A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

Nordeen, Sarah A.; Andersen, Kasper R.; Knockenhauer, Kevin E.; Ingram, Jessica R.; Ploegh, Hidde L.; Schwartz, Thomas U.

doi:10.1038/s41467-020-19884-6

Title: A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

Journal Article · Wed Dec 02 00:00:00 EST 2020 · Nature Communications

DOI:https://doi.org/10.1038/s41467-020-19884-6· OSTI ID:1736296

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^[1]; Knockenhauer, Kevin E. ^[1]; Ingram, Jessica R. ^[2];

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Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Harvard Medical School, Boston, MA (United States)

Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-06CH11357; R01GM77537; T32GM007287; GM103403

OSTI ID:: 1736296

Journal Information:: Nature Communications, Vol. 11, Issue 1; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

References (65)

The nuclear pore complex - structure and function at a glance Kabachinski, G.; Schwartz, T. U. Journal of Cell Science, Vol. 128, Issue 3 https://doi.org/10.1242/jcs.083246	journal	January 2015
Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform Fernandez-Martinez, Javier; Kim, Seung Joong; Shi, Yi Cell, Vol. 167, Issue 5 https://doi.org/10.1016/j.cell.2016.10.028	journal	November 2016
Integrative structure and functional anatomy of a nuclear pore complex Kim, Seung Joong; Fernandez-Martinez, Javier; Nudelman, Ilona Nature, Vol. 555, Issue 7697 https://doi.org/10.1038/nature26003	journal	March 2018
Exploring cellular biochemistry with nanobodies Cheloha, Ross W.; Harmand, Thibault J.; Wijne, Charlotte Journal of Biological Chemistry, Vol. 295, Issue 45 https://doi.org/10.1074/jbc.rev120.012960	journal	August 2020
A general amphipathic α-helical motif for sensing membrane curvature Drin, Guillaume; Casella, Jean-François; Gautier, Romain Nature Structural & Molecular Biology, Vol. 14, Issue 2 https://doi.org/10.1038/nsmb1194	journal	January 2007
Cell type‐specific nuclear pores: a case in point for context‐dependent stoichiometry of molecular machines Ori, Alessandro; Banterle, Niccolò; Iskar, Murat Molecular Systems Biology, Vol. 9, Issue 1 https://doi.org/10.1038/msb.2013.4	journal	January 2013
Structure and gating of the nuclear pore complex Eibauer, Matthias; Pellanda, Mauro; Turgay, Yagmur Nature Communications, Vol. 6, Issue 1 https://doi.org/10.1038/ncomms8532	journal	June 2015
Allosteric activation of apicomplexan calcium-dependent protein kinases Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M. Proceedings of the National Academy of Sciences, Vol. 112, Issue 36 https://doi.org/10.1073/pnas.1505914112	journal	August 2015
Molecular architecture of the inner ring scaffold of the human nuclear pore complex Kosinski, J.; Mosalaganti, S.; von Appen, A. Science, Vol. 352, Issue 6283 https://doi.org/10.1126/science.aaf0643	journal	April 2016
The nuclear pore complex: understanding its function through structural insight Beck, Martin; Hurt, Ed Nature Reviews Molecular Cell Biology, Vol. 18, Issue 2 https://doi.org/10.1038/nrm.2016.147	journal	December 2016
Architecture of the symmetric core of the nuclear pore Lin, D. H.; Stuwe, T.; Schilbach, S. Science, Vol. 352, Issue 6283 https://doi.org/10.1126/science.aaf1015	journal	April 2016
A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops Decanniere, Klaas; Desmyter, Aline; Lauwereys, Marc Structure, Vol. 7, Issue 4 https://doi.org/10.1016/S0969-2126(99)80049-5	journal	April 1999
The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications Götzke, Hansjörg; Kilisch, Markus; Martínez-Carranza, Markel Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-12301-7	journal	September 2019
Molecular architecture of the Nup84–Nup145C–Sec13 edge element in the nuclear pore complex lattice Brohawn, Stephen G.; Schwartz, Thomas U. Nature Structural & Molecular Biology, Vol. 16, Issue 11 https://doi.org/10.1038/nsmb.1713	journal	October 2009
Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p. Grandi, P.; Schlaich, N.; Tekotte, H. The EMBO Journal, Vol. 14, Issue 1 https://doi.org/10.1002/j.1460-2075.1995.tb06977.x	journal	January 1995
Collaboration gets the most out of software Morin, Andrew; Eisenbraun, Ben; Key, Jason eLife, Vol. 2 https://doi.org/10.7554/eLife.01456	journal	September 2013
Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae Longtine, Mark S.; Mckenzie III, Amos; Demarini, Douglas J. Yeast, Vol. 14, Issue 10 https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U	journal	July 1998
Crystal structure of the metazoan Nup62•Nup58•Nup54 nucleoporin complex Chug, Hema; Trakhanov, Sergei; Hülsmann, Bastian B. Science, Vol. 350, Issue 6256 https://doi.org/10.1126/science.aac7420	journal	August 2015
The Nuclear Pore Complex Has Entered the Atomic Age Brohawn, Stephen G.; Partridge, James R.; Whittle, James R. R. Structure, Vol. 17, Issue 9 https://doi.org/10.1016/j.str.2009.07.014	journal	September 2009
The Structure of the Scaffold Nucleoporin Nup120 Reveals a New and Unexpected Domain Architecture Leksa, Nina C.; Brohawn, Stephen G.; Schwartz, Thomas U. Structure, Vol. 17, Issue 8 https://doi.org/10.1016/j.str.2009.06.003	journal	August 2009
The molecular architecture of the nuclear pore complex Alber, Frank; Dokudovskaya, Svetlana; Veenhoff, Liesbeth M. Nature, Vol. 450, Issue 7170 https://doi.org/10.1038/nature06405	journal	November 2007
Camelid nanobodies: killing two birds with one stone Desmyter, Aline; Spinelli, Silvia; Roussel, Alain Current Opinion in Structural Biology, Vol. 32 https://doi.org/10.1016/j.sbi.2015.01.001	journal	June 2015
The Nuclear Pore Complex as a Flexible and Dynamic Gate Knockenhauer, Kevin E.; Schwartz, Thomas U. Cell, Vol. 164, Issue 6 https://doi.org/10.1016/j.cell.2016.01.034	journal	March 2016
The diverse roles of the Nup93/Nic96 complex proteins – structural scaffolds of the nuclear pore complex with additional cellular functions Vollmer, Benjamin; Antonin, Wolfram Biological Chemistry, Vol. 395, Issue 5 https://doi.org/10.1515/hsz-2013-0285	journal	May 2014
Structural Basis of the Nic96 Subcomplex Organization in the Nuclear Pore Channel Schrader, Nils; Stelter, Philipp; Flemming, Dirk Molecular Cell, Vol. 29, Issue 1 https://doi.org/10.1016/j.molcel.2007.10.022	journal	January 2008
Nuclear Pore Composition Regulates Neural Stem/Progenitor Cell Differentiation in the Mouse Embryo Lupu, Floria; Alves, Annabelle; Anderson, Kathryn Developmental Cell, Vol. 14, Issue 6 https://doi.org/10.1016/j.devcel.2008.03.011	journal	June 2008
Structural Evidence for Common Ancestry of the Nuclear Pore Complex and Vesicle Coats Brohawn, S. G.; Leksa, N. C.; Spear, E. D. Science, Vol. 322, Issue 5906 https://doi.org/10.1126/science.1165886	journal	November 2008
Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex Seo, H. -S.; Ma, Y.; Debler, E. W. Proceedings of the National Academy of Sciences, Vol. 106, Issue 34 https://doi.org/10.1073/pnas.0907453106	journal	August 2009
Nanobodies and recombinant binders in cell biology Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge Journal of Cell Biology, Vol. 209, Issue 5 https://doi.org/10.1083/jcb.201409074	journal	June 2015
Atomic force microscopy reveals structural variability amongst nuclear pore complexes Stanley, George J.; Fassati, Ariberto; Hoogenboom, Bart W. Life Science Alliance, Vol. 1, Issue 4 https://doi.org/10.26508/lsa.201800142	journal	July 2018
Yeast surface display platform for rapid discovery of conformationally selective nanobodies McMahon, Conor; Baier, Alexander S.; Pascolutti, Roberta Nature Structural & Molecular Biology, Vol. 25, Issue 3 https://doi.org/10.1038/s41594-018-0028-6	journal	February 2018
Exploiting Nanobodies’ Singular Traits Ingram, Jessica R.; Schmidt, Florian I.; Ploegh, Hidde L. Annual Review of Immunology, Vol. 36, Issue 1 https://doi.org/10.1146/annurev-immunol-042617-053327	journal	April 2018
A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins Frey, Steffen; Görlich, Dirk Journal of Chromatography A, Vol. 1337 https://doi.org/10.1016/j.chroma.2014.02.029	journal	April 2014
Synthetic single domain antibodies for the conformational trapping of membrane proteins Zimmermann, Iwan; Egloff, Pascal; Hutter, Cedric AJ eLife, Vol. 7 https://doi.org/10.7554/eLife.34317	journal	May 2018
Comparative analysis of nanobody sequence and structure data Mitchell, Laura S.; Colwell, Lucy J. Proteins: Structure, Function, and Bioinformatics, Vol. 86, Issue 7 https://doi.org/10.1002/prot.25497	journal	April 2018
Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification : Optimized Andersen, Kasper R.; Leksa, Nina C.; Schwartz, Thomas U. Proteins: Structure, Function, and Bioinformatics, Vol. 81, Issue 11 https://doi.org/10.1002/prot.24364	journal	August 2013
In-cell architecture of the nuclear pore and snapshots of its turnover Allegretti, Matteo; Zimmerli, Christian E.; Rantos, Vasileios Nature, Vol. 586, Issue 7831 https://doi.org/10.1038/s41586-020-2670-5	journal	September 2020
Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A. Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep19211	journal	January 2016
Mechanisms of Membrane Curvature Sensing Antonny, Bruno Annual Review of Biochemistry, Vol. 80, Issue 1 https://doi.org/10.1146/annurev-biochem-052809-155121	journal	July 2011
Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex Kampmann, Martin; Blobel, Günter Nature Structural & Molecular Biology, Vol. 16, Issue 7 https://doi.org/10.1038/nsmb.1618	journal	June 2009
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Fiji: an open-source platform for biological-image analysis Schindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin Nature Methods, Vol. 9, Issue 7 https://doi.org/10.1038/nmeth.2019	journal	June 2012
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Structure and Assembly of the Nuclear Pore Complex Hampoelz, Bernhard; Andres-Pons, Amparo; Kastritis, Panagiotis Annual Review of Biophysics, Vol. 48, Issue 1 https://doi.org/10.1146/annurev-biophys-052118-115308	journal	May 2019
Linker Nups connect the nuclear pore complex inner ring with the outer ring and transport channel Fischer, Jessica; Teimer, Roman; Amlacher, Stefan Nature Structural & Molecular Biology, Vol. 22, Issue 10 https://doi.org/10.1038/nsmb.3084	journal	September 2015
Integrated Structural Analysis of the Human Nuclear Pore Complex Scaffold Bui, Khanh Huy; von Appen, Alexander; DiGuilio, Amanda L. Cell, Vol. 155, Issue 6 https://doi.org/10.1016/j.cell.2013.10.055	journal	December 2013
A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation Beckett, Dorothy; Kovaleva, Elena; Schatz, Peter J. Protein Science, Vol. 8, Issue 4 https://doi.org/10.1110/ps.8.4.921	journal	January 1999
Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy Molecular & Cellular Proteomics, Vol. 13, Issue 11 https://doi.org/10.1074/mcp.M114.040915	journal	August 2014
Structure of the Sec13–Sec16 edge element, a template for assembly of the COPII vesicle coat Whittle, James R. R.; Schwartz, Thomas U. The Journal of Cell Biology, Vol. 190, Issue 3 https://doi.org/10.1083/jcb.201003092	journal	August 2010
The Structure of the Nuclear Pore Complex (An Update) Lin, Daniel H.; Hoelz, André Annual Review of Biochemistry, Vol. 88, Issue 1 https://doi.org/10.1146/annurev-biochem-062917-011901	journal	June 2019
Crystal Structure of Nucleoporin Nic96 Reveals a Novel, Intricate Helical Domain Architecture Jeudy, Sandra; Schwartz, Thomas U. Journal of Biological Chemistry, Vol. 282, Issue 48 https://doi.org/10.1074/jbc.M705479200	journal	September 2007
Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins Lutzmann, Malik; Kunze, Ruth; Buerer, Andrea The EMBO Journal, Vol. 21, Issue 3 https://doi.org/10.1093/emboj/21.3.387	journal	February 2002
SWISS-MODEL: homology modelling of protein structures and complexes Waterhouse, Andrew; Bertoni, Martino; Bienert, Stefan Nucleic Acids Research, Vol. 46, Issue W1 https://doi.org/10.1093/nar/gky427	journal	May 2018
Architecture of the fungal nuclear pore inner ring complex Stuwe, Tobias; Bley, Christopher J.; Thierbach, Karsten Science, Vol. 350, Issue 6256 https://doi.org/10.1126/science.aac9176	journal	August 2015
A Conserved Coatomer-related Complex Containing Sec13 and Seh1 Dynamically Associates With the Vacuole in Saccharomyces cerevisiae Dokudovskaya, Svetlana; Waharte, Francois; Schlessinger, Avner Molecular & Cellular Proteomics, Vol. 10, Issue 6 https://doi.org/10.1074/mcp.M110.006478	journal	March 2011
Structure–function mapping of a heptameric module in the nuclear pore complex Fernandez-Martinez, Javier; Phillips, Jeremy; Sekedat, Matthew D. The Journal of Cell Biology, Vol. 196, Issue 4 https://doi.org/10.1083/jcb.201109008	journal	February 2012
Atomic structure of the Y complex of the nuclear pore Kelley, Kotaro; Knockenhauer, Kevin E.; Kabachinski, Greg Nature Structural & Molecular Biology, Vol. 22, Issue 5 https://doi.org/10.1038/nsmb.2998	journal	March 2015
Architecture of the nuclear pore complex coat Stuwe, T.; Correia, A. R.; Lin, D. H. Science, Vol. 347, Issue 6226 https://doi.org/10.1126/science.aaa4136	journal	February 2015
In situ structural analysis of the human nuclear pore complex von Appen, Alexander; Kosinski, Jan; Sparks, Lenore Nature, Vol. 526, Issue 7571 https://doi.org/10.1038/nature15381	journal	September 2015
[20] Processing of X-ray diffraction data collected in oscillation mode Otwinowski, Zbyszek; Minor, Wladek Macromolecular Crystallography Part A, 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X	book	January 1997
Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex Onischenko, Evgeny; Tang, Jeffrey H.; Andersen, Kasper R. Cell, Vol. 171, Issue 4 https://doi.org/10.1016/j.cell.2017.09.033	journal	November 2017
Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy Rajoo, Sasikumar; Vallotton, Pascal; Onischenko, Evgeny Proceedings of the National Academy of Sciences, Vol. 115, Issue 17 https://doi.org/10.1073/pnas.1719398115	journal	April 2018
COPII — a flexible vesicle formation system Miller, Elizabeth A.; Schekman, Randy Current Opinion in Cell Biology, Vol. 25, Issue 4 https://doi.org/10.1016/j.ceb.2013.04.005	journal	August 2013
A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity Ling, Jingjing; Cheloha, Ross W.; McCaul, Nicholas Molecular Immunology, Vol. 114 https://doi.org/10.1016/j.molimm.2019.08.008	journal	October 2019
The Structure Inventory of the Nuclear Pore Complex Schwartz, Thomas U. Journal of Molecular Biology, Vol. 428, Issue 10 https://doi.org/10.1016/j.jmb.2016.03.015	journal	May 2016

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