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Architecture of the cytoplasmic face of the nuclear pore

Bley, Christopher J. and Nie, Si and Mobbs, George W. and Petrovic, Stefan and Gres, Anna T. and Liu, Xiaoyu and Mukherjee, Somnath and Harvey, Sho and Huber, Ferdinand M. and Lin, Daniel H. and Brown, Bonnie and Tang, Aaron W. and Rundlet, Emily J. and Correia, Ana R. and Chen, Shane and Regmi, Saroj G. and Stevens, Taylor A. and Jette, Claudia A. and Dasso, Mary and Patke, Alina and Palazzo, Alexander F. and Kossiakoff, Anthony A. and Hoelz, André (2022) Architecture of the cytoplasmic face of the nuclear pore. Science, 376 (6598). Art. No. abm9129. ISSN 0036-8075. PMCID PMC9348906. doi:10.1126/science.abm9129. https://resolver.caltech.edu/CaltechAUTHORS:20211102-155046319

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1126/science.abm9129DOIArticle
https://doi.org/10.1101/2021.10.26.465790DOIDiscussion Paper
http://dx.doi.org/10.22002/D1.2209DOIData
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9348906PubMed CentralArticle
ORCID:
AuthorORCID
Bley, Christopher J.0000-0003-2301-1290
Nie, Si0000-0002-1347-0656
Mobbs, George W.0000-0003-2405-3345
Petrovic, Stefan0000-0002-4979-8696
Gres, Anna T.0000-0002-6915-5903
Liu, Xiaoyu0000-0002-6594-9677
Mukherjee, Somnath0000-0001-5447-4496
Harvey, Sho0000-0002-1742-4550
Huber, Ferdinand M.0000-0001-8647-6971
Lin, Daniel H.0000-0002-1393-3374
Tang, Aaron W.0000-0001-8891-6205
Rundlet, Emily J.0000-0003-4041-6206
Regmi, Saroj G.0000-0002-6705-3997
Stevens, Taylor A.0000-0002-6232-5316
Jette, Claudia A.0000-0002-5085-8027
Dasso, Mary0000-0002-5410-1371
Patke, Alina0000-0003-1628-4282
Palazzo, Alexander F.0000-0002-9700-1995
Kossiakoff, Anthony A.0000-0003-3174-9359
Hoelz, André0000-0003-1726-0127
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).
Group:Heritage Medical Research Institute
Funders:
Funding AgencyGrant Number
Donald and Judith VoetUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
NIHACB-12002
NIHAGM-12006
Boehringer Ingelheim FondsUNSPECIFIED
NIH Predoctoral Fellowship5 T32 GM07616
AmgenUNSPECIFIED
NIHZIAHD001902
NIHZIAHD008954
NIHR01-GM117372
NIHP50-GM082545
Camille and Henry Dreyfus FoundationTC-15-082
NIHR01-GM117360
NIHR01-GM111461
Heritage Medical Research InstituteHMRI-15-09-01
Howard Hughes Medical Institute (HHMI)55108534
Issue or Number:6598
PubMed Central ID:PMC9348906
DOI:10.1126/science.abm9129
Record Number:CaltechAUTHORS:20211102-155046319
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20211102-155046319
Official Citation:Architecture of the cytoplasmic face of the nuclear pore. Christopher J. Bley, Si Nie, George W. Mobbs, Stefan Petrovic, Anna T. Gres, Xiaoyu Liu, Somnath Mukherjee, Sho Harvey, Ferdinand M. Huber, Daniel H. Lin, Bonnie Brown, Aaron W. Tang, Emily J. Rundlet, Ana R. Correia, Shane Chen, Saroj G. Regmi, Taylor A. Stevens, Claudia A. Jette, Mary Dasso, Alina Patke, Alexander F. Palazzo, Anthony A. Kossiakoff, André Hoelz. Science, 376 (6598), eabm9129; DOI: 10.1126/science.abm9129
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:111700
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 Nov 2021 16:24
Last Modified:16 Aug 2022 22:14

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