Structural and Biochemical Characterization of the Nuclear Pore Complex Scaffold

 描述（由申请人提供）：核孔复合物（NPC）是一种大型模块化蛋白组装体，主要调节所有真核生物的核质转运。约60- 120 MDa NPC是约30种不同蛋白质的多个拷贝的模块组装体，这些蛋白质排列成亚复合物。由于其大尺寸和固有的灵活性，整个NPC不适合高分辨率结构研究。然而，使用分治方法，我们通过利用NPC的模块化性质并获得各个子复合物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Nic 96复合物的高分辨率结构，并表征其复合物间的相互作用。我们的初步结果提出了一个高分辨率的完整的复合结构的Y-复合物。我们将我们的结果与以前的低分辨率研究进行比较，并强调了支架子复合物的高分辨率结构的重要性。以前的结构研究表明，Y和Nic 96复杂的组件之间有很强的进化关系。因此，通过利用我们在结构上表征良好的Y复合物方面的经验，本提案将集中于研究进化上相似但研究较少的Nic 96复合物的生化特征，并阐明其生物学特性。 高分辨率结构。同时，Y和Nic 96复合物之间的复合物间相互作用将被建立，作为理解它们在NPC中的功能和组织的第一步。Nic 96复合物的详细结构及其与Y复合物的相互作用将揭示NPC功能的结构基础以及与支架成分相关的各种疾病。

 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.