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 ...

We have examined the low-resolution structure of a complete human IgGi using known domain coordinates from crystallographic investigations of immunoglobulin fragment structures. Our results indicate that the Fc ortion of this molecule has a structure similar to that of an isolated Fc fragment, with the carbohydrate moiety playing a central role as the principal contact between the CH2 domains. Carbohydrate also forms a large part of the interface between the Fc and Fab regions. The relative orientations of the variable and constant portions of the Fab regions are intermediate between those reported previously, emphasizing the flexibility of the switch region. These data do not support a two-state allosteric model such as has been proposed for antibody effector functions. Most of the information available at present on the threedimensional structure of immunoglobulins (Igs) has come from studies of isolated fragments (1-3). Progress in the determination of structure of intact Igs has been generally disappointing. The protein Dob, a human IgGl(K) cryoglobulin, was the first crystalline intact Ig to be analyzed by x-ray diffraction (4). The crystals are sensitive to x-irradiation and show evidence of disorder in the diffraction pattern, with few useful reflections beyond 6 A. An electron density map at this resolution showed the overall molecular boundary, on the basis of which a Tshaped model was proposed (5). This model was independently supported by an analysis of electron micrographs of sections of the Dob crystals (6, 7).The human myeloma protein Mcg [IgGl(X)] has also been crystallized, and the crystals diffract to a resolution of 3.5 A (8), but there has been no reported solution of its three-dimensional structure.A third human myeloma protein (IgGi), Kol, has been crystallized and shown to diffract to spacings of 3.5 A (9). An electron density map at 5-A resolution has been reported in which the two Fab arms are clearly visible with an angle of 1200 between them (10). Unfortunately, the Fc part of the molecule is not at all visible in this map, probably because of disorder in the crystal.Progress in the determination of structure of Ig fragments has been more rapid. the structures of two Fabs (11,12), an Fc (13,14), and a number of Bence-Jones proteins (15-17) have been reported. As a result, we now know separately the structures of all the component domains of an IgG molecule. When corresponding domains from different Igs have been compared, they have been found to be similar (refs. 10, 17, and 18; unpublished*). These observations, together with the results of amino acid sequence analyses, strongly suggest that, even in the variable parts of the molecule, the three-dimensional structures of the domains will be highly conserved.The quaternary relationships between domains are not, however, invariant. Substantial differences have been observed in the relative orientation of the variable and constant regions (elbow bend) of the Fabs (ref. 10; unpublished*). Furthermore, solution studies have demonst...

The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr. It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I. The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.