Antigen mobility in the combining site of an anti-peptide antibody.

Abstract

The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 5744), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.One of the most intriguing aspects of antibody recognition is the ability of some antibodies raised against peptide fragments of proteins to crossreact with intact native proteins (1-3). This phenomenon not only has considerable significance from the point of view of protein structure and folding but also has generated much interest in the possibility ofusing small peptides in a pharmacological role, particularly as synthetic vaccines (4,5). Little is known, however, about the underlying principles that determine whether a particular peptide is able to act as a good mimic of a protein epitope, primarily-because of the shortage of structural information pertaining to antibodies complexed with their peptide antigens. To our knowledge there has, for example, been only one report ofthe crystallographic structure of an anti-peptide antibody-peptide complex (6). In contrast a number of structures of anti-protein antibodies complexed with their protein antigens have been determined (7-11). As part of a long-term study in our laboratories of the structural origins of antibody recognition and protein-peptide crossreactivity, a panel of antibodies (Gloopl-GloopS) specific for the loop determinant of hen egg lysozyme was raised against a peptide immunogen that incorporated residues 57-84 of the protein sequence (12)-known as the "loop" peptide. This sequence corresponds to a large surface loop in the native protein structure and contains a disulfide bridge between residues 64 and 80. The NMR studies have focused on one of the antibodies, Gloopl. The antibody binds two forms of the loop peptide, where the disulfide is either reduced or oxidized, with equal affinity (Kd 10-8 M). The antibody is also strongly crossreactive with the native protein; it binds to hen lysozyme itself with only a 10-fold drop in affinity (12, 13). Crystallographic studies have yielded a structure for the Gloopl Fab (R.E.G. a...

Mapping the Epitope of an Inhibitory Monoclonal Antibody to the C-terminal DNA-binding Domain of HIV-1 Integrase

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An NMR Study of the Interaction of 15N‐Labelled Bradykinin with an Antibody Mimic of the Bradykinin B2 Receptor

“…In favourable cases this approach can be extended to difference spectroscopy to yield transferred NOE information on the structure of the binding site (Scherf et al, 1992). In cases where the off rate is slow compared to the NMR chemical shift time scale, dynamic filtering has been used to characterize the binding of different parts of the peptide by detecting flexible residues in doublequantum-filtered COSY spectroscopy (Cheetham et al, 1991 ;Zvi et al, 199.5). When the slow exchange limit is encountered, obtaining conformational information on the residues of the peptide directly involved in binding interactions requires the use of isotope edited and filtered NMR techniques.…”

Identification of B-cell Epitopes on the S4 Subunit of Pertussis Toxin

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