Architecture of the symmetric core of the nuclear pore.
Architecture of the symmetric core of the nuclear pore.
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DOI：
10.1126/science.aaf1015
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发表时间：
2016-04-15
期刊：
影响因子：
--
通讯作者：
Hoelz A
中科院分区：
文献类型：
--
作者：
Lin DH;Stuwe T;Schilbach S;Rundlet EJ;Perriches T;Mobbs G;Fan Y;Thierbach K;Huber FM;Collins LN;Davenport AM;Jeon YE;Hoelz A
核孔复合物（NPC）是大分子在细胞核和细胞质之间转运的主要通道，是基因表达的重要介质和调节剂。NPC是巨大的~120 MDa大分子机器，嵌入核膜中，每个包含~1000个蛋白质亚基，称为核孔蛋白。尽管在通过冷冻电子断层扫描可视化NPC的整体形状和确定核孔蛋白的原子分辨率晶体结构方面取得了实质性进展，但组装NPC的分子结构仍然知之甚少，阻碍了可以研究其在细胞生物学中的许多作用的机制研究的设计。现有的冷冻电子断层重建的NPC仍然太低的分辨率，以允许从头结构的NPC或无偏对接的核孔蛋白片段晶体结构的确定。我们试图通过首先定义NPC的相互作用网络来弥合这一分辨率差距，重点是进化保守的对称核心。我们开发了从纯化的重组蛋白重建NPC原聚体的方案，这使得能够生成NPC对称核心的高分辨率生化相互作用图。接下来，我们确定了关键核孔蛋白相互作用的高分辨率晶体结构，为它们的相对取向提供了空间约束。最后，通过叠加顺序重叠的晶体结构，我们生成了大支架核孔蛋白的精确全长结构。在此生化数据的支持下，我们使用顺序的，无偏的搜索将核孔蛋白晶体结构放置到先前确定的完整的人NPC的冷冻电子断层扫描重建中，从而生成整个NPC对称核心的复合结构。我们的分析表明，NPC的内环和外环利用不同的相互作用机制。虽然外环的结构化外壳核孔蛋白形成广泛的表面接触，但内环的支架蛋白通过接头核孔蛋白中的柔性序列桥接。我们的复合结构揭示了一个定义的辐条架构，具有有限的跨辐条交互。大多数核孔蛋白存在于32个拷贝中，但Nup 170和Nup 188例外。最后，我们观察到通道核孔蛋白的排列，其将其N-末端定向成两个十六元环，确保其N-末端FG重复均匀地投射到中央运输通道中。我们的NPC对称核的复合结构可以用作合理设计实验的平台，以探测NPC的结构和功能。每个核孔蛋白占据多个不同的生物化学环境，解释了这样一个大分子复合物是如何从相对少量的独特基因组装而成的。我们的综合，自下而上的方法提供了一个类似的大型生物巨型组件的生化和结构表征的范例。核孔复合体对称核的复合结构。从细胞质面上方观察，通过将核孔蛋白和核孔蛋白复合物晶体结构顺序无偏对接到完整人NPC的cryoET重建中产生的核孔复合物对称核心的复合结构。核孔蛋白结构显示为彩色卡通，核被膜密度显示为灰色表面。核孔复合物的对称核心的综合分析建立了它的分子结构。
The nuclear pore complex (NPC) is the primary gateway for transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are enormous ~120 MDa macromolecular machines embedded in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite substantial progress in visualizing the overall shape of the NPC by cryoelectron tomography and in determining atomic resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC remains poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology. Existing cryoelectron tomographic reconstructions of the NPC remain too low in resolution to allow for de novo structure determination of the NPC or unbiased docking of nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We developed protocols to reconstitute NPC protomers from purified, recombinant proteins, which enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric core. We next determined high-resolution crystal structures of key nucleoporin interactions, providing spatial restraints for their relative orientation. Lastly, by superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Supported by this biochemical data, we used sequential, unbiased searches to place the nucleoporin crystal structures into a previously determined cryoelectron tomographic reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC symmetric core. Our analysis revealed that the inner and outer rings of the NPC utilize disparate mechanisms of interaction. While the structured coat nucleoporins of the outer ring form extensive surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker nucleoporins. Our composite structure revealed a defined spoke architecture with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with notable exceptions of Nup170 and Nup188. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N-termini into two sixteen-membered rings, ensuring that their N-terminal FG repeats project evenly into the central transport channel. Our composite structure of the NPC symmetric core can be used as a platform for the rational design of experiments to probe NPC structure and function. Each nucleoporin occupies multiple distinct biochemical environments, explaining how such a large macromolecular complex can be assembled from a relatively small number of unique genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemblies. Composite structure of the nuclear pore complex symmetric core. The composite structure of the nuclear pore complex symmetric core generated by sequential, unbiased docking of nucleoporin and nucleoporin complex crystal structures into the cryoET reconstruction of the intact human NPC, viewed from above the cytoplasmic face. Nucleoporin structures are shown as colored cartoons and the nuclear envelope density is shown as a gray surface. An integrated analysis of the symmetric core of the nuclear pore complex established its molecular architecture.
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影响因子：
9.8
作者：
Devos D;Dokudovskaya S;Alber F;Williams R;Chait BT;Sali A;Rout MP
通讯作者：
Rout MP
影响因子：
16.6
作者：
Eibauer, Matthias;Pellanda, Mauro;Turgay, Yagmur;Dubrovsky, Anna;Wild, Annik;Medalia, Ohad
通讯作者：
Medalia, Ohad
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
Cowtan, K
影响因子：
16
作者：
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通讯作者：
Schwartz, Thomas U.
影响因子：
16.8
作者：
Drin, Guillaume;Casella, Jean-Francois;Antonny, Bruno
通讯作者：
Antonny, Bruno