Immunology Today, vol. 8, No. ! ;, ;987

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virus proteins Numerous studies have addressed the nature of antibodyantigen interaction, but only recently have three-dimensional structures of complexes of antibodies with protein antigens been reported, one with lysozyme and one with the influenza virus antigen neuraminidase. Both structures show that there are epitopes involving about 16 amino acids on surface loops of the antigen. In the lysozyme complex the interaction of the components is rigid, but there is a degree of structural flexibility in the formation of the complex with neuraminidase. In this article, Peter Colman, Graeme Laver and their colleagues discuss some speculative implications of the results currently

available. In the past, many methods have been used in attempts to define antigenic determinants (epitopes) on protein molecules1. These m~*,hods have included the use of protein fragments to adsorb antisera2, the production of anti-peptide antisera and their reactions with intact proteins3, proteolytic digestion of antigens in complexes with antibodies as a probe of protected peptide bonds within the epitope4, and the protection of amino acids in the epitope from specific chemical derivitization s. Other methods include the characterization of 'escape mutants' (variants which do not bind neutralizing monoclonal antibodies) and competitive binding studies of monoclonal antibodies for an antigen 6. Although data from some experiments have implicated particular amino acid residues as participants in particular epitopes, none has allowed a complete structural description of an

P.M. ColmanI, G.M. Airz, R.G J.N. Varghesd, A.T. Baked, M.R. P.A. TullochI and W.G. Laved bound to the tetramer. The two antibodies, NC41 and NC10, recognize overlapping epitopes on the neuraminidases. The structure of the NC41 Fab-N9 neuraminidase complex has been solved1° and shows an epitope composed of four surface loops of the neuraminidase (Fig.1). Sequence changes in these loops diminish or abolish antibody binding. The structure also reveals an unusual pairing pattern between the domains of the light and heavy chains in the variable module of the antibody (Fig.2). This implies either that NC41 Fab differs in its three-dimensional structure from other Fab fragments or, as we believe is more likely, that association of antibody with antigen can induce small changes in the quaternary structure of the Fab, through the sliding of domains at the VL--VHinterface. NC41 Fab inhibits neuraminidase activity by about 75% when a small substrate, neuraminyl-lactose (NAL) (molecular weight 500 Da) is used, while NC10 does not

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The first description of the structure of an epitope on a protein molecule was by Arnit et al. z who determined the three-dimensional structure of a complex between lysozyrne and the Fab fragment of a monoclonal antibody. In this complex, 16 amino acids on the lysozyme and 17 on the antibody form a tightly packed interface from which water molecules are excluded. The interaction has been described as conforming to a 'lock-andkey' picture of antibody-antigen interaction, in which, apart from some amino acid side chain movements, no structural changes occur in either the antibody or the antigen. This article describes another antigen-antibody complex in which conformational changes have occurred in the antigen and possibly also in the antibody. We have grown crystals of monoclonal antibody Fab fragments bound to influenza virus neuraminidase of the N9 subtype. Two such crystalline complexes, which diffract X-rays to beyond 3A resolution have been obtained 8. Neuraminidase is a tetramer with circular four-fold symmetry, and in each case four Fab fragments are 1CSIRO, Division of Protein Chemistry, 343 Royal Parade, Parkville, Victoria 3052, Australia; 2Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; 3St Jude Children's ResearchHospital, PO Box 318, Memphis, TN 38101, USA; 4Influenza Research Unit, JCSMR, GPO Box 334, CanberraACT 2601, Australia. ~) 1987, ElsewerPublicahons,Cambridge 0167 4919/87/$0200

Rg. 1. Schematicdiagramof chainfold in N2 and N9 neuraminidasevieweddown the 4-fold axis. (Thissymmetryaxisis bosom right.)Labelledresiduesand adjacentchainsegmentsare referredto in the text. N2 numberingis used. Thesidechainsof aminoacids368-370point towards the viewer,while that of Arg 371 (an activesite residue)pointsawayand into the catalyticsite locatedaboveand to the right of C,,371. Mutationsat positions367, 369, 370, 372, 400 and432 abolishthe bindingof NC41antibodyto neuraminidase,whereasmutations at 368 and 329 reducebinding.A mutationat residue220 (outsidethe NC47epitope)hasno effecton bindingof NC41to neuramlnidase.Solidchainsegmentsshowregionsin contactwith the NC41antibody,contactassignmentsi,'~the brokensolidsegmentsare tentative. 323

Immunology Today, vol. 8, No. 1 I, 1987

the enzyme (Fig.4), distorting the position of an active site arginine (residue 371) 11. This may explain the low neuraminidase activity of this complex, in which there is no steric hindrance to entry of small substrates into the active site pocket. Other explanations which cannot be ruled out include steric hindrance (by the Fab) of the lactose moiety of neuraminyl-lactose and long-range forces originating from the antibody which influence diffusion of the substrate into or out of the active site.

Internal images

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r ~ 2. (4) Schematicdrawing of Vt-Ve pairinq in two different hypothetical antJbod~ The VLand V. domains are represe:]tedas cylinders, and the three complementarity-de,~,rminingregions (CDRs)on each domain are shown. The two antibodies di~cr in the pairing of the variable domains, and as a consequencethe spatialarrangementof the six CDRsis different in the two structures. (B) C, drawing of VL and V, domains of two Fab structures. One lab, Mo°C603, of known structure24is compared with NC41 Fab in a complex with neuraminidase. The two V~ domainsshown here m purple have been superimposed, and the V, domains of free McPC603Fab and bound NC41 Fab are in green and blue respectively. The six CDRsare towards the viewer in brighter colours, and one selected C, from each is labelled. The difference in two VH domains as aligned here is about ~ of rotation. If the two Fabshad the same VL-VHpairing, then both would be supedmposable.Clearly,however, when the Vz domains are superimposed, the V.~domains are not. Thereforeslidit~g has occurred in NC41 Fab on complexformation or the VL-VHpairing in free NC41 Fabdiffe~ from that in McPC603.

324

(Fig. 3). Both antibodies completely inhibit activity for the large substrate, ~etuin (molecular weight 50 000 Da), presumably by blocking access of the substrate to the active site. The crystal structure of the NC41 complex shows that while the Fab does not appear to block access of neuraminyl-lactose to the active site, its binding has altered the conformation of one of the surface loops of

The existence of anti-idiotype antibodies which mimic the function, and presumably therefore also the structure, of the immunizing antigen is weil documented 12. These observations also imply that, in the process of generating a structure (a paratope) on the first antibody (Abl) complementary to the antigen, and a structure on the anti-idiotypic antibody (Ab2) resembling the original antigen, there is little or no distortion of either of the antibodies or of the antigen. A 'lock-and-key' or 'plastercast' picture of the interactions is consistent with the data. On the other hand, many anti-idiotypic antibodies do not resemble the immunizing antigen 13. Here it has been postulated that Ab2 is recognizing a part of the idiotope of Abl that is not used in binding the immunizing antigen. An equally valid description of these phenomena, requiring no such partitioning of the idiotope, is that Abl or the antigen are distorted by their encounter with each other. Abl then carries no faithful negative image of the antigen epitope. Similarly Ab2 may alter its conformation on binding to Abl. If any of these conformational changes occurred, Ab2 would not contain surface structures similar to the immunizing epitope. Thus no part of the idiotope needs to be outside the paratope in order to rationalize the absence of internal images in these experiments. The two different outcomes of these studies on antiidiotypic antibodies are consistent with situations in which significant conformational changes either do or do not accompany the association of antibody with antigen• Both situations are covered by the two immune complexes whose structures are available7.~°.

Escapemutants Some of the earliest experiments to select antigenic variants of influenza virus under antibody pressure were done by Archetti and Horsfall, who passaged the PR8 strain of type A influenza virus in the presence of heterologous polyclonal immune sera14. Gerhard and Webster produced the first escape mutants of influenza virus haemagglutinin using monoclonal antibodies is. Sequence characterization of these latter variants showed, in almost every case, single amino acid sequence changes in the mutant with respect to wild type. Since then, many other escape mutants of viruses have been selected under monoclonal antibody pressure and then characterized. These include the influenza virus neuraminidase antigens and, more recently, rhinovirus and poliovirus16-17. In these cases, where the crystal structures are known, amino acid sequence changes can be located on the known three-dimensional structures. A survey of some of these amino acid changes for the influenza antigens showed that there was no preference for size or character of the replacing or the replaced residue in the antigen, and indicated that even very

Immunology Today, voL 8, No. 11, 1987

subtle changes abolish the binding of an antibody for an antigen 1R. On the other hand, these escape mutants are selected only because antibody binding is abolished, and many cases of more subtle epitope changes which can be accommodated in an antibody-antigen complex go largely unrecorded. Support for this notion comes from the finding that variants selected with one monoclonal antibody bind other monoclonal antibodies with altered affinity. For example, the 32/3 anti-neuraminidase antibody selects a variant with an Asp residue at Asn 329. This mutant binds to another antibody, NC41, with reduced affinity compared with wild type. Indeed, crystals of this complex show that NC41 antibody undergoes no rearrangement on the neuraminidase epitope as a result of this single amino acid sequence changeR. The antibody still inhibits neuraminidase activity with neuraminyl-lactose, but to a lesser extent. The Gibbs free energy of binding of antibody to antigen is the sum of enthalpy and entropy contributions, including those for the desolvation of the antigen epitope and the antibody paratope. Although there may be a large surface area of interaction involving many amino acids (16 on the antigen and 17 on the antibody in the case of the lysozyme complex), the net free energy of the system is often very small, so that the binding constants may also be small. A similar situation is obtained in protein structures where the net free energy of the folded state is the sum of many stabilizing interactions which often barely counterbalance the many positive contributions to the free energy; as a result, single amino acid substitutions may suffice to destabilize the native structure. The loss of one strong hydrogen bond can account for a reduction in the binding constant of nearly three orders of magnitude, which for an antibody-antigen complex with a binding constant in the range 106-107 would effectively abolish the interaction.

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Fig.3.Neuraminidase(NA)inhibition curvesfor monoclonalantibodiesNCI Oand NC41 on neuraminidaseusing neuraminyl-lactose(molecularweight 500 Da) as substrate. Both. antibodies inhibited N9 neuraminidase activity almost totally (95% inhibition) when the large substrate fetuin (molecularweight 50 kDa) was used.

ble only to compare NC41 Fab in the complex with other published free Fab fragments. The VL--VH interface of NC41 Fab in the complex is different to variable modules of other antibodies, and to a first approximation the difference can be described as a sliding of the domains by 8-12 ° at their interface (Fig.2). This results in a displacement of the complementarity-determining regions on one chain by some 4A. If antigen induced a shift of this magnitude, the antibody might contact amino acids on the epitope different from those contacted by an antibody with unchanged structure. ImL:k:6:,~--

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Molecular mimicry

Examples of monocional antibodies crossreacting with antigens that are apparently unrelated to the immunizing antigen are now quite common +9. One explanation of these data is that the crossreacting epitopes are atomically identical. This seems unlikely. Two alternative mechanisms for molecular mimicry should be entertained. In the first, different parts of the paratope are involved in binding the two very different epitopes. The crystal structure of the NC41 Fab-N9 neuraminidase complex shows asymmetry in the binding of the epitope to the antibody. In particular, CDR1 on the light chain makes few, if ,my, contacts with the neuraminidase, while in the lysozyme-Fab complex, CDR2 on the light chain contributes only one amino acid to the interaction. A different set of interacting residues (i.e. a different paratope) may exist for a completely unrelated antigen. Even in such cases, however, a region of atomic identity would be expected for the two epitopes. A second mechanism takes into account the possibility that conformational changes in the antibody result from antigen-induced VL--VH domain sliding. We have proposed that the NC41 antibody undergoes a structural change on binding the N9 neuraminidase. The structure Df free NC41 Fab has not yet been determined (although crystallization trials are in progress). It is therefore possi-

Many different mechanisms for inhibition of enzyme activity by antibodies can be entertained. Early experiments indicated that direct combination o: the antibody with the catalytic site of the enzyme occurred rarely, if at allz°. Indeed, for influenza virus neuraminidase, such. . . . . .

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Fig.4. Stereo drawing of the loop around Arg 37! (an activesite residue, here labelled871) in neuraminidase,showing how the structure in the complex(yellow)hasshifted from that in free neuraminidase(blue)as a result of NC4~lab binding

Immunology Today, vol. 8, No. I 1, 1987

antibodies would protect against all strains of virus, because of the invariance of the neuraminidase catalytic site 21. The absence of antibodies which exclusively see the active site and none of the adjacent strain-variable structures, was e×plained for neuraminidase, in w?~ ch the size and shape of the catalytic cavity could not accommodate the size and shape of the paratope en an antibody 2~. These considerations suggest that an antibody response to the invariant catalytic site is extremely improbable 2~. An identical explanation for the fai'~e of antisera to picornaviruses to bind selectively to putative strain-independent structures has recently been presented under the name 'the canyon hypothesis '22. Steric inhibition by antibody bnund to an epitope ndar the active site of an enzyme has been invoked to explain inhibition towards large substrates, and deformation of the active site by an antibody bound nearby was suggested to inhibit activity on small substrates2°. In cases where conformationa~ changes, or flexibility, in active site structures is important for their function, antibody may also inhibit activity by immobilizing critical structural elements. In the NC41 Fab-N9 neuraminidase complex, an active site amino acid (Arg 371) ~ is slightly displaced from its location in the free neuraminidase structure by the binding of the antibody to adjacent amino acids which are also displaced from their position in the free enzyme structure (Fig.4). The deformation of this loop appears to be related to the multipoint attachment that the NC41 antibody makes to the neuraminidase (see Fig.l), and raises the question of whether conformational changes in the antigen can be caused by antibodies which bind to so-called continuous determinants only. Neutralization of virus infectivity

For many years it was believed that antibodies neutralized viruses by blocking the attachment of the virus particles to receptors on the host cell. However, in some systems it appears that fewer antibodies than are required to block all the receptor binding sites on the virus will neutralize infectivity. More subtle mechanisms are presumably at work in these cases, possibly involving the transmission of conformational changes from the epitope to distant sites23.

Coming soon in ImmunologyToday Among the articles to be featured in forthcoming issues of Immunology Today are: ImmuneprM~e. Wayne Streilein and Thomas Wegm,~nn propose that the immunologic privilege extended to antigens in the anterior chamber of the eye and the pregnant uterus is the result of an active suppression of an immune response. Intefferor~. Jeffrey Browning assessesthe results of clinical trials which suggest that gamma interferon has some efficacy in the treatment of rheumatoid arthritis, and attempts to relate these clinical studies to the known properties of this cytokine. HIV infeclJon. Mark Mlrsh and Angus Dalgleish discuss recent attempts to determine the mechanism of entry of the human immunodeficiency virus type 1 after it has bound its cell-surface receptor, the CD4 antigen. Autoantlbodies.Reinhard Kofler and colleagues review recent work on the genetic origin of self-reactive antibodies, a{ld evaluate the likelihood that abnormal genetic elements or processing mechanisms are responsiblefor the generation of such autoantibodies.

If neutralization, at least in some cases, requires antigen distortion, then in these cases anti-peptide antisera may be found wanting. Binding of antibodies to at least two linearly separated surface loops may be the rr,!~,imal r~.quirement for the establishment of conformational change in the antigen. Even ttlen, however, distortion may not occur, depending on the fit of the antibody to the antigen, and such an antibody may prevent the binding of another distorting (neutralizing?) antibody. Thus the anti-neuraminidase antibody NCl0 does t~c~t inhibit neuraminidase activity towards small substrates (Fig. 3), but can block the attachment of NC41 antibody and 'protect' the enzyme from inhibition by that antibody. NCl0 might therefore be called an 'obstructive antibody', and these should not be encouraged to develop during a response to vaccination. This work was supported by US Public Health Research Grants AI 08831, AI 20591 and AI 19084 from the National Institute of Allergy and Infectious Disease and was greatly facilitated by international telephone facilities provided by the Australian OverseasTelecommunication Commission.

References

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9 Webster, R.G., Air, G.M, Metzger, D.W. etal. (1987) J. Virol. 61, 2910-2916 10 Colman, P.M., Laver,W.G., Varghese,J.N. etal. (1987) Nature 326, 358-363 11 Lentz, M.R., Webster, R.G. and Air, G.M. (1987) Biochembtry 26, 5351-5358 12 Gaulton, G.N. and Greene, M.I. (1986)Annu. Rev. Imrnunol. 4, 253-280. 13 Nisonoff, A. and Lamoyi, E. (1981) Clin. Immunol. 21, 397-400 14 Archetti, L. and Horsfall, F.L.(1950)./. Exp. Med. 92, 441-442 15 Gerhard, W. and Webster, R.G. (1978)J. Exp. Med. 148, 383-392 16 Rossman,M.G., Arnold, E., Erickson, J.W. etal.(1985) Nature 317, 145-153 17 Minor, P.D., Ferguson, M., Evans, D.M.A., Almond, J. and Icenogle, J.P.(1986) J. Gen. Virol. 67, 1283-1291 18 Colman, P.M. and Ward. C.W. (1985) Curr. Top. Microbiol. Immunol. 114, 177-255 19 Srinivasappa,J., Saegusa,J., Prabhakar,B.S.etal. (! 986) J. Virol. 57, 397-401 20 Cinader, B. (1963)Ann. NY. Acad. Sci. 103, 493-1154 21 Colman, P.M., Varghese,J.N. and Laver, W.G. (1983)Nature 303, 41-44 22 Luo, M., Vriend, G., Kamer, G. etal.(1987) Science235, 182-191 23 Dimmock, N.J. (1984)J. Gen. Virol. 65, 1015-1022 24 Satow, Y., Cohen, G.H., Padlan, E.A. and Davies, D.R. (1986)J. Moleco Biol. 190, 593-604