Site-specific mutagenesis of a recombinant anti-single-stranded DNA Fab - Role of heavy chain complementarity-determining region 3 residues in antigen interaction

The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol, Chem, 271, 12241-12246), in the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg(98) and Asp(108)) and in the middle (Tyr(101)-Arg-Pro-Tyr-Tyr(105)) Of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT)(15) using two independent methods, All mutations in the middle of HCDR3 led to either abolished or diminished affinity, Tyr(101) likely participates in hydrogen bonding, while Tyr(104) and Tyr(105) may be involved in aromatic-aromatic interactions with the ligand. The residues Arg(102) and Pro(103) were not as critical as the tyrosines. It is speculated that HCDR3 interacts with the thymines, rather than the phosphates, of the ligand. A 3-fold increase in affinity was observed by mutation of Asp(108) to alanine. The highly conserved Arg(98) and Asp(108) do not appear to form a salt bridge.