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 papillom avirus (HPV) E2 proteins is responsible for regulation of Vi rus transcript ion. The dimeric E2 DNA-binding domain of HPV-16 (E2c) dissociates into a p artially folded state under high hydrostatic pressure. We show here that pr essure-induced monomers of E2c are highly structured, as evidenced by NMR h ydrogen-deuterium exchange measurements. On binding to both specific and no nspecific DNA. E2c becomes stable against pressure. Competitive binding stu dies using fluorescence polarization of fluorescein-labeled DNA demonstrate the reversibility of the specific binding. To assess the thermodynamic par ameters 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 denat uration fell linearly with increase in pressure for both protein-DNA comple xes, and the measured Volume change was similar to that obtained for E2c al one. The data show that the free energy of dissociation increases when E2c binds to a nonspecific: DNA sequence but increases even more when the prote in binds to the specific DNA sequence. Thus, specific complexes are tighter but do not entail variation in the volume change. The thermodynamic data i ndicate that DNA-bound E2c dissociates into monomers bound to DNA. The exis tence 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.