利用电子显微学研究I型AAA+ ATPase酶Vps4蛋白的结构与功能

Vps4 belongs to the type-I AAA family of proteins. Vps4 functions together with ESCRTIII in membrane deformation and fission events. These cellular functions include vesicle formation at the endosome, cytokinesis and viral budding. Thus, Vps4 plays an important role in the biological processes inside cells. ESCRT-III is composed of four proteins. Once these soluble, cytoplasmic proteins are recruited to the membrane, they oligomerize into a polymer that seems to have the shape of concentric rings or spirals to facilitate the membrane deformation and fission events. Vps4 disassembles ESCRT-III polymers by using the energy from ATP hydrolysis, thereby recycling the ESCRT-III proteins for further rounds of membrane sorting. Previous studies indicate that Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III on the membrane, where Vps4 oligomerizes into an active ATPase. However, the mechanism underlying the disassembly of ESCRTIII by Vps4 is still unknown. In this project, we are aiming to study the structures and functions of Vps4 oligomers, and the Vps4-ESCRTIII complex by using the electron microscopy and the methods of biochemistry and molecular biology, in order to elucidate the molecular mechanism of how ESCRTIII is disassembled by Vps4.

Vps4属于I型AAA+ ATPase酶家族蛋白,它和内体蛋白分选转运装置III（ESCRTIII）一起在多囊体的形成、细胞质分裂和逆转录病毒如HIV的出芽等膜分裂过程中起关键作用，因此具有重要的生理功能。ESCRTIII复合体由4个蛋白组成，它们被招募到膜上后组装形成复合体帮助膜的弯曲并促进膜分裂。ESCRTIII复合体需要解开成单体以被循环使用。Vps4的作用就是利用水解ATP的能量将ESCRTIII解开。当细胞质中的Vps4二体或单体被招募到膜上与ESCRTIII结合后，会形成具有很高ATP酶活的寡聚体，将ESCRTIII解聚。然而，Vps4是如何利用水解ATP的能量将ESCRTIII复合体解开的问题一直未得到解决。本项目旨在通过研究Vps4活性寡聚体和Vps4与底物ESCRTIII形成的复合体的结构并结合生物化学和分子生物学的分析，揭示Vp4解聚ESCRTIII复合体的分子机制。

细胞内，内体蛋白分选转运装置III（ESCRT-III）和Vps4蛋白一起组成的蛋白机器介导多种膜重塑过程。ESCRT-III复合体由4个蛋白组成，它们被招募到膜上后组装形成复合体帮助膜的弯曲并促进膜分裂。ESCRT-III复合体需要解开成单体以被循环使用。Vps4的作用就是利用水解ATP的能量将ESCRT-III 解开。在本项目的支持下，我们利用冷冻电镜解析了Vps4蛋白结合ATP状态下的六聚体结构以及进一步结合辅助因子Vta1后形成的复合体的结构，分辨率分别为3.9埃和4.2埃。结构显示Vps4六聚体呈现明显的不对称性，整体是一个开口螺旋的环状结构；Vta1结合在Vps4六聚体的边缘，并且每一个Vta1二聚体通过两种不同的方式结合相邻的两个Vps4单体，从而促进ESCRT-III解聚过程中Vps4活性形式寡聚体的形成。我们的结构以及在结构指导下的生化和单分子实验结果为揭示Vps4解聚ESCRT-III的分子机制提供了重要的见解。