Structural Dynamics Of The E6Ap/Ube3A-E6-P53 Enzyme-Substrate Complex

Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. We therefore developed and applied different approaches in structural mass spectrometry to show that binding of E6 induces conformational rearrangements in E6AP, which result in the positioning of E6 and p53 in the immediate vicinity of the catalytic centre of E6AP. Our data provides structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex and reveals how E6 can both stimulate the ubiquitin ligase activity of E6AP and facilitate the transfer of ubiquitin from E6AP onto p53.泛素连接酶E6AP（ubiquitin ligase E6AP）的失调与宫颈癌等人类疾病的发生存在因果关联。当其与人乳头瘤病毒（human papillomaviruses）的E6癌蛋白结合形成复合物后，E6AP会靶向肿瘤抑制蛋白p53（tumor suppressor p53）并使其降解，从而推动肿瘤发生进程。此外，E6可通过尚未阐明的分子机制强效激活E6AP。然而，目前仍缺乏能够阐明E6AP-E6-p53酶-底物复合物组装路径，以及E6激活E6AP具体机制的结构学数据。为此，我们开发并应用了多种结构质谱法（structural mass spectrometry）技术，证实E6的结合会诱导E6AP发生构象重排，最终使E6与p53精准定位于E6AP催化中心（catalytic centre）的紧邻区域。我们的研究为全长E6AP-E6-p53酶-底物复合物的动态特性提供了结构与功能层面的深入认知，并揭示了E6可同时增强E6AP的泛素连接酶活性（ubiquitin ligase activity），以及促进泛素（ubiquitin）从E6AP向p53转移的分子机制。