Article | Published:

Crystal structure of the human COP9 signalosome

Nature volume 512, pages 161165 (14 August 2014) | Download Citation

Abstract

Ubiquitination is a crucial cellular signalling process, and is controlled on multiple levels. Cullin–RING E3 ubiquitin ligases (CRLs) are regulated by the eight-subunit COP9 signalosome (CSN). CSN inactivates CRLs by removing their covalently attached activator, NEDD8. NEDD8 cleavage by CSN is catalysed by CSN5, a Zn2+-dependent isopeptidase that is inactive in isolation. Here we present the crystal structure of the entire 350-kDa human CSN holoenzyme at 3.8 Å resolution, detailing the molecular architecture of the complex. CSN has two organizational centres: a horseshoe-shaped ring created by its six proteasome lid–CSN–initiation factor 3 (PCI) domain proteins, and a large bundle formed by the carboxy-terminal α-helices of every subunit. CSN5 and its dimerization partner, CSN6, are intricately embedded at the core of the helical bundle. In the substrate-free holoenzyme, CSN5 is autoinhibited, which precludes access to the active site. We find that neddylated CRL binding to CSN is sensed by CSN4, and communicated to CSN5 with the assistance of CSN6, resulting in activation of the deneddylase.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 4D10 and 4D18 for two CSN unit cell variants, and 4D0P for CSN4.

References

  1. 1.

    , & Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development. Cell 78, 117–124 (1994)

  2. 2.

    & The COP9 signalosome. Annu. Rev. Cell Dev. Biol. 19, 261–286 (2003)

  3. 3.

    et al. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science 292, 1382–1385 (2001)

  4. 4.

    et al. Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Science 292, 1379–1382 (2001)

  5. 5.

    et al. The fission yeast COP9/signalosome is involved in cullin modification by ubiquitin-related Ned8p. BMC Biochem. 2, 7 (2001)

  6. 6.

    , & Building and remodelling Cullin–RING E3 ubiquitin ligases. EMBO Rep. 14, 1050–1061 (2013)

  7. 7.

    et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736 (2009)

  8. 8.

    et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298, 608–611 (2002)

  9. 9.

    et al. The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation. Cell 147, 1024–1039 (2011)

  10. 10.

    et al. Structural basis for a reciprocal regulation between SCF and CSN. Cell Rep. 2, 616–627 (2012)

  11. 11.

    , & Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism. J. Biol. Chem. 287, 29679–29689 (2012)

  12. 12.

    , & Structural assembly of cullin-RING ubiquitin ligase complexes. Curr. Opin. Struct. Biol. 20, 714–721 (2010)

  13. 13.

    , , , & The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1. Mol. Cell. Biol. 20, 8185–8197 (2000)

  14. 14.

    et al. A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination. Proc. Natl Acad. Sci. USA 97, 4579–4584 (2000)

  15. 15.

    et al. Nedd8 modification of Cul-1 activates SCFβTrCP-dependent ubiquitination of IκBα. Mol. Cell. Biol. 20, 2326–2333 (2000)

  16. 16.

    , & Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1–CUL1 complex to promote ubiquitin polymerization. J. Biol. Chem. 275, 32317–32324 (2000)

  17. 17.

    , , & Modification of cullin-1 by ubiquitin-like protein Nedd8 enhances the activity of SCFskp2 toward p27kip1. Biochem. Biophys. Res. Commun. 270, 1093–1096 (2000)

  18. 18.

    et al. Symmetrical modularity of the COP9 signalosome complex suggests its multifunctionality. Structure 17, 31–40 (2009)

  19. 19.

    , & The COP9 signalosome: more than a protease. Trends Biochem. Sci. 33, 592–600 (2008)

  20. 20.

    et al. Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers. J. Clin. Invest. 121, 851–865 (2011)

  21. 21.

    , , & Roles of COP9 signalosome in cancer. Cell Cycle 10, 3057–3066 (2011)

  22. 22.

    et al. Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1. Proc. Natl Acad. Sci. USA 110, 1273–1278 (2013)

  23. 23.

    , , & Structural insights into the COP9 signalosome and its common architecture with the 26S proteasome lid and eIF3. Structure 18, 518–527 (2010)

  24. 24.

    et al. Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl Acad. Sci. USA 109, 14870–14875 (2012)

  25. 25.

    et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012)

  26. 26.

    et al. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc. Natl Acad. Sci. USA 109, 1380–1387 (2012)

  27. 27.

    , & Molecular model of the human 26S proteasome. Mol. Cell 46, 54–66 (2012)

  28. 28.

    , & Conformational switching of the 26S proteasome enables substrate degradation. Nature Struct. Mol. Biol. 20, 781–788 (2013)

  29. 29.

    , , , & Structural roles for human translation factor eIF3 in initiation of protein synthesis. Science 310, 1513–1515 (2005)

  30. 30.

    et al. Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3). Proc. Natl Acad. Sci. USA 108, 20473–20478 (2011)

  31. 31.

    et al. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153, 1108–1119 (2013)

  32. 32.

    et al. Architecture of human translation initiation factor 3. Structure 21, 920–928 (2013)

  33. 33.

    et al. Crystal structure of human eIF3k, the first structure of eIF3 subunits. J. Biol. Chem. 279, 34983–34990 (2004)

  34. 34.

    et al. The Arabidopsis COP9 signalosome subunit 7 is a model PCI domain protein with subdomains involved in COP9 signalosome assembly. Plant Cell 20, 2815–2834 (2008)

  35. 35.

    et al. The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together. Proc. Natl Acad. Sci. USA 109, 149–154 (2012)

  36. 36.

    et al. The crystal structure of the MPN domain from the COP9 signalosome subunit CSN6. FEBS Lett. 586, 1147–1153 (2012)

  37. 37.

    et al. Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation. Biochem. J. 448, 55–65 (2012)

  38. 38.

    et al. Crystal structure and versatile functional roles of the COP9 signalosome subunit 1. Proc. Natl Acad. Sci. USA 110, 11845–11850 (2013)

  39. 39.

    et al. Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes. Nucleic Acids Res. 42, 4123–4139 (2014)

  40. 40.

    , & Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation. Nature Struct. Mol. Biol. 21, 220–227 (2014)

  41. 41.

    et al. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11. Proc. Natl Acad. Sci. USA 111, 2984–2989 (2014)

  42. 42.

    & The PCI domain: a common theme in three multiprotein complexes. Trends Biochem. Sci. 23, 204–205 (1998)

  43. 43.

    & Structural biology of the PCI-protein fold. BioArchitecture 2, 118–123 (2012)

  44. 44.

    , , & Structure of the Jab1/MPN domain and its implications for proteasome function. Biochemistry 42, 11460–11465 (2003)

  45. 45.

    , & JAMM: a metalloprotease-like zinc site in the proteasome and signalosome. PLoS Biol. 2, e2 (2004)

  46. 46.

    et al. The minimal deneddylase core of the COP9 signalosome excludes the Csn6 MPN domain. PLoS ONE 7, e43980 (2012)

  47. 47.

    et al. Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains. Nature 455, 358–362 (2008)

  48. 48.

    et al. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008)

  49. 49.

    et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002)

  50. 50.

    et al. Structural basis of the Cks1-dependent recognition of p27Kip1 by the SCFSkp2 ubiquitin ligase. Mol. Cell 20, 9–19 (2005)

  51. 51.

    , & Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells. Protein Sci. 16, 2023–2029 (2007)

  52. 52.

    , & The subunit 1 of the COP9 signalosome suppresses gene expression through its N-terminal domain and incorporates into the complex through the PCI domain. J. Mol. Biol. 305, 1–9 (2001)

  53. 53.

    , , , & Purification of the COP9 signalosome from porcine spleen, human cell lines, and Arabidopsis thaliana plants. Methods Enzymol. 398, 468–481 (2005)

  54. 54.

    XDS. Acta Crystallogr. D 66, 125–132 (2010)

  55. 55.

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

  56. 56.

    , , , & Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

  57. 57.

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

  58. 58.

    & Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

  59. 59.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  60. 60.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  61. 61.

    , , , & BUSTER Version 2.11.5 (Global Phasing, 2011)

  62. 62.

    et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

  63. 63.

    , & Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218–1222 (2010)

  64. 64.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

  65. 65.

    , & Low-resolution refinement tools in REFMAC5. Acta Crystallogr. D 68, 404–417 (2012)

  66. 66.

    & Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)

  67. 67.

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

  68. 68.

    et al. Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements. Assay Drug Dev. Technol. 3, 613–622 (2005)

  69. 69.

    , , & Formation of an intricate helical bundle dictates the assembly of the 26S proteasome lid. Structure 21, 1624–1635 (2013)

  70. 70.

    et al. Proenzyme structure and activation of astacin metallopeptidase. J. Biol. Chem. 285, 13958–13965 (2010)

  71. 71.

    , , , & Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases. Nature 358, 164–167 (1992)

  72. 72.

    , & PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 36, 2295–2300 (2008)

Download references

Acknowledgements

This work was supported by the Novartis Research Foundation, the Swiss National Science Foundation (31003A_144020) and the European Research Council (MoBa-CS #260481). G.M.L. is the recipient of a Novartis (NIBR) presidential postdoctoral fellowship. Part of this work was performed at beamline X06DA and X10SA of the Swiss Light Source. We thank H. Wu for sharing the A. thaliana CSN1 coordinates before publication; H. Gut, J. Keusch and M. Jones for support; and C. Vonrhein and the BUSTER development group, R. Read, A. McCoy, T. Terwilliger, R. Nicholls, R. Kingston and K. Diederichs for technical advice. We are also grateful for the assistance of B. Martoglio, R. Assenberg, C. Logel and I. Bechtold in developing the PT22 CSN assays.

Author information

Author notes

    • Gondichatnahalli M. Lingaraju
    •  & Richard D. Bunker

    These authors contributed equally to this work.

Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland

    • Gondichatnahalli M. Lingaraju
    • , Richard D. Bunker
    • , Simone Cavadini
    • , Daniel Hess
    • , Eric S. Fischer
    •  & Nicolas H. Thomä
  2. University of Basel, Petersplatz 10, 4003 Basel, Switzerland

    • Gondichatnahalli M. Lingaraju
    • , Richard D. Bunker
    • , Simone Cavadini
    • , Eric S. Fischer
    •  & Nicolas H. Thomä
  3. Novartis Pharma AG, Institutes for Biomedical Research, Novartis Campus, 4056 Basel, Switzerland

    • Ulrich Hassiepen
    •  & Martin Renatus

Authors

  1. Search for Gondichatnahalli M. Lingaraju in:

  2. Search for Richard D. Bunker in:

  3. Search for Simone Cavadini in:

  4. Search for Daniel Hess in:

  5. Search for Ulrich Hassiepen in:

  6. Search for Martin Renatus in:

  7. Search for Eric S. Fischer in:

  8. Search for Nicolas H. Thomä in:

Contributions

G.M.L., M.R. and N.H.T. initiated the project. G.M.L. established the purification methods for CSN and CSN4, produced most proteins, performed the gel-based assays, and obtained the crystals. G.M.L. improved the crystals with input from R.D.B. G.M.L. and R.D.B. collected the diffraction data. R.D.B. carried out the crystallographic analyses and interpreted the results. S.C. performed electron microscopy analysis. D.H. carried out protein analysis. E.S.F. developed the binding and activity assays with input from U.H.; E.S.F. performed the assays and analysed the results. N.H.T. supervised all aspects of the project and analysed the results. R.D.B. and N.H.T. wrote the manuscript with important contributions from G.M.L., M.R., E.S.F. and S.C.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Nicolas H. Thomä.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Data, a Supplementary Discussion and Supplementary References.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13566

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.