title: DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer.
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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Torizawa
authors.forename: N
authors.initials: N
authors.lastname: Yamamoto
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Nobuoka
authors.forename: Y
authors.initials: Y
authors.lastname: Komatsu
authors.forename: H
authors.initials: H
authors.lastname: Morioka
authors.forename: O
authors.initials: O
authors.lastname: Nikaido
authors.forename: E
authors.initials: E
authors.lastname: Ohtsuka
authors.forename: K
authors.initials: K
authors.lastname: Kato
authors.forename: I
authors.initials: I
authors.lastname: Shimada
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D010759:Phosphorus Isotopes; D011485:Protein Binding; D011740:Pyrimidine Dimers / Q000276:immunology*; D013113:Spin Labels
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010759:Phosphorus Isotopes; D011740:Pyrimidine Dimers; D013113:Spin Labels; D004247:DNA