Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.
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.
Cerutti
María Laura
ML
Fundación Instituto Leloir, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
Ferreiro
Diego U
DU
Sanguineti
Santiago
S
Goldbaum
Fernando A
FA
de Prat-Gay
Gonzalo
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D001666:Binding Sites, Antibody; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D052162:Human papillomavirus 16 / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D009856:Oncogene Proteins, Viral; D004247:DNA