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Architecture of the symmetric core of the nuclear pore

Lin, Daniel H. and Stuwe, Tobias and Schilbach, Sandra and Rundlet, Emily J. and Perriches, Thibaud and Mobbs, George and Fan, Yanbin and Thierbach, Karsten and Huber, Ferdinand M. and Collins, Leslie N. and Davenport, Andrew M. and Jeon, Young E. and Hoelz, André (2016) Architecture of the symmetric core of the nuclear pore. Science, 352 (6283). Art. No. aaf1015. ISSN 0036-8075. PMCID PMC5207208. http://resolver.caltech.edu/CaltechAUTHORS:20160414-150533638

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Abstract

INTRODUCTION: The nuclear pore complex (NPC) is the primary gateway for the transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are very large (~120 MDa) macromolecular machines embedded in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite substantial progress in visualizing the overall shape of the NPC by means of cryoelectron tomography (cryo-ET) and in determining atomic-resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC has thus far remained poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology. RATIONALE: Existing cryo-ET reconstructions of the NPC are too low in resolution to allow for de novo structure determination of the NPC or unbiased docking of nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We developed protocols to reconstitute NPC protomers from purified recombinant proteins, which enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric core. We next determined high-resolution crystal structures of key nucleoporin interactions, providing spatial restraints for their relative orientation. By superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Lastly, we used sequential unbiased searches, supported by the biochemical data, to place the nucleoporin crystal structures into a previously determined cryo-ET reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC symmetric core. RESULTS: Our analysis revealed that the inner and outer rings of the NPC use disparate mechanisms of interaction. Whereas the structured coat nucleoporins of the outer ring form extensive surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker nucleoporins. Our composite structure revealed a defined spoke architecture in which each of the eight spokes spans the nuclear envelope, with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with the exceptions of Nup170 and Nup188, which are present in 48 and 16 copies, respectively. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N termini into two 16-membered rings, thus ensuring that their N-terminal FG repeats project evenly into the central transport channel. CONCLUSION: Our composite structure of the NPC symmetric core can be used as a platform for the rational design of experiments to investigate NPC structure and function. Each nucleoporin occupies multiple distinct biochemical environments, explaining how such a large macromolecular complex can be assembled from a relatively small number of genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemblies.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1126/science.aaf1015DOIArticle
http://science.sciencemag.org/content/352/6283/aaf1015PublisherArticle
http://science.sciencemag.org/content/352/6283/aaf1015/suppl/DC1PublisherSupplementary Materials
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207208PubMed CentralArticle
Additional Information:© 2016 American Association for the Advancement of Science. 17 December 2015; accepted 19 March 2016. We thank J.-Y. Mock, S. Petrovic, and A. Patke for critical reading of the manuscript. We thank M. Beck for sharing the cryo-ET reconstruction of the intact human NPC before publication; E. Hurt for providing material and discussions; and C. Bayes, S. Butkovich, C. Frick, A. Koehl, H. Kuberczyk, R. Tran, and S. Zimmerman for experimental support. We also acknowledge J. Kaiser and the scientific staff of the SSRL beamline 12-2, the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at the APS, and the ALS beamline 8.2.1 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, ALS, 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 (grant ACB-12002) and the National Institute of General Medical Sciences (grant AGM-12006). D.H.L and A.M.D. were supported by a NIH Research Service Award (5 T32 GM07616). D.H.L. was also supported by an Amgen Graduate Fellowship through the Caltech-Amgen Research Collaboration. T.S. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. F.M.H. was supported by a doctoral fellowship from the Boehringer Ingelheim Fonds. Y.F. was supported by a visiting doctoral student scholarship from the China Scholarship Council. A.H. was supported by Caltech startup funds, the Albert Wyrick V Scholar Award from the V Foundation for Cancer Research, the 54th Mallinckrodt Scholar Award from the Edward Mallinckrodt Jr. Foundation, a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research, a Camille-Dreyfus Teacher Scholar Award, and NIH grant R01-GM111461, and he is an inaugural Heritage Principal Investigator of the Heritage Medical Research Institute. The coordinates and structure factors have been deposited in the PDB with accession numbers 5HAX (Nup170NTD•Nup53R3), 5HAY (Nup170CTD), 5HAZ (Nup170CTD), 5HB0 (Nup170CTD•Nup145NR3), 5HB1 (Nup170SOL), 5HB2 (Nic96SOL), 5HB3 (Nic96SOL•Nup53R2), 5HB4 (Nup192∆HEAD), 5HB5 (Nup145NAPD), 5HB6 (Nup145NAPD•Nup145CN), 5HB7 (Nup53RRM, space group P212121), and 5HB8 (Nup53RRM, space group P3121). A PyMol session file containing the composite structure of the NPC symmetric core can be obtained from our webpage (http://ahweb.caltech.edu). The authors declare no financial conflicts of interest. This work is dedicated to the memory of Mandy Hoelz.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
NIHR01-GM111461
National Cancer InstituteACB-12002
National Institute of General Medical SciencesAGM-12006
NIH5 T32 GM07616
Caltech-Amgen Research CollaborationUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Boehringer Ingelheim FondsUNSPECIFIED
China Scholarship CouncilUNSPECIFIED
CaltechUNSPECIFIED
V Foundation for Cancer ResearchUNSPECIFIED
Edward Mallinckrodt Jr. FoundationUNSPECIFIED
Sidney Kimmel Foundation for Cancer ResearchUNSPECIFIED
Camille and Henry Dreyfus FoundationUNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
Subject Keywords:Reconstitution, spectroscopy, and crystallography allow the construction of a model of the human nuclear pore
PubMed Central ID:PMC5207208
Record Number:CaltechAUTHORS:20160414-150533638
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160414-150533638
Official Citation:Architecture of the symmetric core of the nuclear pore BY DANIEL H. LIN, TOBIAS STUWE, SANDRA SCHILBACH, EMILY J. RUNDLET, THIBAUD PERRICHES, GEORGE MOBBS, YANBIN FAN, KARSTEN THIERBACH, FERDINAND M. HUBER, LESLIE N. COLLINS, ANDREW M. DAVENPORT, YOUNG E. JEON, ANDRÉ HOELZ SCIENCE 15 APR 2016
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66192
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:14 Apr 2016 22:42
Last Modified:18 Jul 2017 21:30

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