Three-dimensional structure of an antibody-antigen complex.

Abstract

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 ...

The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment

“…While loop 3 was identified by the escape mutations, no such mutations have been found on loops 1 or 2, which participate in the remainder of the footprint on symmetry-related VP2 subunits. This is consistent with the study of lysozyme-Fab complexes, where mutations have identified only a select subset of interface residues [18,19]. It is also consistent with studies of Reynolds et al [21] who showed that restrictions of the viability of polioviruses containing mutations within an antigenic site limit the range of escape mutations which can be observed by normal selection procedures.…”

“…This compares well with the lysozyme surface area that is buried when complexed with HyHEL-5 [18,19]. In general, Fab-protein contacts cover surface areas of between 690Å 2 and 916Å 2 using a 1.7Å probe [20].…”

Calculation of the free energy of association for protein complexes

“…Interestingly, using volumes to obtain ASP values (cal/A3) produces the best correlation, suggesting that cavity formation is an important parameter in calculating the free The experimental association energies were obtained from the literature. a, Vincent and Lazdunski (1972); b, Vincent et al (1974); c, Chen and Bode (1983); d, Hass and Ryan (1980); e, Bunting and Myers (1975); f, Read et al (1983); g, Ascenzi et al (1988); h, Empie and Laskowski (1982); i, Bode (1979) and Bolognes et al (1982); j , Pekar and Frank (1972); k , Akasaka et al (1982); I, Sheriff et al (1987); m, Ross et al (1977). Chothia and Janin (1975) first suggested that the free energy of association was related to the amount of surface area that was buried in the interface.…”

Conformation of the hypervariable region L3 without the key proline residue

“…1B). The amide group of the adjacent framework residue Gln 90, the only L3 key residue present in CRIS-1, forms four hydrogen bonds: three with main-chain atoms from residues 92,93, and 94, and one with Oy from side chain of Thr 97 (Suh et al, 1986), and HyHel-5 (Sheriff et al, 1987), which represent, respectively, canonical forms 1, 2, and 3 for this hypervariable region. As a reference, the side chain of residue 90 is also shown in some detail.…”