Atomic Structure of the Nuclear Pore Complex

核孔复合体的原子结构

• 批准号: 10438837
• 负责人: Andre Hoelz
• 金额: $ 37.73万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10438837
• 关键词: Accounting; Address; Anatomy; Architecture; Binding; Biochemical; Biogenesis; Biological; C-terminal; Caliber; Cell Cycle; Cell Nucleus; Cell physiology; Cells; Characteristics; Complex; Cryo-electron tomography; Crystallization; Cytoplasmic Filaments; Disease; Eukaryotic Cell; Event; Evolution; Functional disorder; Gene Expression Regulation; Genetic Transcription; Grant; Health; Human; Inherited; Integral Membrane Protein; Knowledge; Life; Link; Maps; Membrane; Methodology; Mitosis; Molecular; N-terminal; Nature; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Structure; Nucleic Acids; Organelles; Outcome; Physiological; Post-Translational Protein Processing; Proteins; Recombinants; Research; Role; Structure; Validation; Work; Yeasts; flexibility; genetic information; human disease; in vivo; insight; leukemia; mRNA Export; macromolecular assembly; macromolecule; molecular mass; nucleocytoplasmic transport; protein folding; public health relevance; reconstitution; reconstruction; scaffold; success

MODIFIED ABSTRACT The enclosure of genetic information in the nucleus is one of the great hallmarks of evolution, but creates the necessity for dedicated portals through which folded proteins and protein/nucleic acid complexes can cross the nuclear envelope (NE). The nuclear pore complex (NPC), a cylindrical supramolecular structure embedded in circular pores permeating the NE, is the sole gateway for passage through the NE and can accomplish the selective bidirectional transport of macromolecules of up to ~40 nm in diameter at a rate of several hundred events per second. Beyond its primary role in nucleocytoplasmic transport, the NPC also contributes to additional modes of gene regulation for example through direct interaction with the transcription and mRNA export machineries. The NPC thus represents an essential organelle for all eukaryotic life and, accordingly, NPC dysfunction has been associated with various forms of human disease. Architecturally, the NPC consists of a central symmetric core to which asymmetric components called cytoplasmic filaments and nuclear basket are attached. The NPC is built from ~34 different proteins termed nucleoporins that are each present in multiple copies such that the entire assembly reaches the extraordinary mass of ~110 MDa in humans. Nucleoporins are organized into distinct subcomplexes which constitute physiological building blocks of the intact NPC in vivo. To determine the atomic architecture of the NPC, my group has been pursuing a divide-and-conquer approach, in which we have mapped nucleoporin interactions, reconstituted recombinant nucleoporin complexes and determined their crystal structures to be fit into cryo-electron tomographic reconstructions of the intact NPC. In this way, we achieved a near-atomic composite structure of the ~60MDa human NPC symmetric core in the previous grant period. Building on this progress, we now propose to expand our structural characterization to still unresolved parts of the NPC and to use our already gained knowledge to address fundamental NPC-associated cell biological questions. Specifically, we plan to elucidate the molecular interactions in the NPC’s inner ring that are essential for the formation of its central transport channel, and between the symmetric core and transmembrane NPC components that are essential for NPC anchoring in the NE pores. The outcome of the proposed research is expected to greatly increase our understanding of the molecular mechanisms by which the NPC regulates nucleocytoplasmic transport and associated cellular processes, while simultaneously creating a mechanistic basis for currently untreatable “nup diseases.” Furthermore, the methodologies developed herein will serve as a paradigm for the characterization of other essential cellular mega-assemblies as large, flexible and complex as the NPC whose functional mechanisms have remained elusive due to lack of structural insight.

修改摘要 遗传信息在细胞核中的封闭是进化的重要标志之一，但这也为折叠蛋白质和蛋白质/核酸复合物穿过核膜（NE）创造了专门的门户。核孔复合物（NPC）是一种嵌入渗透NE的圆孔中的圆柱形超分子结构，是通过NE的唯一通道，可以以每秒数百个事件的速率完成直径高达约40 nm的大分子的选择性双向运输。除了其在核质转运中的主要作用外，NPC还有助于基因调控的其他模式，例如通过与转录和mRNA输出机制的直接相互作用。因此，NPC代表了所有真核生物的基本细胞器，因此，NPC功能障碍与各种形式的人类疾病有关。在结构上，NPC由一个中心对称的核心组成，称为细胞质丝和核篮的不对称组件连接到该核心。NPC由约34种不同的蛋白质组成，称为核孔蛋白，每种蛋白质都以多个拷贝存在，使得整个组装体在人类中达到约110 MDa的非凡质量。核孔蛋白被组织成不同的亚复合物，其构成体内完整NPC的生理构建块。为了确定NPC的原子结构，我的团队一直在追求一种分而治之的方法，在这种方法中，我们绘制了核孔蛋白相互作用，重组核孔蛋白复合物，并确定了它们的晶体结构，以适应完整NPC的冷冻电子断层扫描重建。通过这种方式，我们在前一个资助期实现了~ 60 MDa人类NPC对称核的近原子复合结构。基于这一进展，我们现在建议将我们的结构表征扩展到NPC中尚未解决的部分，并利用我们已经获得的知识来解决基本的NPC相关细胞生物学问题。具体来说，我们计划阐明的分子相互作用在NPC的内环，是必不可少的中央运输通道的形成，以及之间的对称核心和跨膜NPC组件，是必不可少的NPC锚定在NE孔。这项研究的结果有望大大增加我们对NPC调节核质转运和相关细胞过程的分子机制的理解，同时为目前无法治疗的“nup疾病”创造一个机制基础。此外，本文开发的方法将作为表征其他必要的细胞大型组装体的范例，这些组装体与NPC一样大、灵活和复杂，其功能机制由于缺乏结构洞察力而仍然难以捉摸。