High Resolution Assembly Structure of the Nuclear Pore Complex

核孔复合体的高分辨率组装结构

• 批准号: 7339888
• 负责人: Thomas Schwartz
• 金额: $ 28.51万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7339888
• 关键词: Affect; Affinity; Autoimmune Process; Binding; CDC2L1 gene; Capsid; Caspase-1; Cell Nucleus; Cell division; Cells; Class; Coiled-Coil Domain; Complex; Condition; Crystallization; Cytoplasm; DDX6 gene; Data; Diffusion; Docking; Drug Design; Electrons; Equipment and supply inventories; Eukaryota; Eukaryotic Cell; Facility Construction Funding Category; Future; Genetic Transcription; Glycine; Goals; Growth; HIV; Head Start Program; Hepatitis B Virus; Higher Order Chromatin Structure; Homeostasis; Human; Human Pathology; In Vitro; Individual; Ions; Karyopherins; Knowledge; Lead; Length; Liver diseases; Mediating; Membrane; Membrane Transport Proteins; Methionine; Methods; Microscopic; Molecular; Molecular Machines; N-terminal; Nature; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Pathogenicity; Phenylalanine; Play; Positioning Attribute; Primary biliary cirrhosis; Principal Investigator; Process; Prohibit; Proteins; Publishing; RNA Splicing; Range; Recruitment Activity; Research; Research Personnel; Research Proposals; Resolution; Roentgen Rays; Role; Side; Site; Site-Directed Mutagenesis; Source; Specificity; Structure; Tail; Techniques; Translations; Variant; Vertebrates; Viral; Virus; X-Ray Crystallography; Yeasts; analytical ultracentrifugation; base; design; drug development; finesse; human PRIM2A protein; improved; in vivo; infancy; insight; leukemia; light scattering; mRNA Export; member; microcalorimetry; milligram; mutant; nuclear pore complex protein p107; nuclear pore protein p62; nucleocytoplasmic transport; pathogen; polypeptide; prevent; reconstitution; research study; scaffold; size; transcription factor

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.

描述（由申请人提供）：核孔复合物（NPC）是一个60-80 MDa的模块化蛋白质组装体，是通往细胞核的唯一通道。在一个典型的人类细胞中，数千个npc嵌入了包裹细胞核的双层膜——核膜（NE）中。由于转录和翻译在细胞核和细胞质之间是有空间分隔的，因此npc必须完成大量的转运任务，主要包括从细胞核输出mRNA和核糖体亚基，并输入转录因子等核蛋白。然而，除了它们的常规功能外，鼻咽癌及其组成部分核孔蛋白在人类病理中也起着特别突出的作用。个别核孔蛋白与几种类型的白血病和严重肝病，原发性胆汁性肝硬化有关。此外，与这一建议最相关的是，许多致病性病毒，包括艾滋病毒和乙型肝炎病毒，都使用NPC作为它们进入细胞核的位点。一些病毒也编码蛋白质，阻断特定底物的核运输，作为劫持细胞策略的一部分。为了充分了解这些病毒过程，从而能够破坏病毒与NPC的相互作用，我们非常希望能够从原子细节上破译其结构。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，我们提出了使用x射线晶体学和电子显微镜技术的双管齐下的方法。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件，由沿2倍和8倍旋转对称排列的单个子复合物组成。在脊椎动物中，npc在细胞分裂过程中分解成这些亚复合物，然后再由它们重新组装。我们的假设是，这些亚配合物是适用于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子组件组成，即表征良好的异三聚体p62和异三聚体Nup160复合物以及表征较差的Nup205复合物。我们的第一个目标是解决p62亚复合物的晶体结构。据预测，它的核心结构将是一个螺旋状的线圈组合，这种排列方式将解开晶体结构。这种结构将为理解人大会议的内部互动网络提供基础。我们的第二个目标是解决非美国Nup160复合体的结构，它被认为是后来向NPC招募附属组件的平台。重要的是，它可以从异二聚体亚基重构，然后适用于x射线晶体学。从我们的实验中获得的结构信息应该为像NPC这样的大型大分子机器的构建提供详细的分子洞察，并应该指导药物的合理设计，例如，专门破坏病毒衣壳与NPC的对接。