Atomic Structure of the Nuclear Pore Complex

修改后的摘要 遗传信息被包围在细胞核中是进化的最大标志之一，但也创造了专用门户的必要性，折叠的蛋白质和蛋白质/核酸复合体可以通过这些门户跨越核膜(NE)。核孔复合体(NPC)是嵌入在贯穿NE的圆形孔中的圆柱形超分子结构，是穿过NE的唯一通道，可以以每秒数百个事件的速度完成直径达40 nm的大分子的选择性双向传输。除了在核质运输中的主要作用外，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，鼻咽癌代表着所有真核生物生命的重要细胞器，因此，鼻咽癌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，其上附着着被称为细胞质细丝和核篮子的不对称成分。NPC是由大约34种不同的称为核孔蛋白的蛋白质组成的，每种蛋白质都以多个副本存在，以至于整个组装在人类体内达到了~110丙二醛的非凡质量。核孔蛋白被组织成不同的亚复合体，这些亚复合体构成了体内完整的NPC的生理构件。为了确定NPC的原子结构，我的团队一直在采用分而治之的方法，在这种方法中，我们绘制了核孔素相互作用图，重组了重组核孔素复合体，并确定了它们的晶体结构，以适合于完整NPC的冷冻电子断层重建。通过这种方式，我们在之前的授权期内实现了~60MDa人NPC对称核的近原子复合结构。在这一进展的基础上，我们现在建议将我们的结构特征扩大到NPC仍未解决的部分，并利用我们已经获得的知识来解决与NPC相关的基本细胞生物学问题。具体地说，我们计划阐明NPC内环中的分子相互作用对于形成其中心运输通道是必不可少的，以及对称核心和跨膜NPC组件之间的相互作用对于NPC锚定在NE孔中是必不可少的。这项拟议的研究结果有望极大地提高我们对NPC调节核质运输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，本文开发的方法学将成为描述像NPC这样大型、灵活和复杂的其他基本细胞巨型组件的范例，由于缺乏结构洞察力，这些组件的功能机制仍然难以捉摸。

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.