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 (Arg98 and Asp108) and in the middle (Tyr101-Arg-Pro-Tyr-Tyr105) 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. Tyr101 likely participates in hydrogen bonding, while Tyr104 and Tyr105 may be involved in aromatic-aromatic interactions with the ligand. The residues Arg102 and Pro103 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 Asp108 to alanine. The highly conserved Arg98 and Asp108 do not appear to form a salt bridge.
Komissarov
A A
AA
Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65212, USA.
Marchbank
M T
MT
Calcutt
M J
MJ
Quinn
T P
TP
Deutscher
S L
SL
D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex; D001666:Binding Sites, Antibody; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D000936:Antigen-Antibody Complex; D004277:DNA, Single-Stranded; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C027903:oligo (dT)