Structural and Biochemical Characterization of the Nuclear Pore Complex Scaffold

 描述(申请人提供)：核孔复合体(NPC)是一种大的、模块化的蛋白质组合，主要调节所有真核生物的核质运输。~60-120MDa NPC是由~30个不同蛋白质的多个拷贝组成的模块化组装，这些拷贝排列成亚复合体。由于其庞大的规模和内在的灵活性，整个NPC不适合进行高分辨率的结构研究。然而，使用分而治之的方法，我们通过利用NPC的模块化性质并获得单个子复合体的高分辨率结构来解决这个问题。作为了解NPC详细组织的第一步，我们建议解决NPC结构支架Y和Nic96复合体的高分辨率结构，并表征它们之间的相互作用。我们的初步结果显示了一个高分辨率的Y-络合物的完全复合结构。我们将我们的结果与以前的低分辨率研究进行了比较，并强调了支架亚复合体的高分辨率结构的重要性。以前的结构研究表明，Y和Nic96的一些复杂成分之间存在很强的进化关系。因此，通过利用我们在结构上特征良好的Y复合体的经验，这一建议将集中在研究进化上相似但较少研究的Nic96复合体的生化特征，并阐明其 高分辨率结构。同时，将建立Y和Nic96复合体之间的复合体之间的相互作用，作为了解它们在NPC中的功能和组织的第一步。Nic96复合体的详细结构及其与Y-复合体的相互作用将揭示NPC功能的结构基础以及与支架组件相关的各种疾病。

 DESCRIPTION (provided by applicant): The nuclear pore complex (NPC) is a large, modular protein assembly that principally regulates nucleocytoplasmic transport in all eukaryotes. The ~60-120MDa NPC is a modular assembly of multiple copies of ~30 distinct proteins that are arranged into sub-complexes. The entire NPC is not amenable to high- resolution structural studies, due to its large size and intrinsic flexibility. However, using a divide and conquer approach we tackle this problem by taking advantage of the modular nature of the NPC and obtaining high- resolution structures of individual sub-complexes. As a first step towards understanding the detailed organization of the NPC, we propose to solve high-resolution structures of the NPC structural scaffold Y and Nic96 complex and to characterize their inter-complex interactions. Our preliminary results present a high-resolution complete composite structure of the Y-complex. We compare our results to previous low-resolution studies and highlight the importance of high-resolution structures of the scaffold sub-complexes. Previous structural studies have shown a strong evolutionary relationship between a number of Y and Nic96 complex components. Thus, by leveraging our experience with the structurally well-characterized Y complex, this proposal will focus on investigating the biochemical characteristics of the evolutionarily similar but less investigated Nic96 complex and elucidate its high-resolution structure. Concurrently, the inter-complex interactions between the Y and Nic96 complex will be established as a first step towards understanding their function and organization into the NPC. A detailed structure of the Nic96 complex and its interplay with the Y-complex will uncover the structural basis for the function of the NPC and the various diseases associated with scaffold components.