Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2

M. L. CERUTTI, D. U. FERREIRO, S. SANGUINETI, F. A. GOLDBAUM* AND GONZALO DE PRAT-GAY.

BIOCHEMISTRY

Año: 2006 vol. 45 p. 15520 - 15528

0006-2960

We have obtained a monoclonal antibody (ED15) against the C-terminal DNA-binding domainof the high-risk human papillomavirus strain-16 E2 protein that strongly interferes with its DNA-bindingactivity. We here characterize the recognition mechanism of this antibody and find that the ED15-E2interaction has a strong electrostatic component, which correlates with the high proportion of acidic residuesfound in the antibody combining site. Further circular dichroism experiments in the presence of phosphateshow that, in addition to electrostatic screening of key potential interactions, ionic strength affects theconformation of the epitope. In addition, the interaction is strongly modulated by pH, which correlateswith the local flexibility of the epitope rather than the presence of pH sensitive residues at the interface.Noticeably, this finding is well correlated with the strong entropic component of the interaction. Sitedirected mutagenesis indicates that the ED15 epitope involves at least part of the DNA-binding helix ofE2, explaining the mAb inhibitory activity. At physiological salt concentrations, the equilibrium dissociationconstant of the E2-ED15 interaction is 10-7 M and the association rate is 104 M-1 s-1, at least 1 orderof magnitude slower than those generally reported in the most extensively described “nonflexible”antibody-protein interactions, indicating the presence of a slow conformational rearrangement on theantigen as the rate-limiting step. The crucial role of antigen flexibility in antibody-protein recognition isdiscussed.