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Published June 10, 2022 | Supplemental Material + Accepted Version + Submitted
Journal Article Open

Architecture of the linker-scaffold in the nuclear pore


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.

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© 2022 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. https://www.science.org/about/science-licenses-journal-article-reuse Received: 26 October 2021. Accepted: 15 April 2022. Published in print: 10 June 2022. We thank A. Patke for critical reading and editing of the manuscript and insightful discussions; M. Beck for sharing an ~12-Å cryo-ET reconstruction of the intact human HeLa cell NPC before publication [Electron Microscopy Data Bank (EMDB) ID EMD-14322]; E. Hurt, S. Wente, B. Fountura, D. Baltimore, and the Kazusa DNA Research Institute for providing material; and F. Liang, A. Lyons, A. Tang, and J. Thai for experimental support. We are grateful to D. Borek, J. Kollman, G. Lander, D. Lin, S. Saladi, and members of the Hoelz lab for insightful discussion and expertise. We acknowledge J. Kaiser, the scientific staff of the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2, and the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at the Advanced Photon Source (APS) for their support with x-ray diffraction measurements; S. Chen and A. Malyutin of the Beckman Institute Resource Center for Transmission Electron Microscopy at the California Institute of Technology (Caltech) for support with cryo-EM microscopy imaging; J. Myers and the scientific staff of the Pacific Northwest CryoEM Center (PNCC) at the Oregon Health and Science University (OHSU) and the Environmental Molecular Sciences Laboratory (EMSL) for their support with cryo-EM imaging; and the Cold Spring Harbor Laboratory (CSHL) cryo-EM course, the CSHL X-ray Methods in Structural Biology course, and the Michigan Life Science Institute cryo-EM workshop and their instructors M. Cianfrocco, W. Furey, G. Gilliland, J. Kollman, G. Lander, M. Ohi, J. Pflugrath, A. McPherson, and M. Vos, along with all the course staff and lecturers for valuable expert training. The Molecular Observatory at Caltech is supported by D. and J. Voet, the Gordon and Betty Moore Foundation, and the Beckman Institute. The Center for Molecular Medicine at Caltech is supported by the Gordon and Betty Moore Foundation. The operations at the SSRL and APS are supported by the US Department of Energy (DOE) and the National Institutes of Health (NIH). GM/CA has been funded in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. A.H. was supported by a Camille-Dreyfus Teacher Scholar Award (TC-15-082) and NIH grants R01-GM117360 and R01-GM111461, is an investigator of the Heritage Medical Research Institute (HMRI-15-09-01), and is a faculty scholar of the Howard Hughes Medical Institute (55108534). S.P. was supported by a predoctoral fellowship from the Boehringer Ingelheim Fonds and by an Amgen Graduate Fellowship through the Caltech-Amgen Research Collaboration. Author contributions: A.H. conceived and coordinated the study. S.P., D.S., T.P., C.J.B., K.T., and A.H. designed the research. S.P., D.S., T.P., C.J.B., K.T., B.B., G.P.T., and L.S. performed the research. S.P., D.S., T.P., C.J.B., K.T., B.B., S.N., G.W.M., T.A.S., X.L., G.P.T., L.S., and A.H. analyzed data. S.P., D.S., T.P., C.J.B., and A.H. integrated and conceptualized the results. D.S. and T.P. contributed momentously and equally to this work. S.P., C.J.B., S.N., G.W.M., and A.H. wrote and revised the manuscript, with contributions from all authors. Data and materials availability: Materials generated in this study are available on request from the corresponding author. The coordinates and structure factors of crystal structures have been deposited in the Protein Data Bank (PDB) with accession numbers 7MVT (Nup192ΔHead•Nic96187-301), 7MVW (Nup188NTD), 7MVX (Nup188•Nic96R2), 7MW0 (NUP93SOL), and 7MW0 (NUP93SOL•NUP53R2). The coordinates of single particle cryo-EM structures have been deposited in the PDB with accession numbers 7MVU (Nup192•Nic96R2), 7MVV (Nup192•Nic96R2•Nup145NR1•Nup53R1), 7MVY (Nup188•Nic96R2), and 7MVZ (Nup188•Nic96R2•Nup145NR2). The maps of single particle cryo-EM structures have been deposited in the EMDB with accession numbers EMD-24056 (Nup192•Nic96R2), EMD-24057 (Nup192•Nic96R2•Nup145NR1•Nup53R1), EMD-24058 (Nup188•Nic96R2), and EMD-24059 (Nup188•Nic96R2•Nup145NR2). PyMol and Chimera sessions containing the composite structures of the constricted human, dilated human, and dilated S. cerevisiae NPC symmetric core can be obtained from our webpage (http://ahweb.caltech.edu), and coordinates are deposited in the PDB with the respective accession numbers 7TBJ, 7TBK, and 7TBI. Quantitative docking data, workflow code, PyMol, and Chimera sessions were deposited on CaltechDATA (89). The authors declare no conflicts of interest.

Attached Files

Accepted Version - nihms-1824449.pdf

Submitted - 2021.10.26.465796v1.full.pdf

Supplemental Material - science.abm9798_mdar_reproducibility_checklist.pdf

Supplemental Material - science.abm9798_sm.pdf


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