High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners
High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners
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DOI：
10.1021/bi025579n
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发表时间：
2002-08-20
期刊：
影响因子：
2.9
通讯作者：
de Prat-Gay, G
中科院分区：
文献类型：
--
作者：
Alonso, LG;García-Alai, MM;de Prat-Gay, G
已知高危乳头瘤病毒主要通过E7（其两种癌蛋白之一）发挥其转化活性。尽管其相关性，但尚未获得可解释E7的表观广泛结合特异性的结构信息。从HPV-16纯化到接近同质的重组E7在凝胶过滤色谱中显示出两种物质，其中一种对应于分子量为22 kDa的二聚体，通过多角度光散射测定。E7二聚体被分离用于表征，并且显示当从pH 7.0改变至5.0时经历实质性的构象转变，螺旋结构增加并且疏水性表面的溶剂可及性增加。即使在SDS的存在下，该蛋白质是耐热变性，我们表明，在单体中的持久残留结构是负责其报告的异常电泳行为。二聚体还显示出非小叶的流体动力学体积的基础上凝胶过滤实验，并变得更加球形的存在下，0.3 M的盐酸胍，与疏水表面变得可访问的溶剂，如所示的大幅增加ANS结合。在低蛋白浓度下，观察到球状E7二聚体的解离，在结构化和扩展单体的合作展开之前。虽然E7具有类似于天然未折叠多肽的特性，但其远紫外圆二色谱、协同展开和ANS结合位点的暴露支持折叠和延伸，而不是无序和波动的构象。在生理范围内和温和变性剂浓度下，当pH值降低时，溶剂对疏水表面的可及性大幅增加，这表明构象特性可能已经演变为使蛋白质-蛋白质识别大量报告的细胞结合伴侣。
High-risk papillomaviruses are known to exert their transforming activity mainly through E7, one of their two oncoproteins. Despite its relevance, no structural information has been obtained that could explain the apparent broad binding specificity of E7. Recombinant E7 from HPV-16 purified to near homogeneity showed two species in gel filtration chromatography, one of these corresponding to a dimer with a molecular weight of 22 kDa, determined by multiangle light scattering. The E7 dimer was isolated for characterization and was shown to undergo a substantial conformational transition when changing from pH 7.0 to 5.0, with an increase in helical structure and increased solvent accessibility to hydrophobic surfaces. The protein was resistant to thermal denaturation even in the presence of SDS, and we show that persistent residual structure in the monomer is responsible for its reported anomalous electrophoretic behavior. The dimer also displays a nonglobular hydrodynamic volume based on gel filtration experiments and becomes more globular in the presence of 0.3 M guanidinium chloride, with hydrophobic surfaces becoming accessible to the solvent, as indicated by the large increase in ANS binding. At low protein concentration, dissociation of the globular E7 dimer was observed, preceding the cooperative unfolding of the structured and extended monomer. Although E7 bears properties that resemble natively unfolded polypeptides, its far-UV circular dichroism spectrum, cooperative unfolding, and exposure of ANS binding sites support a folded and extended, as opposed to disordered and fluctuating, conformation. The large increase in solvent accessibility to hydrophobic surfaces upon small pH decrease within physiological range and in mild denaturant concentrations suggests conformational properties that could have evolved to enable protein-protein recognition of the large number of cellular binding partners reported.