Protein Engineering, Vol. 13, No. 5, 339-344,
May 2000
© 2000 Oxford University Press
Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis
1 Departments of Microbiology, Oncology 3 Pathology, Saskatoon Cancer Center, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0W0 4 Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada
A B72.3 Fab/sTn2 complex was modeled from the known structure of B72.3 Fab and the dimeric Tn-serine cluster (sTn2). 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 sTn2 epitope. Among 15 CDR residues which contact sTn2 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 sTn2. Of these 12 mutants, eight that are all from the heavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala, Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn2 affinities, and four consisting of three light-chain mutations (Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation (His35Ala) retained wild-type sTn2 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.