Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.

On the basis of comparative studies of known antibody structures and sequences it has been argued that there is a small repertoire of main-chain conformations for at least five of the six hypervariable regions of antibodies, and that the particular conformation adopted is determined by a few key conserved residues. These hypotheses are now supported by reasonably successful predictions of the structures of most hypervariable regions of various antibodies, as revealed by comparison with their subsequently determined structures.

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Structure of an antibody-antigen complex: Crystal structure of the HyHEL-10 Fab-lysozyme complex (x-ray ABSTRACTThe crystal structure of the complex of the anti-lysozyme HyHEL-10 Fab and hen egg white lysozyme has been determined to a nominal resolution of3.0 A. The antigenic determinant (epitope) on the lysozyme is discontinuous, consisting of residues from four different regions of the linear sequence. It consists of the exposed residues of an a-helix together with surrounding amino acids. The epitope crosses the active-site cleft and includes a tryptophan located within this cleft. The combining site of the antibody is mostly flat with a protuberance made up of two tyrosines that penetrate the cleft.All six complementarity-determining regions of the Fab con-

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementaritydetermining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. Until recently knowledge of the structural aspects of antibody-antigen interactions has been based on the x-ray analysis of four Fab structures and on some complexes with hapten (1-5). Haptens were observed to bind in grooves or pockets in the combining sites of the New and McPC603 Fabs, and these occupied a small fraction of the total available area of these sites. When haptens bind to these Fabs, no large conformational change occurs. However, one cannot rule out the possibility that the behavior of antibodies would be different when they are bound to larger antigens, such as proteins. For example, the interaction with a much greater fraction of the combining site might in itself be sufficient to induce conformational changes in the antibody. Also, the interacting surfaces might not possess the grooves and pockets observed for haptens, but might resemble more closely the kind of surface observed in other protein-protein interfaces, where exclusion ofbound water is believed to play a key role. For this reason we undertook several years ago to investigate the crystal structures of complexes of the Fabs of several monoclonal antibodies to hen egg white lysozyme complexed with the lysozyme (6). In this paper we report the analysis of two different crystal forms of one of these complexes.The site on the lysozyme to which the antibody binds has been the subject of an extensive serological analysis (7) through a study of cross-reactivity with different avian lysozymes. The results of that analysis are in striking agreement with the crystal structure observations and will be discussed.During the course of this analysis two reports of related x-ray studies of Fab-antigen complexes have appeared (8, 9), one being a description of another lysozyme-antilysozyme complex, although to a different epitope of the lysozyme, and the other describing a complex with the neuraminidase of influenza virus. The observations and conclusions from these two investigations differ in important ways from one another, and we describe below how our results can be related to them. MATERIALS AND METHODSMonoclonal antibody (mAb) HyHEL-5 and the Fab-lysozyme complex were prepared as described previously (6, 7). Crystals were grown by vapor diffusion against 20% (wt/vol) polyethylene glycol 3400 (Aldrich) in 0.1 M imidazole hydrochloride, pH 7.0, 10 mM spermine with ...

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