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 Dasso, Mary and Patke, Alina and Palazzo, Alexander F. and Kossiakoff, Anthony A. and Hoelz, André (2021) Architecture of the cytoplasmic face of the nuclear pore. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20211102-155046319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 mRNA export and a hotspot for nucleoporin-associated diseases, has remained elusive. Here, we report a composite structure of the entire human cytoplasmic face obtained by combining biochemical reconstitution, crystal structure determination, docking into cryo-electron tomographic reconstructions, and physiological validation, accounting for a third of the NPC’s mass. Whereas an evolutionarily conserved ∼540 kDa hetero-hexameric cytoplasmic filament nucleoporin complex is anchored by species-specific motifs above the central transport channel, attachment of the pentameric NUP358 bundles depends on the double-ring arrangement of the coat nucleoporin complex. Our results and the predictive power of our composite structure provide a rich foundation for elucidating the molecular basis of mRNA export and nucleoporin diseases.
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| Additional Information: | The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This version posted October 26, 2021. We thank the members of the Hoelz laboratory for insightful discussions, Martin Beck for sharing of the ~12Å cryoET reconstruction of the intact human NPC prior to publication, Valerie Doye, Ulrike Kutay, Ed Hurt, and Iain Mattaj for providing material, Alex Cohen, Jacqueline Chou, Reeti Gulati, Young Jeon, Claudia Jette, Hannah Margolis, Taylor Stevens, Evelyn Stuwe, Tobias Stuwe, and Jimmy Thai for experimental support, and Valerie Altounian for the preparation of NPC schematics. We acknowledge Jens 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. The Molecular Observatory at the California Institute of Technology (Caltech) is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program. The operations at the SSRL and APS are supported by the U.S. 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). 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.P and F.M.H. were supported by a Ph.D. fellowship of the Boehringer Ingelheim Fonds. A.A.K. was supported by NIH grants R01-GM117372 and P50-GM082545. A.H. was supported by a Camille-Dreyfus Teacher Scholar Award and NIH grants R01-GM117360 and R01-GM111461, is an Investigator of the Heritage Medical Research Institute, and a Faculty Scholar of the Howard Hughes Medical Institute. The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 7MNJ (NUP358145-673), 7MNK (NUP358OE), 7MNI (NUP88NTD•NUP98APD), 7MNL (NUP358NTD•Fab14), 7MNM (NUP358NTD T585M•sAB-14), 7MNN (NUP358NTD T653I•Fab14), 7MNO (NUP358NTD I656V•Fab14), 7MNP (NUP358-ZnF2•Ran(GDP)), 7MNQ (NUP358-ZnF2•Ran(GDP)), 7MNR (NUP358-ZnF3•Ran(GDP)), 7MNS (NUP358-ZnF4•Ran(GDP)), 7MNT (NUP358-ZnF5/6•Ran(GDP)), 7MNU (NUP358-ZnF7•Ran(GDP)),7MNV (NUP358-ZnF8•Ran(GDP)), 7MNW (NUP358-RanBDI•Ran(GMPPNP)), 7MNX (Nup358-RanBD-II•Ran(GMPPNP)), 7MNY (NUP358-RanBD-III•Ran(GMPPNP)), 7MNZ (NUP358-RanBD-VQNYDNKQV•Ran(GPPNHP)), 7MO0 (NUP50-RanBD•Ran(GMPPNP)), 7MO1 (NUP153-ZnF1•Ran(GDP)), 7MO2 (NUP153-ZnF2•Ran(GDP)), 7MO3 (NUP153-ZnF3•Ran(GDP), 2.05Å), 7MO4 (NUP153-ZnF3•Ran(GDP), 2.4Å), 7MO5 (NUP153-ZnF4•Ran(GDP)). PyMol and Chimera sessions containing the composite structure of the human NPC cytoplasmic face can be obtained from our webpage (http://ahweb.caltech.edu), as well as deposited on PDB-dev (XXXX) and CaltechDATA (XXXX). The authors declare no financial conflicts of interest. | ||||||||||||||||||||||||||||||||
| Group: | Heritage Medical Research Institute | ||||||||||||||||||||||||||||||||
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| DOI: | 10.1101/2021.10.26.465790 | ||||||||||||||||||||||||||||||||
| 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, Mary Dasso, Alina Patke, Alexander F. Palazzo, Anthony A. Kossiakoff, André Hoelz. bioRxiv 2021.10.26.465790; doi: https://doi.org/10.1101/2021.10.26.465790 | ||||||||||||||||||||||||||||||||
| 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: | 10 May 2022 22:44 |
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