Analysis of antibodies of known structure suggests a lack of correspondence between the residues in contact with the antigen and those modified by somatic hypermutation

Forty unique murine antibody–antigen complexes determined at 2.5 Å or less resolution are analyzed to determine whether the residues in direct contact with the antigen are modified by somatic hypermutation. This was done by taking advantage of the recent characterization of the pool of Vκ germline genes of the mouse. The average number of residues in contact with the antigen in the VL gene, which contains the CDRL‐1, CDRL‐2, and all but one residue of CDRL‐3, was six. The average number of somatic mutations was similar (around five). However, as many as 53% of the antibodies did not show somatic replacements of residues in contact with the antigen. Another 28% had only one. Overall, the frequency of antibodies with increasing number of somatic replacements in residues in contact with the antigen decreased exponentially. A possible explanation of this finding is that mutations in the contacting residues have an adverse effect on the antigen–antibody interaction. This implies that most of the observed mutations are those remaining after negative (purifying) selection. Therefore, efficient strategies of site‐directed mutagenesis to improve the affinity of antibodies should be focused on residues other than those directly interacting with the antigen. Proteins 2001;45:199–206. © 2001 Wiley‐Liss, Inc.