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

Nature Chemical Biology volume 10, pages 156–163 (2014)Cite this article

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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.

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Figure 1: CC0651 potentiates the interaction between Cdc34A and [15N]ubiquitin.
Figure 2: CC0651 potentiates the interaction between [15N]-Cdc34A and ubiquitin.
Figure 3: Crystal structure of a CC0651-Cdc34A-ubiquitin complex.
Figure 4: Mutations in Cdc34A that disrupt interaction with ubiquitin impair sensitivity to CC0651.
Figure 5: Effect of CC0651 and ubiquitin on the interaction of [15N]Rbx1 with Cdc34A.

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References

  1. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    CAS  PubMed  Google Scholar 

  2. Hoeller, D. & Dikic, I. Targeting the ubiquitin system in cancer therapy. Nature 458, 438–444 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Soucy, T.A., Dick, L.R., Smith, P.G., Milhollen, M.A. & Brownell, J.E. The NEDD8 conjugation pathway and its relevance in cancer biology and therapy. Genes Cancer 1, 708–716 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. MacGurn, J.A., Hsu, P.C. & Emr, S.D. Ubiquitin and membrane protein turnover: from cradle to grave. Annu. Rev. Biochem. 81, 231–259 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Matyskiela, M.E. & Martin, A. Design principles of a universal protein degradation machine. J. Mol. Biol. 425, 199–213 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Ye, Y. & Rape, M. Building ubiquitin chains: E2 enzymes at work. Nat. Rev. Mol. Cell Biol. 10, 755–764 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wenzel, D.M., Stoll, K.E. & Klevit, R.E. E2s: structurally economical and functionally replete. Biochem. J. 433, 31–42 (2011).

    Article  CAS  PubMed  Google Scholar 

  8. Saha, A., Lewis, S., Kleiger, G., Kuhlman, B. & Deshaies, R.J. Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Mol. Cell 42, 75–83 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wickliffe, K.E., Lorenz, S., Wemmer, D.E., Kuriyan, J. & Rape, M. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pruneda, J.N. et al. Structure of an E3:E2-Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Deshaies, R.J. & Joazeiro, C.A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Emanuele, M.J. et al. Global identification of modular cullin-RING ligase substrates. Cell 147, 459–474 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liao, H. et al. Quantitative proteomic analysis of cellular protein modulation upon inhibition of the NEDD8-activating enzyme by MLN4924. Mol. Cell Proteomics 10, M111.009183 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Kleiger, G., Saha, A., Lewis, S., Kuhlman, B. & Deshaies, R.J. Rapid E2–E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell 139, 957–968 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Duda, D.M. et al. Structure of a glomulin-RBX1–CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface. Mol. Cell 47, 371–382 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Brownell, J.E. et al. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Mol. Cell 37, 102–111 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Ceccarelli, D.F. et al. An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell 145, 1075–1087 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Spratt, D.E. & Shaw, G.S. Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin. J. Mol. Biol. 407, 425–438 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Choi, Y.S. et al. The human Cdc34 carboxyl terminus contains a non-covalent ubiquitin binding activity that contributes to SCF-dependent ubiquitination. J. Biol. Chem. 285, 17754–17762 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hamilton, K.S. et al. Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail. Structure 9, 897–904 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Plechanovová, A., Jaffray, E.G., Tatham, M.H., Naismith, J.H. & Hay, R.T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Dou, H., Buetow, L., Sibbet, G.J., Cameron, K. & Huang, D.T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat. Struct. Mol. Biol. 19, 876–883 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Spratt, D.E., Wu, K., Kovacev, J., Pan, Z.Q. & Shaw, G.S. Selective recruitment of an E2–ubiquitin complex by an E3 ubiquitin ligase. J. Biol. Chem. 287, 17374–17385 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Capili, A.D. & Lima, C.D. Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction. J. Mol. Biol. 369, 608–618 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Knipscheer, P., van Dijk, W.J., Olsen, J.V., Mann, M. & Sixma, T.K. Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation. EMBO J. 26, 2797–2807 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Seol, J.H. et al. Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev. 13, 1614–1626 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Saha, A. & Deshaies, R.J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bracher, P.J., Snyder, P.W., Bohall, B.R. & Whitesides, G.M. The relative rates of thiol-thioester exchange and hydrolysis for alkyl and aryl thioalkanoates in water. Orig. Life Evol. Biosph. 41, 399–412 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Song, J. et al. Stability of thioester intermediates in ubiquitin-like modifications. Protein Sci. 18, 2492–2499 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pierce, N.W., Kleiger, G., Shan, S.O. & Deshaies, R.J. Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462, 615–619 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Burroughs, A.M., Jaffee, M., Iyer, L.M. & Aravind, L. Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation. J. Struct. Biol. 162, 205–218 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Thiel, P., Kaiser, M. & Ottmann, C. Small-molecule stabilization of protein-protein interactions: an underestimated concept in drug discovery? Angew. Chem. Int. Edn Engl. 51, 2012–2018 (2012).

    Article  CAS  Google Scholar 

  35. Schulman, B.A. & Harper, J.W. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat. Rev. Mol. Cell Biol. 10, 319–331 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pashkova, N. et al. WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins. Mol. Cell 40, 433–443 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Reyes-Turcu, F.E., Ventii, K.H. & Wilkinson, K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Husnjak, K. & Dikic, I. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Ernst, A. et al. A strategy for modulation of enzymes in the ubiquitin system. Science 339, 590–595 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Zheng, N., Wang, P., Jeffrey, P.D. & Pavletich, N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102, 533–539 (2000).

    Article  CAS  PubMed  Google Scholar 

  41. Tang, X. et al. Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination. Cell 129, 1165–1176 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  43. Johnson, B.A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004).

    CAS  PubMed  Google Scholar 

  44. Wang, A.C., Grzesiek, S., Tschudin, R., Lodi, P.J. & Bax, A. Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N. J. Biomol. NMR 5, 376–382 (1995).

    CAS  PubMed  Google Scholar 

  45. Fielding, L. NMR methods for the determination of protein–ligand dissociation constants. Prog. Nucl. Mag. Reson. Spectrosc. 51, 219–242 (2007).

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

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Qian, X. et al. Discovery of the first potent and selective inhibitor of centromere-associated protein E: GSK923295. ACS Med. Chem. Lett. 1, 30–34 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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.

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Author notes

  1. Hao Huang, Derek F Ceccarelli and Stephen Orlicky: These authors contributed equally to this work.

Authors and 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.

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Correspondence to Mike Tyers or Frank Sicheri.

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Competing interests

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

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Huang, H., Ceccarelli, D., Orlicky, S. et al. E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin. Nat Chem Biol 10, 156–163 (2014). https://doi.org/10.1038/nchembio.1412

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