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Atomic structure of the Y complex of the nuclear pore

Nature Structural & Molecular Biology volume 22, pages 425–431 (2015)Cite this article

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Abstract

The nuclear pore complex (NPC) is the principal gateway for transport into and out of the nucleus. Selectivity is achieved through the hydrogel-like core of the NPC. The structural integrity of the NPC depends on ~15 architectural proteins, which are organized in distinct subcomplexes to form the >40-MDa ring-like structure. Here we present the 4.1-Å crystal structure of a heterotetrameric core element ('hub') of the Y complex, the essential NPC building block, from Myceliophthora thermophila. Using the hub structure together with known Y-complex fragments, we built the entire ~0.5-MDa Y complex. Our data reveal that the conserved core of the Y complex has six rather than seven members. Evolutionarily distant Y-complex assemblies share a conserved core that is very similar in shape and dimension, thus suggesting that there are closely related architectural codes for constructing the NPC in all eukaryotes.

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Figure 1: Structure of the M. thermophila Y-complex hub at 4.1-Å resolution.
Figure 2: Fitness analysis of hub interactions.
Figure 3: Composite high-resolution structure of the Y complex.
Figure 4: Comparison of the X-ray–based, composite Y-complex structure with published 3D EM reconstruction structures.
Figure 5: Flexibility of the Y complex.
Figure 6: Fitting of the composite H. sapiens Y complex into the cryo-ET map of the entire NPC.

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References

  1. Hetzer, M.W. & Wente, S.R. Border control at the nucleus: biogenesis and organization of the nuclear membrane and pore complexes. Dev. Cell 17, 606–616 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Strambio-De-Castillia, C., Niepel, M. & Rout, M.P. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat. Rev. Mol. Cell Biol. 11, 490–501 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Brohawn, S.G., Partridge, J.R., Whittle, J.R.R. & Schwartz, T.U. The nuclear pore complex has entered the atomic age. Structure 17, 1156–1168 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Reichelt, R. et al. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J. Cell Biol. 110, 883–894 (1990).

    Article  CAS  PubMed  Google Scholar 

  5. Yang, Q., Rout, M.P. & Akey, C.W. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Mol. Cell 1, 223–234 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Rout, M.P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Walther, T.C. et al. The conserved Nup107–160 complex is critical for nuclear pore complex assembly. Cell 113, 195–206 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Harel, A. et al. Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores. Mol. Cell 11, 853–864 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Lutzmann, M., Kunze, R., Buerer, A., Aebi, U. & Hurt, E. Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. EMBO J. 21, 387–397 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Belgareh, N. et al. An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. J. Cell Biol. 154, 1147–1160 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vasu, S. et al. Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export. J. Cell Biol. 155, 339–354 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Neumann, N., Lundin, D. & Poole, A.M. Comparative genomic evidence for a complete nuclear pore complex in the last eukaryotic common ancestor. PLoS ONE 5, e13241 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Vollmer, B. & Antonin, W. The diverse roles of the Nup93/Nic96 complex proteins: structural scaffolds of the nuclear pore complex with additional cellular functions. Biol. Chem. 395, 515–528 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Alber, F. et al. The molecular architecture of the nuclear pore complex. Nature 450, 695–701 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Bui, K.H. et al. Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233–1243 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Berke, I.C., Boehmer, T., Blobel, G. & Schwartz, T.U. Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex. J. Cell Biol. 167, 591–597 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Boehmer, T., Jeudy, S., Berke, I.C. & Schwartz, T.U. Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex. Mol. Cell 30, 721–731 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brohawn, S.G., Leksa, N.C., Spear, E.D., Rajashankar, K.R. & Schwartz, T.U. Structural evidence for common ancestry of the nuclear pore complex and vesicle coats. Science 322, 1369–1373 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Brohawn, S.G. & Schwartz, T.U. Molecular architecture of the Nup84–Nup145C–Sec13 edge element in the nuclear pore complex lattice. Nat. Struct. Mol. Biol. 16, 1173–1177 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Leksa, N.C., Brohawn, S.G. & Schwartz, T.U. The structure of the scaffold nucleoporin Nup120 reveals a new and unexpected domain architecture. Structure 17, 1082–1091 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Whittle, J.R.R. & Schwartz, T.U. Architectural nucleoporins Nup157/170 and Nup133 are structurally related and descend from a second ancestral element. J. Biol. Chem. 284, 28442–28452 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bilokapic, S. & Schwartz, T.U. Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex. Proc. Natl. Acad. Sci. USA 109, 15241–15246 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hsia, K.-C., Stavropoulos, P., Blobel, G. & Hoelz, A. Architecture of a coat for the nuclear pore membrane. Cell 131, 1313–1326 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Debler, E.W. et al. A fence-like coat for the nuclear pore membrane. Mol. Cell 32, 815–826 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Nagy, V. et al. Structure of a trimeric nucleoporin complex reveals alternate oligomerization states. Proc. Natl. Acad. Sci. USA 106, 17693–17698 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Seo, H.-S. et al. Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex. Proc. Natl. Acad. Sci. USA 106, 14281–14286 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sampathkumar, P. et al. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex. Proteins 79, 1672–1677 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fernandez-Martinez, J. et al. Structure-function mapping of a heptameric module in the nuclear pore complex. J. Cell Biol. 196, 419–434 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Whittle, J.R.R. & Schwartz, T.U. Structure of the Sec13-Sec16 edge element, a template for assembly of the COPII vesicle coat. J. Cell Biol. 190, 347–361 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Thierbach, K. et al. Protein interfaces of the conserved Nup84 complex from Chaetomium thermophilum shown by crosslinking mass spectrometry and electron microscopy. Structure 21, 1672–1682 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Algret, R. et al. Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway. Mol. Cell. Proteomics 13, 2855–2870 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Siniossoglou, S. et al. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell 84, 265–275 (1996).

    Article  CAS  PubMed  Google Scholar 

  34. Stuwe, T. et al. Architecture of the nuclear pore complex coat. Science 347, 1148–1152 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kim, S.J. et al. Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex. Mol. Cell. Proteomics 13, 2911–2926 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Liu, X., Mitchell, J.M., Wozniak, R.W., Blobel, G. & Fan, J. Structural evolution of the membrane-coating module of the nuclear pore complex. Proc. Natl. Acad. Sci. USA 109, 16498–16503 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kampmann, M. & Blobel, G. Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex. Nat. Struct. Mol. Biol. 16, 782–788 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Süel, K.E., Cansizoglu, A.E. & Chook, Y.M. Atomic resolution structures in nuclear transport. Methods 39, 342–355 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Cook, A., Bono, F., Jinek, M. & Conti, E. Structural biology of nucleocytoplasmic transport. Annu. Rev. Biochem. 76, 647–671 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013).

    CAS  PubMed  Google Scholar 

  41. Ori, A. et al. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol. Syst. Biol. 9, 648 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Cronshaw, J.M., Krutchinsky, A.N., Zhang, W., Chait, B.T. & Matunis, M.J. Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915–927 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Andersen, K.R., Leksa, N.C. & Schwartz, T.U. Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81, 1857–1861 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  47. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Terwilliger, T.C. et al. phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta. J. Struct. Funct. Genomics 13, 81–90 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  52. Jeudy, S. & Schwartz, T.U. Crystal structure of nucleoporin Nic96 reveals a novel, intricate helical domain architecture. J. Biol. Chem. 282, 34904–34912 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Schrader, N. et al. Structural basis of the nic96 subcomplex organization in the nuclear pore channel. Mol. Cell 29, 46–55 (2008).

    Article  CAS  PubMed  Google Scholar 

  54. Kelley, L.A. & Sternberg, M.J.E. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).

    Article  CAS  PubMed  Google Scholar 

  55. Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Ciccarelli, F.D. et al. Toward automatic reconstruction of a highly resolved tree of life. Science 311, 1283–1287 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Fritz-Laylin, L.K. et al. The genome of Naegleria gruberi illuminates early eukaryotic versatility. Cell 140, 631–642 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Waterhouse, A.M. et al. Jalview Version 2: a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The X-ray crystallography was conducted at the Advanced Photon Source Northeastern Collaborative Access Team (APS NE-CAT) beamlines, which are supported by award GM103403 from the US National Institute of General Medical Sciences, US National Institutes of Health (NIH). Use of the APS is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract DE-AC02-06CH11357. We thank K. Rajashankar (APS NE-CAT) for help in phasing the structure; E. Brignole (MIT) for help with generating the cryo-ET consensus map; and L. Berchowitz and A. Amon (MIT) for help with the in vivo fitness analysis. Research was supported by the US NIH under grants R01GM77537 (T.U.S.) and T32GM007287 (K.K. and K.E.K.) and the US National Science Foundation Graduate Research Fellowship under grant 1122374 (K.E.K.).

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  1. Kotaro Kelley and Kevin E Knockenhauer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA

    Kotaro Kelley, Kevin E Knockenhauer, Greg Kabachinski & Thomas U Schwartz

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  1. Kotaro Kelley
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Contributions

T.U.S., K.K. and K.E.K. designed the study. K.K. and K.E.K. performed the experiments. K.K., K.E.K. and T.U.S. analyzed the data. G.K. performed and analyzed the fitness tests. K.K., K.E.K., G.K. and T.U.S. interpreted the structure and wrote the manuscript.

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Correspondence to Thomas U Schwartz.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Electron density map for hub structure.

Final 2Fo-Fc electron density map for the hub contoured at 1.5 σ, shown in the same view as Fig. 1b,c. (Inset, top) zoom-in on the interaction region of the hub. (Inset, bottom) the hub structure is placed into the electron density.

Supplementary Figure 2 Superposition of M. thermophila and S. cerevisiae ACE1 proteins of the hub.

(a) Overlay of mtNup85 (orange) and scNup85 (gray) reveals structural conservation, despite low (14%) sequence identity. N and C termini of the mtNups are labeled. (b) 90° rotation of the superposed structures. (c) Overlay of mtNup145C (cyan) and scNup145C (gray) (20% identity). (d) 90° rotation of the superposed structures.

Supplementary Figure 3 mtNup85 lacks an Seh1-binding motif.

(a) mtNup85, where the structural element N-terminal of α1 is an additional helix, α0 (green). The crown and trunk elements, which align to the solved scNup85 fragment, are shown in gray. The tail element, not solved in S. cerevisiae, is shown in orange. (b) scNup85, aligned to mtNup85, where the Seh1 insertion blade, β7 (green), is N-terminal to α1. Seh1 is labeled and scNup85 is shown in gray.

Supplementary Figure 4 Composite Y-complex structure generated through overlapping crystal-structure fragments.

Overlapping elements between the hub and previously solved structures are shown in green. Gray elements are non-redundant crystal structures used to generate the composite. The only portion of the composite that is modeled is a four-helix bundle in Nup84 (blue).

Supplementary Figure 5 Fitting of the composite H. sapiens Y complex into an inner-ring position in the 3D EM tomography map suffers from significant steric clashes with the outer ring fits.

Inner ring fit (3rd top scoring solution) is colored green. One of the outer ring fits (1st solution) is colored gray and regions of steric clash with Nup133 of the inner ring fit are colored red. The arrow (top view) shows where the Y complex stem needs to move in order to match the position suggested by Bui et al.15 and avoid steric clashes with the adjacent outer ring. Such stem placement would involve rotation at or around the Nup96-Nup107 interface, which is unlikely due to the energetic cost of disrupting the complex’s hydrophobic core.

Supplementary information

Supplementary Figures and Text

Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Notes 1–4 (PDF 11769 kb)

Supplementary Data Set 1

Composite structure of the human Y complex (PDB 3055 kb)

Supplementary Data Set 2

Composite structure of the S. cerevisae Y complex (PDB 2882 kb)

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Kelley, K., Knockenhauer, K., Kabachinski, G. et al. Atomic structure of the Y complex of the nuclear pore. Nat Struct Mol Biol 22, 425–431 (2015). https://doi.org/10.1038/nsmb.2998

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