DNA tightens the dimeric DNA-binding domain of human papillomavirus E2 protein without changes in volume.
The recognition of palindromic specific DNA sequences by the human papillomavirus (HPV) E2 proteins is responsible for regulation of virus transcription. The dimeric E2 DNA-binding domain of HPV-16 (E2c) dissociates into a partially folded state under high hydrostatic pressure. We show here that pressure-induced monomers of E2c are highly structured, as evidenced by NMR hydrogen-deuterium exchange measurements. On binding to both specific and nonspecific DNA, E2c becomes stable against pressure. Competitive binding studies using fluorescence polarization of fluorescein-labeled DNA demonstrate the reversibility of the specific binding. To assess the thermodynamic parameters for the linkage between protein dissociation and DNA binding, urea denaturation curves were obtained at different pressures in the presence of specific and nonspecific DNA sequences. The change in free energy on denaturation fell linearly with increase in pressure for both protein-DNA complexes, and the measured volume change was similar to that obtained for E2c alone. The data show that the free energy of dissociation increases when E2c binds to a nonspecific DNA sequence but increases even more when the protein binds to the specific DNA sequence. Thus, specific complexes are tighter but do not entail variation in the volume change. The thermodynamic data indicate that DNA-bound E2c dissociates into monomers bound to DNA. The existence of monomeric units of E2c bound to DNA may have implications for the formation of DNA loops, as an additional target for viral and host factors binding to the loosely associated dimer of the N-terminal module of the E2 protein.
Lima
L M
LM
Programa de Biologia Estrutural, Departamento de Bioquimica Médica-Instituto de Ciências Biomédicas, Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Rio de Janeiro, Brazil.
Foguel
D
D
Silva
J L
JL
D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D019281:Dimerization; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea
D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D014508:Urea; D004247:DNA