利用纳米盘技术结合电子显微学方法研究20S复合体的结构与功能

The molecules produced by the cell are transported around the cell via vesicle traffic. This major transport system occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment. The last step of the vesicle traffic is the membrane fusion, which requires the evolutionarily conserved SNARE proteins consisting of v-SNARE on vesicle membranes and t-SNARE on target membranes. During membrane fusion, SNARE proteins on opposing membranes coil together to form a highly stable four-helix bundle. After membrane fusion, SNARE complexes must be disassembled into individual SNARE proteins for reuse, and NSF and its adaptor protein, SNAP, are responsible for the disassembly process. A functional complex consisting of NSF, SNAP and the SNARE complex, termed the 20S complex, was discovered and purified by sedimentation. In the 20S complex, NSF disassociates the SNARE complex upon ATP hydrolysis, making the proteins available for recycling. The mechanism of how the stable SNARE complex is disassembled, however, is still unknown. The lack of structural information of 20S complex has limited our understanding of this mechanism. The purpose of this proposal is to reveal the structure of the 20S complex on the membrane using the newly developed nanodics technology and the cryo-electron microscopy, to elucidate the interactions among the component proteins in the 20S complex, and ultimately to provide important insight into the disassembly process of the SNARE complex by NSF.

细胞内的物质运输是通过囊泡转运实现的。融合是囊泡转运的最后一步，它是由位于两个膜上的SNARE蛋白通过形成稳定的SNARE复合体实现的。SNARE复合体必须被解开才能循环使用，这是通过NSF蛋白和SNAP蛋白完成的。NSF、SNAP和SNARE复合体一起形成沉降系数为20S的复合体。在20S复合体中，NSF水解ATP提供能量将SNARE复合体解开，使它们能够被循环使用。然而，NSF是如何利用水解ATP释放的能量解聚SNARE复合体的这一关键问题，仍未得到解决。目前20S复合体的结构信息非常匮乏，限制了对NSF解聚SNARE复合体的机制的认识。本项目拟利用纳米盘技术和冷冻电子显微学对膜上的20S复合体的结构进行解析，并在此基础上对复合体中各成分之间的相互作用进行研究，阐明明NSF蛋白与α-SNAP和SNARE之间的相互作作用机制，为进一步揭示NSF解聚SNARE的分子机制提供重要的结构基础。

细胞内的物质运输是通过囊泡转运实现的。融合是囊泡转运的最后一步，它是由位于两个膜上的SNARE蛋白通过形成稳定的SNARE复合体实现的。SNARE复合体必须被解开才能循环使用，这是通过NSF蛋白和SNAP蛋白完成的。NSF、α-SNAP和SNARE复合体一起形成沉降系数为20S的复合体。在20S复合体中，NSF水解ATP提供能量将SNARE复合体解开，使它们能够被循环使用。然而，NSF是如何利用水解ATP的能量解聚SNARE复合体的这一关键问题，仍未得到解决。在本项目的支持下，我们创新性的将20S重组到纳米盘中，解决了20S聚沉的问题，从而得到了分散均一的样品。通过冷冻电镜和局部重构算法我们解析了20S中SNARE-SNAP亚复合体的结构。结构显示四个α-SNAP互相结合形成一个右手螺旋的桶状结构，紧密包裹着中心的左手螺旋的SNARE复合体。在α-SNAP上一个保守的疏水尖端和SNARE有着非常紧密的结合，甚至深入到了SNARE螺旋中。生化实验证明这个疏水尖端对于SNARE的解聚是至关重要的。根据这些结果我们以及以前的研究发现，我们提出了NSF协同α-SNAP解聚SNARE复合体的工作模型。