title: Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis.
abstract: A B72.3 Fab/sTn(2) complex was modeled from the known structure of B72.3 Fab and the dimeric Tn-serine cluster (sTn(2)). In the complex model, the side chains of 15 heavy- and light-chain complementarity-determining region (CDR) residues and the main chains of two light-chain CDR residues contact the sTn(2) epitope. Among 15 CDR residues which contact sTn(2) in the model, two heavy-chain residues (Ser95 and Tyr97) and light-chain CDR residue (Tyr96) have been confirmed in a previous study. To test the accuracy of the computational model, further site-directed mutagenesis was performed by alanine scanning on the remaining 12 residues that are predicted in the model to have side-chain interactions with sTn(2). Of these 12 mutants, eight that are all from the heavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala, Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn(2) affinities, and four consisting of three light-chain mutations (Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation (His35Ala) retained wild-type sTn(2) affinity. On the whole, this evidence suggests that the complex model, although not perfect, is correct in many of its features. In a more general vein, these results lend credibility to the computational modeling approach for the study of the molecular basis of antigen-antibody complexes.
authors.affiliation: Departments of Microbiology, Oncology. Pathology, Saskatoon Cancer Center, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0W0, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Xiang
authors.forename: M
authors.initials: M
authors.lastname: Srivamadan
authors.forename: R
authors.initials: R
authors.lastname: Rajala
authors.forename: Z
authors.initials: Z
authors.lastname: Jia
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; C091202:B72.3 antibody