Disulphide dimerised peptide creates a crystal contact in an anti-peptide antibody.
Although many Fab derived from human and murine antibodies have been crystallized, several such fragments, including anti-peptide Fab 4 x 11, remain problematic for crystallisation. Diffracting crystals have been obtained for this Fab only in complex with a cysteine containing peptide CGGIRGERA. The IRGERA portion contained in the C-terminus of histone H3 is recognised by the monoclonal antibody and the CGG portion is an essential spacer. Crystallisation experiments and seeding studies show that disulphide bond formation is essential for obtaining crystals. Data has been collected to 2.7 A resolution, and the structure solved by molecular replacement. The asymmetric unit contains two antibodies whose binding sites are face-to-face in an non-crystallographic approximate two-fold rotational axis. We find two Fab in the asymmetric unit with the two antigen combining sites facing each other. This result was unexpected since it has been common practice to avoid the cysteine-containing peptides because of heterogeneity, since the peptide solution is likely to contain both dimeric and monomeric peptides. This result suggests that the cysteine containing peptides used in immunisation for coupling them to a carrier protein could also be used to screen for crystals. Structural data obtained for the same peptides as those used in the immunisation is valuable to evaluate to what extent the linker to the carrier protein may have contributed to the shaping of the antibody binding site. The introduction of exposed cysteines on the surface of proteins by site directed mutagenesis could help resolve difficult crystallisation cases. Should these not form a disulphide bridge in the crystal but nonetheless crystallize, the free cysteine could be used to make a heavy atom derivative for isomorphous replacement.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D009842:Oligopeptides; D003545:Cysteine