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.

人类乳头瘤病毒（human papillomavirus, HPV）E2蛋白对回文特异性DNA序列的识别，负责调控病毒的转录过程。HPV16型的二聚体E2 DNA结合结构域（E2c）在高静水压作用下，会解离为部分折叠的中间状态。本研究通过核磁共振（nuclear magnetic resonance, NMR）氢氘交换（hydrogen–deuterium exchange）实验证实，高压诱导产生的E2c单体仍保持高度结构化。当与特异性或非特异性DNA结合后，E2c即可获得抗高压稳定性。采用荧光素标记DNA的荧光偏振（fluorescence polarization）竞争结合实验，证明了该特异性结合具有可逆性。为探究蛋白质解离与DNA结合之间的偶联热力学参数（thermodynamic parameters），研究人员在不同压力条件下，分别在特异性与非特异性DNA序列存在的环境中获取了尿素变性（urea denaturation）曲线。结果显示，两类蛋白质-DNA复合物的变性自由能（free energy）变化均随压力升高呈线性下降，且测得的体积变化（volume change）与单独存在的E2c相近。实验数据表明，当E2c与非特异性DNA序列结合时，其解离自由能会升高；而当与特异性DNA序列结合时，解离自由能的升高幅度更为显著。由此可见，特异性复合物的结合更为紧密，但并未带来体积变化的差异。热力学数据显示，与DNA结合的E2c会解离为结合于DNA的单体。结合于DNA的E2c单体的存在，可能对DNA环的形成具有重要意义——这为结合于E2蛋白N端结构域松散二聚体的病毒与宿主因子提供了额外的结合靶点。