Introduction 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. Rationale 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. Results 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. Conclusion 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-assemb...
The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kDa inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin hetero-trimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.
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.
The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the arrangement of the structured scaffold building blocks in the NPC symmetric core, their cohesion by multivalent unstructured linker proteins remained elusive. Combining biochemical reconstitution, high resolution structure determination, docking into cryo-electron tomographic reconstructions, and physiological validation, we elucidated the architecture of the entire linker-scaffold, yielding a near-atomic composite structure of the symmetric core accounting for ∼77 MDa of the human NPC. Whereas linkers generally play a rigidifying role, the linker-scaffold of the NPC provides the plasticity and robustness necessary for the reversible constriction and dilation of its central transport channel. Our results complete the structural characterization of the NPC symmetric core, providing a rich foundation for future functional studies.One sentence summaryAn interdisciplinary analysis established the near-atomic molecular architecture and evolutionary conservation of the linker-scaffold of the human nuclear pore complex.
Background: Ndc10 is a DNA-binding protein in yeast that is responsible for centromere formation. Results: The structure of the protein unexpectedly shows that it contains a type IB topoisomerase/-integrase fold. Conclusion:The structure demonstrates how the IB/Int fold has been adapted to a new cellular role. Significance: The structure is the first example of the IB/Int fold being used in a non-catalytic protein.
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