Article | Published:

E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin

Nature Chemical Biology volume 10, pages 156163 (2014) | Download Citation

Abstract

Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small-molecule inhibitor of the E2 ubiquitin-conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin-binding site. A structure of the ternary CC0651–Cdc34A–ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester without decreasing the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities.

  • Compound C20H21Cl2NO6

    (2R,3S,4S)-5-(3',5'-Dichloro-[1,1'-biphenyl]-4-yl)-2,3-dihydroxy-4-(2-methoxyacetamido)pentanoic acid

  • Compound C22H27NO6

    (2R,3S,4S)-5-(3',5'-Dimethyl-[1,1'-biphenyl]-4-yl)-2,3-dihydroxy-4-(2-methoxyacetamido)pentanoic acid

  • Compound C20H21F2NO6

    (2R,3S,4S)-5-(3',5'-Difluoro-[1,1'-biphenyl]-4-yl)-2,3-dihydroxy-4-(2-methoxyacetamido)pentanoic acid

  • Compound C22H27NO6

    (2R,3S,4S)-5-(3',4'-Dimethyl-[1,1'-biphenyl]-4-yl)-2,3-dihydroxy-4-(2-methoxyacetamido)pentanoic acid

  • Compound C23H29NO9

    (2R,3S,4S)-2,3-Dihydroxy-4-(2-methoxyacetamido)-5-(3',4',5'-trimethoxy-[1,1'-biphenyl]-4-yl)pentanoic acid

  • Compound C27H29NO7

    (2R,3S,4S)-5-(3'-(Benzyloxy)-[1,1'-biphenyl]-4-yl)-2,3-dihydroxy-4-(2-methoxyacetamido)pentanoic acid

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Acknowledgements

We thank A. Ernst, R. Deshaies, A. van der Sloot, R. Grunberg, J.-F. Lavallée, C. James, K. Chan and F. Mercurio for helpful discussions. We also thank I. Kourinov and staff at the Argonne National Laboratory for assistance with microdiffraction experiments at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, as supported by award RR-15301 from the National Center for Research Resources at the US National Institutes of Health (NIH) and contract DE-AC02-06CH11357 from the US Department of Energy. G.K. and A.Z. are funded by grants from the National Center for Research Resources (5P20RR016464-11) and the National Institute of General Medical Sciences (8 P20 GM103440-11) from the NIH. This work was supported by grants to F.S. and M.T. from the Canadian Institutes of Health Research (MOP-126129, MOP-57795); by an award from the Ministère de l'enseignement supérieur, de la recherche, de la science et de la technologie du Québec through Génome Québec to M.T.; by a Canadian Institutes of Health Research Postdoctoral Fellowship to D.J.S.-C.; by a Canada Research Chair in structural biology to F.S.; and by a Canada Research Chair in systems and synthetic biology to M.T.

Author information

Author notes

    • Hao Huang
    • , Derek F Ceccarelli
    •  & Stephen Orlicky

    These authors contributed equally to this work.

Affiliations

  1. Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.

    • Hao Huang
    • , Derek F Ceccarelli
    • , Stephen Orlicky
    •  & Frank Sicheri
  2. Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Québec, Canada.

    • Daniel J St-Cyr
    • , Serge Plamondon
    • , Anne Marinier
    •  & Mike Tyers
  3. Department of Chemistry, University of Nevada–Las Vegas, Las Vegas, Nevada, USA.

    • Amy Ziemba
    •  & Gary Kleiger
  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

    • Pankaj Garg
    • , Sachdev Sidhu
    •  & Frank Sicheri
  5. Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.

    • Pankaj Garg
    •  & Sachdev Sidhu
  6. School of Biological Sciences, University of Edinburgh, Edinburgh, UK.

    • Manfred Auer
  7. School of Biomedical Sciences, University of Edinburgh, Edinburgh, UK.

    • Manfred Auer
  8. Department of Chemistry, University of Montreal, Montreal, Québec, Canada.

    • Anne Marinier
  9. Department of Medicine, University of Montreal, Montreal, Québec, Canada.

    • Mike Tyers

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Contributions

H.H. performed NMR; D.F.C. performed X-ray crystallography; S.O. and A.Z. performed ubiquitination assays; P.G. generated protein reagents; S.P. and D.J.S.-C. synthesized CC0651 and its analogs; M.A., S.S., A.M., G.K., M.T. and F.S. designed experiments and interpreted results; and M.T. and F.S. wrote the manuscript with contributions from all other authors.

Competing interests

A provisional patent application for E2 enzyme inhibition has been filed, with M.T. and F.S. as inventors.

Corresponding authors

Correspondence to Mike Tyers or Frank Sicheri.

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–12.

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DOI

https://doi.org/10.1038/nchembio.1412

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