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Architecture of the linker-scaffold in the nuclear pore

Petrovic, Stefan and Samanta, Dipanjan and Perriches, Thibaud and Bley, Christopher J. and Thierbach, Karsten and Brown, Bonnie and Nie, Si and Mobbs, George W. and Stevens, Taylor A. and Liu, Xiaoyu and Hoelz, André (2021) Architecture of the linker-scaffold in the nuclear pore. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20211102-164838429

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Abstract

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.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2021.10.26.465796DOIDiscussion Paper
ORCID:
AuthorORCID
Bley, Christopher J.0000-0003-2301-1290
Nie, Si0000-0002-1347-0656
Mobbs, George W.0000-0003-2405-3345
Hoelz, André0000-0003-1726-0127
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 Alina Patke for critical reading and editing of the manuscript and insightful discussions; Martin Beck for sharing an ~12 Å cryo-ET reconstruction of the intact human HeLa cell NPC prior to publication; Ed Hurt, Susan Wente, Beatriz Fountura, David Baltimore and the Kazusa DNA Research Institute for providing material, and Felice Liang, Alex Lyons, Aaron Tang, Jimmy Thai, Giovani Pinton Tomaleri, and Lucas Schaus for experimental support. We are grateful to Dominika Borek, Shyam Saladi, and members of the Hoelz lab for discussion and suggestions and Valerie Altounian for the preparation of NPC schematics. We acknowledge Jens Kaiser, the scientific staff of the 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. We acknowledge the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program for their support of the Molecular Observatory at the California Institute of Technology (Caltech). The operations at the SSRL are supported by the U.S. Department of Energy (DOE) and the National Institutes of Health (NIH). We acknowledge Songye Chen and Andrey Malyutin of the Beckman Institute Resource Center for Transmission Electron Microscopy at the Caltech for their support with cryo-electron microscopy (cryo-EM) imaging. We also acknowledge Janette 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. 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. We acknowledge the Center for Molecular Medicine at Caltech for access to an isothermal titration calorimetry instrument. The establishment of the Center for Molecular Medicine was made possible by a generous grant from the Gordon and Betty Moore Foundation. We acknowledge 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 Michael Cianfrocco, William Furey, Gary Gilliland, Justin Kollman, Gabe Lander, Melanie Ohi, James Pflugrath, Alexander McPherson, and Matthijn Vos, along with and all the course staff and lecturers for valuable expert training. S.P. was supported by a pre-doctoral fellowship from the Boehringer Ingelheim Fonds and by an Amgen Graduate Fellowship through the Caltech-Amgen Research Collaboration. A.H. was supported by a Camille-Dreyfus Teacher Scholar Award, NIH grants R01-GM117360 and R01-GM111461, and is a Faculty Scholar with the Howard Hughes Medical Institute and an Investigator with the Heritage Medical Research Institute. The coordinates and structure factors of crystal structures have been deposited in the PDB with accession numbers 7MVT (Nup192 residues 185-1756; Nic96 residues 187-301), 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), EMD-24059 (Nup188•Nic96R2•Nup145NR2). PyMol and Chimera sessions containing the composite structures of the dilated S. cerevisiae, constricted human and dilated human NPC symmetric core can be obtained from our webpage (http://ahweb.caltech.edu) and are deposited in the PDB with the respective accession numbers XXXX, XXXX, XXXX. All quantitative docking data and code were deposited on CaltechDATA (XXXX, XXXX). The authors declare no financial conflicts of interest. A.H. is a co-founder of AMPlify Biosciences, Inc., a biotech company working in the hearing field. The authors have declared no competing interest.
Group:Heritage Medical Research Institute, Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
NIHU24GM129547
Boehringer Ingelheim FondsUNSPECIFIED
AmgenUNSPECIFIED
Camille and Henry Dreyfus FoundationUNSPECIFIED
NIHR01-GM117360
NIHR01-GM111461
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
DOI:10.1101/2021.10.26.465796
Record Number:CaltechAUTHORS:20211102-164838429
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20211102-164838429
Official Citation:Architecture of the linker-scaffold in the nuclear pore. Stefan Petrovic, Dipanjan Samanta, Thibaud Perriches, Christopher J. Bley, Karsten Thierbach, Bonnie Brown, Si Nie, George W. Mobbs, Taylor A. Stevens, Xiaoyu Liu, André Hoelz. bioRxiv 2021.10.26.465796; doi: https://doi.org/10.1101/2021.10.26.465796
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:111701
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 Nov 2021 21:52
Last Modified:02 Nov 2021 21:52

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