Ecole Supérieure de Biotechnologie de Strasbourg (IREBS, FRE 3211), Boulevard Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France.
J Mol Biol. 2010 Feb 12;396(1):90-104. doi: 10.1016/j.jmb.2009.11.022. Epub 2009 Nov 13.
Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.
乳头瘤病毒 E6 癌蛋白结合并经常引发许多对控制细胞增殖和/或细胞死亡至关重要的细胞蛋白降解。E6 蛋白的结构研究长期以来一直受到难以获得高浓度重组 E6 样品的阻碍。在这里，我们表明来自八种人乳头瘤病毒株和一种牛乳头瘤病毒株的重组 E6 蛋白作为寡聚体和多聚体存在。这些物质使用多种生化和生物物理技术进行了表征，包括分析凝胶过滤、活性测定、表面等离子体共振、电子显微镜和傅里叶变换红外光谱。通过与麦芽糖结合蛋白融合，E6 寡聚体的特征得以简化，这减缓了高级多聚体的形成。每种寡聚形式的比例取决于所考虑的病毒株而异。寡聚体似乎由折叠单元组成，在高危黏膜人乳头瘤病毒 E6 的情况下，这些单元保留与泛素连接酶 E6 相关蛋白的结合能力，并具有降解促凋亡蛋白 p53 的能力。除了小尺寸的寡聚体外，E6 蛋白还自发组装成大型组织多聚体结构，这一过程伴随着β-折叠二级结构含量的显著增加。最后，使用带有不同标签的 E6 进行的共定位实验进一步证明了真核细胞中 E6 自身缔合的发生。这些数据的综合表明，自缔合是 E6 蛋白的一种普遍特性，既发生在体外也发生在体内，因此可能具有功能相关性。
