Structural basis for specific DNA recognition by an antibody.

SANGUINETI, S., CENTENO CROWLEY, J.M., LODEIRO MERLO, M.F., CERUTTI, M.L., GOLDBAUM, F., STANFIELD, R. AND PRAT GAY, G. DE

Pinamar, Argentina. Diciembre 3-6, 2005

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2005

SAIB

STRUCTURAL BASIS FOR SPECIFIC DNA RECOGNITION BY AN ANTIBODY Santiago Sanguineti1, Juan M. Centeno Crowley1, María F. Lodeiro Merlo1, María L. Cerutti1, Fernando A. Goldbaum1, Robyn Stanfield2 & Gonzalo de Prat Gay1 1 Fundación Instituto Leloir and CONICET, 2 The Scripps Research Institute. e-mail: ssanguineti@leloir.org.ar We have obtained high affinity monoclonal antibodies against the 18mer duplex DNA site of the human papillomavirus E2 protein, and characterized their unusual thermodynamic properties. We found that the antibody ED10 can recognize one of the strands of the duplex DNA with subnanomolar affinity, but not the other. Shortening the oligonucleotide down to 4 bases while leaving the 5´ intact, display a binding affinity as high as the 18mer single stranded DNA. We crystallized the free Fab and bound to a 6mer single stranded oligonucleotide, which diffracted to 2.7 and 1.9 Å, respectively. Only the first two bases from the 5´ end (dTdC) display electronic densities at the antibody binding site, the rest are flexible. We observe at least 4 hydrogen bonds and the bases make substantial hydrophobic interactions with aromatic residues. There is a substantial conformational change to allow DNA binding. The antibody can thus recognize two bases at the 5´end, even in the 18mer duplex, but recognition of duplexes without dTdC at the 5´ end strongly suggest two recognition modes, a preferential high affinity single-stranded mode we show here and a double stranded mode for which we have no structural evidence. The flexibility of the antibody or the existence of conformers in solution can explain this mode. Section: Enzymology and Structural Biology