INVESTIGADORES
FERREIRO Diego Ulises
artículos
Título:
Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.
Autor/es:
CERUTTI ML, FERREIRO DU, SANGUINETI S, GOLDBAUM FA, DE PRAT-GAY G
Revista:
BIOCHEMISTRY
Referencias:
Año: 2006 vol. 45 p. 15520 - 15528
ISSN:
0006-2960
Resumen:
We have obtained a monoclonal antibody (ED15)
against the C-terminal DNA-binding domain of the high-risk human
papillomavirus strain-16 E2 protein that strongly interferes with its
DNA-binding activity. We here characterize the recognition mechanism of
this antibody and find that the ED15-E2 interaction has a strong
electrostatic component, which correlates with the high proportion of
acidic residues found in the antibody combining site. Further circular
dichroism experiments in the presence of phosphate show that, in
addition to electrostatic screening of key potential interactions,
ionic strength affects the conformation of the epitope. In addition,
the interaction is strongly modulated by pH, which correlates with 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. Site
directed mutagenesis indicates that the ED15 epitope involves at least
part of the DNA-binding helix of E2, explaining the mAb inhibitory
activity. At physiological salt concentrations, the equilibrium
dissociation constant of the E2-ED15 interaction is 10(-7) M and the
association rate is 10(4) M-1 s-1, at least 1 order of magnitude slower
than those generally reported in the most extensively described
"nonflexible" antibody-protein interactions, indicating the presence of
a slow conformational rearrangement on the antigen as the rate-limiting
step. The crucial role of antigen flexibility in antibody-protein
recognition is discussed.