Abstract
Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.