title: Mapping and modification of an antibody hapten binding site: a site-directed mutagenesis study of McPC603.
abstract: The quantitative contributions of various amino acid residues to hapten binding in the Fv fragment of the antibody McPC603 were investigated by site-directed mutagenesis. The three-dimensional structure of the Fab' fragment of McPC603 is known to atomic resolution. The haptens phosphocholine, choline sulfate, 3-(trimethylammonium)propane-1-sulfonate, 4-(trimethylammonium)butyric acid, and 4-(trimethyl-ammonium)butyric acid methyl ester were tested for binding. It was found that the phosphate group but not the sulfate and sulfonate groups, interacts with the hydroxyl group of Tyr33(h). The required positive charge for the binding of the phosphate must be contributed by Arg52(h); a lysine at this position or an additional positive charge at position 33(h) abolishes the binding to a phosphocholine affinity column. The interaction between Tyr100(l) and Glu35(h) was found to be essential and could not be functionally replaced by any other pair of residues tested. Binding of the quaternary ammonium ion needs a negative charge; it can reside in either Asp97(l) or Asp101(h), but both together prevent binding to the affinity column. These data may serve as the basis for the development of quantitative treatments of antigen-antibody interactions.
authors.affiliation: Genzentrum der Universität München, Max-Planck-Institut für Biochemie, Martinsried, West Germany.
authors.forename: R
authors.initials: R
authors.lastname: Glockshuber
authors.forename: J
authors.initials: J
authors.lastname: Stadlmüller
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000596:Amino Acids / Q000235:genetics; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D006241:Haptens / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000276:immunology*; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010767:Phosphorylcholine / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D000906:Antibodies; D000941:Antigens; D006241:Haptens; D007128:Immunoglobulin Fragments; D010767:Phosphorylcholine; D008239:Lysine