Somatic mutations in immunoglobulin V gene determine the structure and function of the protein − an evidence from homology modeling

The antigen binding site of IgG immunoglobulins consists of two separate peptide domains,VH and VL. This part of combined domains contains three loops in each and it provides unique structure to distinguish antigens. The unique sequences encode the antigen binding site are determined by recombination of V region genes in both VH and VL during the differentiation of B−lymphocyte and successive somatic mutations. To investigate the correlation between the site of somatic hypermutations in immunoglobulin V region genes and the affinity maturation of antibodies, single clone derived anti−double stranded DNA antibody−producing hybridomas showing different DNA−binding activities were selected from an autoimmune disease−prone, (NZB x NZW)F1 female mouse. DNA sequence analyses showed that mutational points in V region genes were distributed in both complementality determining regions (CDRs) and framework regions and that there were no common mutational positions that account for the affinity maturation of the clones. The number of cationic amino acid residues did not simply explain the difference in the affinity among these clones too. Studies using homology modeling on a graphic computer showed that changes in the tertiary structure of Fv were minimal between the low and high affinity clones. The molecular dynamics simulation of the antigen−binding sites, however, revealed a significant increase in the flexibility of antigen−binding loops in high affinity clones. There was a case in that a single mutation in CDR1 of light chain from aspartic acid to glycine markedly increased the vibration of heavy chain CDR3, in association with the increase in DNA−binding activity. The interaction between VH CDR3 and VL CDR1 was only able to be found on the three dimensional graphics by estimating the distance between two separate peptide chains over the period of molcular dynamics simulation. Together with findings by others as well as our own, it is likely that the affinity maturation of anti−DNA antibodies is mediated by two molecular mechanisms caused by somatic hypermutations in V region genes, one by amino acid substitutions to electrophilic residues along the surface of antigen−binding CDR loops particularly of heavy chain CDR3, and one by their increased flexibility particularly between light chain CDR1 and heavy chain CDR3. This explanation is consistent with the induced−fit hypothesis of antigen−antibody interaction. Thus a honology modeling is useful tool for the analysis of the effect of mutaitions in sequences to the function of large bio−molecules.