mimicry lacks experimental evidence for protein antigens and is unlikely for carbohydrate epitopes. However, crystallographic studies of antiidiotypic antibodies in anti-protein systems show that complementarity can be accomplished without an accurate internal image. This leads to the question: what other functional properties of proteins can be mimicked by antiidiotypic antibodies? Bentley el ai. describe two types of cross-reactivity based on structural studies of antibody-antigen complexes : cross-reactivity with closely homologous antigens (Fab D1.3 and Fv D11.15 with various lysozymes) and with structurally distinct antigens (D1.3 with lysozyme and the anti-idiotope E225). In both cases, the versatility of water molecules in perfecting antigen-antibody complementarity and the occurrence of side-chain conformational changes are observed. The D1.3 idiotope recognized by E225 overlaps only partially with the paratope which binds lysozyme, and the shared idiotope-paratope residues are often exploited in different ways. These observations are also relevant to the discussion of mimicry by antiidiotypic antibodies, emphasizing functional mimicry. Colman discusses crystallographic studies of amino acid substitutions in the antibody-antigen interface, the role of substitutions in affinity maturation of the immune response, and implications concerning the size of the antibody repertoire. Analysis of the NC41 antibody-complex system indicates that escape mutants of the influenza virus neuraminidase result in shape changes of the antigen which could interfere with docking to a rigid antibody structure. In contrast, point mutations are shown to induce movement of antibody side chains to accommodate specific changes. Cygler describes structural mechanisms of carbohydrate recognition : the importance of stacking in-

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teractions between aromatic residues and the hydrophobic patches on the sugars, and specificity achieved through hydrogen bonds. In the Se155-4 Fab-oligosaccharide complex, shape complimentarity provides a significant contribution of van der Waals interactions. In addition to direct hydrogen bonds in the complex, ordered water molecules form bridging hydrogen bonds between the sugars and the Fab. Structural studies of mutants and other complexes show that binding to antibody induces conformational changes in the oligosaccharide, whereas comparison of free and complexed structures do not show conformational changes in the antibody. Edmundson et al. discuss extensive studies of ligand binding to the Meg dimer ; induced fit mechanisms, the concept of a larger main binding site cavity and deep pocket, and binding to overlapping sites. The authors also describe two types of conformational changes in Fab NC6.8 upon complexation with NC174 hapten : local changes consistent with induced fit, and transmitted conformational changes brought about by differences in elbow bend angle. The change in elbow angle leads to a large shift in the position of C-terminal residues, with implications for transmitted signal to the Fc region. However, could this shift be due to crystal packing in different space groups? Examples of crystal structures have been reported of (admittedly much smaller) elbow angle differences in Fab related by non-crystallographic symmetry. Mariuzza et al. describe the results of calorimetric measurements of the FvD1.3-HEL association reaction and correlate these results with the high resolution three-dimensional structure of the complex, showing that the formation of hydrogen bonds and van der Waals interactions provide the dominant forces stabilizing the antigen-antibody complex. The crystallographic results show, in addition to direct antibody-antigen interactions, the importance of water molecules in providing hydrogen bonds bridging antigen to antibody. Although entropic effects (in particular, what goes on in the bulk solvent upon complex formation) are more difficult to describe based on crystallographic studies, the calorimetric results suggest quantitatively that these effects do not drive the reaction. Smith-Gill describes structural epitopes as defined by X-ray studies and functional epitopes defined by immunochemical assays. Experimental studies have identified a small subset of critical residues within the structural and functional epitopes, and theoretical calculations suggest the importance of the energetic epitope (which may be predicted and calculated from the structural data). As also noted in other contributions to this Forum, functional assays have detected significant contributions of non-contact residues as well as long-range contributions to antibody-antigen interactions. Study of the nature of

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such contributions may lead to deeper insight into antibody structure-function relationships. Spinelli and Alzari discuss structural implications of somatic mutations in the anti-phenyloxazolone system. Correlation of sequence changes in the antiphOx system with the three-dimensional structure of Fab NQ10/12.5 provides insight into the role of somatic mutations during maturation of the immune response. In the Fab NQ10/12.5-ph0x complex, the hapten occupies a deep cavity, displacing water molecules and slightly modifying side chains. This provides another example of the influence of water molecules and conformational changes on hapten binding. Again, it is interesting to note the occurrence of long range or indirect antigen-binding effects. Wilson et al. analyse structures of antibodypeptide complexes ; in some cases, antibody-bound peptide is found to adopt a different conformation, and conformational changes occur in anti-peptide antibodies on ligand binding. The size of the antigenic determinant is remarkably constant (epitopes vary from 7-12 residues), and the buried interacting surfaces of antibodies are remarkably similar for different antigen-antibody complexes. This work also describes use of anti-peptide antibodies against the V3 loop of gp120, with applications to vaccine and drug development, and the novel use of anti-peptide antibodies to determine the structure of the V3 loop. S. Smith-Gill

Comments

:

on article by Mariuzza

et al.

The thermodynamics of association of HEL with the antibody HyHEL-5 has also recently been characterized by isothermical titration calorimetry (K.A. Hibbits, D.S. Gill, and R.C. Willson, 1994, J. Biol. Chem., in press). They found that, like D1.3, the HyHEL-S/HEL association is enthalpically driven at all temperatures examined, and is also accompanied by unfavourable entropy changes. Mariuzza et al. emphasize the potential importance of van der Waals contacts in the entropic interactions. Hibbits et al. also concluded that hydrogen bonds and van der Waals contacts likely make the main contributions to the negative entropy, but in addition suggested that an additional important source of unfavourable entropy may be constraints of vibrational motions in regions within the epitope which are highly mobile when uncomplexed, but which exhibit much lower mobility when complexed. It is interesting that both D1.3 and HyHEL-5 are directed against the same protein; will these two complexes prove to be the exception or the rule, for anti-protein antibodies in general, or for anti-HEL antibodies specifically? A survey of protein/protein interactions by Hibbits et al. suggests unfavourable entropy is unusual among

IN IMMUNOLOGY protein/protein interactions. Unlike the D1.3 complex, there are very few water molecules in the HyHEL-5 interface, so the nature of potential hydrophobic effects may be very different in these two antibodies. The two complexes also differ in the relative importance of electrostatic interactions. The HyHEL-lO/HEL complex has more van der Waals contacts than either D1.3 or HyHEL-5, and is intermediate between the two with respect to electrostatics and water within the interface. It will be very interesting to compare the thermodynamics of HyHEL-10 and other recently characterized anti-HEL antibodies with that of other anti-protein antibodies. Comments

on articles by Wilson et al. and Cygler

There doesn’t appear to be a real distinction between the types of structural motifs used by antiprotein antibodies in contrast to anti-hapten or antipeptide antibodies, as suggested by Wilson et al. The groove and pocket motifs are quite prominent in the anti-HEL mAb HyHEL-5 and D1.3, respectively. In HyHEL-5 a distinctive groove accommodates the HEL side chains of Arg68 and Arg45, which form a prominent ridge on the surface of HEL. The important pocket motif is critical to complex formation by D1.3, to accommodate the side chain of HEL Gln 121; mutation of HEL Gin 121 to His drastically reduces binding, because the His will not fit in the pocket (Bhat et al., 1990). On the other hand, the carbohydrate-specific 5539 has a relatively flat pocket or depression on its combining site in contrast to distinctive pockets seen in some other carbohydrate- or hapten-specific antibodies. Thus, a range of similar basic motifs is seen in all 3 types of antibodies (antihapten, anti-peptide, and anti-protein); the only real difference is in the total amount of surface area (i.e. surface in addition to the groove, pocket, or depression) involved in antigen complementarity. As reviewed by both Wilson et al. and Cygler (see also Edmundson et al., this Forum, and Benjamin et al., 1992, J. Biol. Chem., 31, 9539-9545), all 3 types of complexes (hapten, peptide, and protein) appear to also undergo induced fits of both antigen and antibody, although to date those described for antiprotein complexes are less dramatic. Comments

on paper by Colman

et al.

Structural data has been obtained on a mutant within the HyHEL-5 epitope (D. Davies and S. Chako (1993), Accounts Chem. Res., 26, 421-427), Arg68 + Lys, which reduces binding affinity by three orders of magnitude. The results support the conclusion that local charges at and immediately adjacent to the mutated residue account for the disruption in binding. In the mutant complex, the interface region

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is very similar to the native complex, with significant changes in the positioning of water molecules to provide complementarity in the mutant structure, and all major changes localized to the mutation site. Local changes in hydrogen bonding patterns are sufficient to explain the affinity differences. In contrast, results with engineered mutations on the antibody HyHEL-10 (Lavoie et al., 1992) support the argument that indirect effects of antibody residues may significantly contribute to effective repertoire size. Somatic mutation of the germ-line Jn-encoded residue 101 increased the affinity of the antibody by over 5 orders of magnitude, and also changed fine specificity. In the HyHEL-lO/HEL complex, this residue is neither in contact with antigen nor in contact with other antibody residues that contact antigen. The same study also showed that point mutations in light chain non-contact residues also indirectly affect fine specificity patterns; in this case, the affected residues are immediately adjacent to contact residues. These experiments also demonstrated that single amino acid point changes can contribute to sensitivity of the antibody to mutations in the antigen, without significantly altering affinity for the original antigen. HyHEL-10 and the closely related antibody HyHEL-8 use different members of the same V, family with the same V,; they have nearly identical affinities for the same epitope on HEL, but significantly different patterns of fine specificity. Such subtle effects on fine-specificity or crossreactivity patterns add another dimension to repertoire considerations.

this variant is about 500-fold less than for the homologous antigen, it is still in the micromolar range. Indeed, the complex between D1.3 and this lysozyme variant has been crystallized and its structure determination is underway.

R. Mariuzza :

For S.J. Smith-Gill

For P.M.

The author addresses the important question of whether productive antigen-antibody binding is largely mediated by a small subset of the total number of contacting residues, as suggested by certain theoretical calculations, or whether complex cooperative interactions across the entire contacting surface are responsible for the observed affinities. Recent X-ray crystallographic and thermodynamic studies in our laboratory of site-directed mutants of the antilysozyme antibody D1.3 indicate that the latter is the more accurate view (unpublished). In general, we find that the number of contacts made by a particular residue in the X-ray structure is a fairly good indication of its relative contribution to the binding reaction.

Colman

With respect to antigenic variants which no longer seem to bind antibody, it should be pointed out that the distinction between “binding” and “not binding” is an arbitrary one in that affinities which are below the detection threshold of one technique may be measurable by another. For example, the reported failure of D1.3 to bind to a lysozyme variant in which Gin is replaced by His was based on the lack of reactivity observed by ELISA. More recently, we have been able to measure binding by two other physical techniques, ultracentrifugation and titration calorimetry, which are more suited for studying lowaffinity interactions. While the affinity of D1.3 for Key-words:

Antibody,

Antigen,

Structure,

Epitope;

For A.B.

Edmundson

et al.

The finding of conformational differences between free and hapten-complexed NC6.8 Fab reinforces the notion of an induced fit mechanism of binding. While the differences observed in the combining site may be readily attributed to the binding process, interpreting changes in the elbow bend angle may be more difficult. This region of the antibody molecule is known to be quite flexible due to the small number of longitudinal contacts between V and C domains. In fact, cases are known in which different crystal forms of the same Fab display different elbow bend angles independently of whether or not antigen is bound. The explanation usually given is that this simply reflects differences in crystal packing, whereby one particular conformation out of many which exist in solution is stabilized by a particular set of intermolecular contacts in the crystal lattice. Nevertheless, the idea of allosteric changes in antibody conformation remains an attractive one, as it would provide an elegant structural explanation for phenomena such as B-cell activation and complement fixation.

Forum.