High Resolution Assembly Structure of the Nuclear Pore Complex

描述（由申请人提供）：核孔复合物（NPC）是一种60-80 MDa的模块化蛋白组装体，是细胞核的唯一通道。在一个典型的人类细胞中，数千个NPC嵌入核被膜（NE）中，这是包裹细胞核的双层膜。由于转录和翻译在空间上被划分在细胞核和细胞质之间，NPC必须完成巨大的运输任务，主要包括从细胞核输出mRNA和核糖体亚基以及输入核蛋白如转录因子。然而，除了它们的常规功能外，NPC及其成分核孔蛋白在人类病理学中也起着特别突出的作用。单个核孔蛋白与几种类型的白血病和严重的肝病、原发性胆汁性肝硬化有关。此外，与该提议最相关的是，许多致病病毒，包括HIV和肝炎B病毒，使用NPC作为它们进入细胞核的位点。一些病毒还编码蛋白质，这些蛋白质中断特定底物的核转运，作为其劫持细胞策略的一部分。为了充分理解这些病毒过程并因此能够破坏病毒与NPC的相互作用，非常希望以原子细节来破译其结构。这些信息将为针对病毒病原体的靶向药物开发提供基础，这是我们的长期目标。由于NPC的复杂性和大小，我们提出了一个双管齐下的方法，使用X射线晶体学和电子显微镜技术。我们的建议是基于这样的观察，即NPC是一个高度模块化的组装，由单独的子复合体组成，这些子复合体沿着沿着二重和八重旋转对称排列。在脊椎动物中，NPC在细胞分裂期间分解成这些亚复合物，然后重新组装。我们的假设是，这些子复合物是服从X射线晶体学分析。脊椎动物NPC的建筑核心结构基本上由三个独特的、充分表征的异源三聚体p62和异源聚体Nup 160复合物以及较少表征的Nup 205复合物组成。我们的第一个目标是解决p62亚复合物的晶体结构。它的核心结构被预测为螺旋线圈的螺旋组装，其晶体结构的排列将解开。该结构将为理解NPC组件内部的相互作用网络提供基础。我们的第二个目标是解决九聚体Nup 160复合物的结构，该复合物被认为是后来为NPC招募附属成分的平台。重要的是，它可以从异二聚体亚基中重建，然后可以进行X射线晶体学分析。从我们的实验中获得的结构信息应该为构建大型大分子机器（如NPC）提供详细的分子见解，并且应该进一步指导药物的合理设计，例如，特异性破坏病毒衣壳与NPC的对接。

DESCRIPTION (provided by applicant): The nuclear pore complex (NPC), a 60-80 MDa modular protein assembly, serves as the exclusive gateway to the nucleus of the cell. In a typical human cell, several thousand NPCs are embedded in the nuclear envelope (NE), the double-layered membrane that wraps the nucleus. Since transcription and translation are spatially divided between nucleus and cytoplasm, NPCs have to fulfill an enormous transport task, mainly consisting of exporting mRNA and ribosomal subunits from the nucleus and importing nuclear pro- teins such as transcription factors. Apart from their regular function, however, the NPC and its constituents, nucleoporins, also play a particularly prominent role in human pathology. Individual nucleoporins are implicated in several types of leukemia and severe liver disease, primary biliary cirrhosis. Furthermore, and most pertinent to this proposal, many pathogenic viruses, including HIV and hepatitis B virus, use the NPC as their entry site to the nucleus. Several viruses also encode proteins, which interrupt nuclear transport of specific substrates as part of their strategy to hijack the cell. In order to fully understand these viral pro- cesses and consequently be able to disrupt viral interaction with the NPC, it is highly desirable to decipher its structure in atomic detail. Such information will provide a basis for targeted drug development against viral pathogens, which is our long-term objective. Due to the complexity and size of the NPC, we propose a two-pronged approach using X-ray crystallographic and electron-microscopic techniques. Our proposal is based on the observation that the NPC is a highly modular assembly composed of individual subcomplexes that arrange along two- and eight-fold rotational symmetries. In vertebrates, NPCs disassemble into these subcomplexes during cell division and reassemble from them afterwards. Our hypothesis is that these sub- complexes are amenable to X-ray crystallographic analysis. The architectural core structure of the vertebrate NPC is essentially composed of three subassemblies, well-characterized heterotrimeric p62 and heterononameric Nup160 complexes and in addition the less-characterized Nup205 complex. Our first aim is to solve the crystal structure of the p62 subcomplex. Its core structure is predicted to be a helical assembly of coiled coils, the arrangement of which the crystal structure will unravel. This structure will provide a basis for understanding the inner interaction network of the NPC assembly. Our second aim is to solve the structure of nonameric Nup160 complex, which is considered to be the platform that later recruits accessory components to the NPC. Importantly, it can be reconstituted in wfrofrom heterodimeric subunits and is then amenable to X-ray crystallography. Structural information gained from our experiments should provide de- tailed molecular insight into construction of a large macromoloecular machine like the NPC and should fur- ther guide rational design of drugs that, for example, specifically disrupt docking of viral capsids to the NPC.