利用冷冻电子显微学研究Vps4寡聚体及其与底物形成的复合体的结构和分子机理

ESCRT-III复合物和I型AAA+ ATPase酶Vps4介导膜远离细胞质方向的形变，参与细胞内多种重要的生命活动，如多囊体的形成、病毒的出芽、细胞分裂、细胞膜的损伤修复等。它们的缺陷引起多种人类疾病，包括神经退行性疾病和癌症等。然而，它们发挥作用的机理却知之甚少。Vps4在辅助因子Vta1的帮助下结合ATP形成稳定的寡聚体，并利用水解ATP产生的能量将ESCRT-III多聚体解开成单体循环利用。目前Vps4寡聚体及其与底物形成的复合体的结构信息非常匮乏，限制了对ESCRT系统的认识和应用。本项目拟利用冷冻电子显微学对Vta1-Vps4复合物及其结合了模型底物Vps24-2嵌合蛋白的复合物的结构进行解析，并在此基础上对复合物中各成分之间的相互作用进行研究， 从而为阐明Vps4蛋白解聚ESCRT-III复合体的分子机制提供重要信息，同时更深入理解I型AAA+ ATPase酶的工作机制。

ESCRT-III complex and Vps4, a type-I AAA+ ATPase, mediate membrane budding away from the cytosol. They are involved in many important biological processes including the biogenesis of multivesicular bodies, the budding of HIV-1 and other viruses from the plasma membrane of infected cells, the membrane abscission step in cytokinesis, and the repair of plasma membrane wounds, etc. Their defects are involved in human diseases including neurodegeneration and cancer. However, the molecular mechanisms of ESCRT-III/Vps4-dependent membrane remodelling are only partially understood. In the cell, with the help of the cofactor Vta1, Vps4 assembles into a higher order oligomer upon binding with ATP, and utilizes the energy from ATP hydrolysis to disassemble ESCRT-III polymer, making individual ESCRT-III subunits available for recycling. The lack of structural information of Vps4 oligomer and its complex with the substrate limited our understanding of the ESCRT system and the application base on it. The purpose of this proposal is to reveal the structures of the Vta1-Vps4 complex and its complex with the model substrate of the MBP-Vps24-2 chimeric protein by using cryo-electron microscopy and to elucidate the interactions among the component proteins in complexes. Our research will provide important insights into the understanding of the mechanism of the ESCRT-III polymer disassembly by Vps4, and the knowledge of how the type-I AAA+ ATPase functions.