INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC’s eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC’s symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases. RATIONALE Although the arrangement of most scaffold nucleoporins in the NPC’s symmetric core was determined by quantitative docking of crystal structures into cryo–electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC’s inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo–electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC’s integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark–shaped keystones of two evolutionarily conserved hetero‑octameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structure‑guided functional analysis in Saccharomyces cerevisiae , we confirmed the robustness of linker‑scaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and S. cerevisiae cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linker‑scaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring. CONCLUSION We have substantially advanced the structural and biochemical characterization of the symmetric core of the S. cerevisiae and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linker‑scaffold establishes the NPC’s integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPC‑associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins. Linker-scaffold architecture in the human NPC’s symmetric core. Near‑atomic composite structure of the NPC’s symmetric core obtained by quantitative docking of high-resolution crystal and single-particle cryo-EM structures into a cryo-ET reconstruction of the intact human NPC. Schematic representations of the intricate linker-scaffold topology of the cytoplasmic outer ring, inner ring, and nuclear outer ring (clockwise from top) are depicted for the boxed regions. C, C terminus; N, N terminus.

中文翻译：

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核孔中连接支架的结构

简介 在真核细胞中，大分子在细胞核和细胞质之间的选择性双向转运是通过核孔复合体 (NPC) 发生的。嵌入核膜孔中的 ~110-MDa 人类 NPC 是 ~1200-Å 宽和 ~750-Å-高的 ~1000 种蛋白质的集合，统称为核孔蛋白。由于 NPC 沿核质轴的八重旋转对称性，~34 种不同的核孔蛋白中的每一种都以八的倍数出现。在结构上，NPC 的对称核心由一个环绕中央传输通道的内环和两个固定在核壳两侧的外环组成。由于它在从 DNA 到 RNA 再到蛋白质的遗传信息流动中起着核心作用，NPC 通常是病毒感染的目标，其核孔蛋白成分与多种疾病有关。基本原理 虽然大多数支架核孔蛋白在 NPC 对称核心中的排列是通过将晶体结构定量对接到完整 NPC 的低温电子断层扫描 (cryo-ET) 图中来确定的，但多价连接体核孔蛋白的拓扑结构和它们内聚的分子细节仍然存在难以捉摸。最近，来自不同物种的 NPC 的原位低温 ET 重建表明，NPC 的内环能够响应核膜膜张力的变化进行可逆收缩和扩张，从而将中央传输通道的直径调节约 200 Å . 我们结合生化重构，高分辨率晶体和单粒子冷冻电子显微镜 (cryo-EM) 结构测定、对接冷冻 ET 图和生理学验证，以阐明连接子-支架相互作用网络的分子结构，这不仅对 NPC 至关重要完整性，但也赋予允许和承受这种大规模构象变化所必需的可塑性和稳健性。结果通过对所有真菌和人类连接体核孔蛋白的支架结合区域进行生物化学定位，并确定连接体-支架复合物的晶体和单粒子冷冻电镜结构，我们完成了对生物化学易处理的连接体-支架网络的表征，并建立了其进化保守性，尽管存在相当大的序列差异。我们确定了完整的 Nup188 和 Nup192 支架中枢与其 Nic96、Nup145N 和 Nup53 接头核孔蛋白结合区域结合的一系列晶体和单粒子冷冻电镜结构，揭示了这两种蛋白质形成了两个进化过程中截然不同的问号形基石保守的异八聚体内环复合物。接头通过短定义的基序与支架表面口袋结合，侧翼区域通常形成额外的分散相互作用以加强结合。使用结构导向的功能分析 接头通过短定义的基序与支架表面口袋结合，侧翼区域通常形成额外的分散相互作用以加强结合。使用结构导向的功能分析 接头通过短定义的基序与支架表面口袋结合，侧翼区域通常形成额外的分散相互作用以加强结合。使用结构导向的功能分析酿酒酵母，我们证实了接头-支架相互作用的稳健性，并确定了我们的生化和结构发现的生理相关性。实验结构定量对接人类和人类的近原子复合结构酿酒酵母收缩和扩张的 NPC 的低温 ET 图在结构上消除了 Nup188 和 Nup192 中枢在完整真菌和人类 NPC 中的定位，并揭示了连接器-支架网络的拓扑结构。连接支架产生八个相对刚性的内环辐条，这些辐条灵活地相互连接以允许形成横向通道。出乎意料的是，我们发现接头-支架相互作用通过在八个核和细胞质外环辐条之间形成紧密的交联主食而在外环中发挥相反的作用，从而限制了对内环的扩张运动。结论我们已经大大推进了对称核心的结构和生化表征酿酒酵母和人类 NPC，并确定了近原子复合结构。复合结构揭示了进化上保守的连接支架建立 NPC 完整性的分子机制，同时允许观察到的中央传输通道的可塑性。复合结构是 NPC 组装和拆卸的机械解剖、NPC 相关疾病的病因学、NPC 扩张在可溶性和完整膜蛋白货物的核质转运中的作用以及不对称核孔蛋白的锚定的路线图。 人类 NPC 对称核心中的链接器支架架构。通过将高分辨率晶体和单粒子低温 EM 结构定量对接到完整人类 NPC 的低温 ET 重建中获得的 NPC 对称核心的近原子复合结构。盒装区域描绘了细胞质外环、内环和核外环（从顶部顺时针方向）的复杂接头-支架拓扑结构的示意图。C、C总站；N，N总站。