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

Architecture of the cytoplasmic face of the nuclear pore

Abstract

The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the NPC symmetric core architecture, the asymmetrically decorated cytoplasmic face, essential for messenger RNA (mRNA) export and a hotspot for nucleoporin-associated diseases, has remained elusive. Here we report a composite structure of the human cytoplasmic face obtained by combining biochemical reconstitution, crystal structure determination, docking into cryo–electron tomographic reconstructions, and physiological validation. Whereas species-specific motifs anchor an evolutionarily conserved ~540-kilodalton heterohexameric cytoplasmic filament nucleoporin complex above the central transport channel, attachment of the NUP358 pentameric bundles depends on the double-ring arrangement of the coat nucleoporin complex. Our composite structure and its predictive power provide a rich foundation for elucidating the molecular basis of mRNA export and nucleoporin diseases.

Additional Information

© 2022 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. Submitted 22 October 2021; accepted 15 April 2022. We thank the members of the Hoelz laboratory, M. Guttman, and S. Shan for insightful discussions; A. Cohen, J. Chou, R. Gulati, Y. Jeon, H. Margolis, E. Stuwe, T. Stuwe, and J. Thai for experimental support; M. Beck for sharing the ~12-Å cryo-ET reconstruction of the intact human NPC prior to publication; and V. Doye, U. Kutay, E. Hurt, and I. Mattaj for providing material. We acknowledge J. Kaiser and the scientific staff of the Stanford Synchrotron Radiation Laboratory (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. Funding: The Molecular Observatory at the California Institute of Technology (Caltech) is supported by Donald and Judith Voet, the Gordon and Betty Moore Foundation, and the Beckman Institute. The operations at the SSRL and APS are supported by the US Department of Energy 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). S.P. and F.M.H. were supported by a PhD fellowship of the Boehringer Ingelheim Fonds. D.H.L. was supported by an NIH Research Service Award (5 T32 GM07616) and Amgen Graduate Fellowship through the Caltech-Amgen Research Collaboration. S.C., S.G.R., and M.D. were supported by National Institute of Child Health and Human Development Division of Intramural Research funding ZIAHD001902 and ZIAHD008954. A.A.K. was supported by NIH grants R01-GM117372 and P50-GM082545. A.H. was supported by a Camille-Dreyfus Teacher Scholar Award (TC-15-082) and NIH grants R01-GM117360 and R01-GM111461 and is an Investigator of the Heritage Medical Research Institute (HMRI-15-09-01) and a Faculty Scholar of the Howard Hughes Medical Institute (55108534). Author contributions: A.H. conceived and coordinated the study. C.J.B., S.N., G.W.M., S.P., A.T.G., X.L., S.M., S.H., F.M.H., D.H.L., B.B., A.W.T., E.J.R., A.R.C., S.C., S.G.R., T.A.S., C.A.J., M.D., A.P., A.F.P., A.A.K., and A.H. designed the research. C.J.B., S.N., G.W.M., S.P., A.T.G., X.L., S.M., S.H., F.M.H., D.H.L., B.B., A.W.T., E.J.R., A.R.C., S.C., S.G.R., T.A.S., C.A.J., and A.H. performed the research. C.J.B., S.N., G.W.M., S.P., A.T.G., X.L., S.M., S.H., F.M.H., D.H.L., B.B., A.W.T., E.J.R., A.R.C., S.C., S.G.R., T.A.S., C.A.J., M.D., A.P., A.F.P., A.A.K., and A.H. analyzed the data. S.M. and A.A.K. (synthetic antibodies) and S.C., S.G.R., and M.D. (auxin-degron cell lines) provided new reagents. A.P. and A.F.P. provided reagents and experimental guidance. C.J.B., S.N., G.W.M., S.P., A.T.G., X.L., A.P., and A.H. integrated and conceptualized the results. C.J.B., S.N., G.W.M., S.P., A.P., and A.H. wrote and revised the manuscript, with contributions from all authors. The authors declare no conflicts of interest. Data and materials availability: Materials generated in this study are available on request from the corresponding author. The auxin-inducible AID::NUP358 HCT116, AID::NUP358 DLD1, and NUP160::NG AID DLD1 degron cell lines are subject to a materials transfer agreement, which is available upon request. The coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession codes 7MNJ (NUP358145-673), 7MNK (NUP358OE), 7MNI (NUP88NTD•NUP98APD), 7MNL (NUP358NTD•sAB-14), 7MNM (NUP358NTD T585M•sAB-14), 7MNN (NUP358NTD T653I•sAB-14), 7MNO (NUP358NTD I656V•sAB-14), 7MNP (NUP358ZnF2•Ran(GDP)), 7MNQ (NUP358ZnF2•Ran(GDP)), 7MNR (NUP358ZnF3•Ran(GDP)), 7MNS (NUP358ZnF4•Ran(GDP)), 7MNT (NUP358ZnF5/6•Ran(GDP)), 7MNU (NUP358ZnF7•Ran(GDP)), 7MNV (NUP358ZnF8•Ran(GDP)), 7MNW (NUP358RanBD-I•Ran(GMPPNP)), 7MNX (Nup358RanBD-II•Ran(GMPPNP)), 7MNY (NUP358RanBD-III•Ran(GMPPNP)), 7MNZ (NUP358RanBD-IV•Ran(GMPPNP)), 7MO0 (NUP50RanBD•Ran(GMPPNP)), 7MO1 (NUP153ZnF1•Ran(GDP)), 7MO2 (NUP153ZnF2•Ran(GDP)), 7MO3 (NUP153ZnF3•Ran(GDP), 2.05 Å), 7MO4 (NUP153ZnF3•Ran(GDP), 2.4 Å), and 7MO5 (NUP153ZnF4•Ran(GDP)). PyMol and Chimera sessions containing the composite structures of the constricted and dilated human NPCs can be obtained from our website (http://ahweb.caltech.edu), and coordinates are deposited in the PDB with accession numbers 7TBL and 7TBM, respectively. Quantitative docking data, workflow code, and PyMol and Chimera sessions have been deposited in the CaltechDATA repository (101).

Attached Files

Accepted Version - nihms-1824452.pdf

Submitted - 2021.10.26.465790v1.full.pdf

Supplemental Material - science.abm9129_mdar_reproducibility_checklist.pdf

Supplemental Material - science.abm9129_sm.pdf

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Additional details

Created:
August 20, 2023
Modified:
December 22, 2023