Abstact

The phenomenon in which the antibody affinity for T cell-dependent antigens increases through multiple rounds of somatic hypermutation (SHM) is referred to as affinity maturation. The elucidation of the structural and physical properties of antibodies obtained at various stages of the affinity maturation process can help us understand the molecular recognition mechanism of proteins in general. For this purpose, we used anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) single-chain (scFv) antibodies, prepared from the parent antibodies F8, B2, C6, and E11, and analyzed the crystal structures either in the absence or presence of NP or (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP). Comparison of the structures revealed that the antibodies shared a common antigen recognition architecture consisting of residues with basic side chains, Arg50(H) and Lys58(H)/Arg58(H), in addition to those at the junctional positions of gene segments, Trp96(L) and Tyr95(H) or His100B(H). These residues are responsible for the recognition of antigenic determinants, nitro-, hydroxyl- and phenylacetyl-groups, through hydrogen bond formation. Second, the Trp33(H) to Leu33(H) mutation seemed to strengthen the interaction because the antigen was closer to the combining site. Finally, analysis of NP and NNP complexes showed little difference in the antigen recognition modes and in the overall structures of the complementarity-determining regions between C6 and E11 scFvs. It was suggested that the replacement of residues by SHM provided a unique binding site for each antibody by fine tuning the microenvironment without disturbing specificity.