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The mechanism of OTUB1-mediated inhibition of ubiquitination

Abstract

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin1. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A2 (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13Ub thiolester4. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding4, are required for binding to UBC13Ub and inhibition of K63Ub synthesis5. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1–UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer6,7. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes4 in a non-catalytic manner.

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Figure 1: Allosteric regulation of OTUB1 by ubiquitin.
Figure 2: Structure OTUB1–UBC13 and OTUB1–Ubal–UBC13 DCA Ub.
Figure 3: Conformational changes in the OTU domain triggered by Ubal binding.
Figure 4: OTUB1 N-terminal arm and the mechanism of E2 inhibition.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and diffraction amplitudes are deposited in the Protein Data Bank under accession numbers 4DHI (worm OTUB1–UBC13), 4DHJ (worm OTUB1–Ubal–UBC13DCAUb) and 4DHZ (human/worm OTUB1–Ubal– UBC13DCAUb).

References

  1. Al-Hakim, A. et al. The ubiquitous role of ubiquitin in the DNA damage response. DNA Repair (Amst.) 9, 1229–1240 (2010)

    Article  CAS  Google Scholar 

  2. Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000)

    Article  CAS  Google Scholar 

  3. Stewart, G. S. et al. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136, 420–434 (2009)

    Article  CAS  Google Scholar 

  4. Nakada, S. et al. Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1. Nature 466, 941–946 (2010)

    Article  ADS  CAS  Google Scholar 

  5. Wang, T. et al. Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. J. Mol. Biol. 386, 1011–1023 (2009)

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

  7. 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  Google Scholar 

  8. Messick, T. E. et al. Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein. J. Biol. Chem. 283, 11038–11049 (2008)

    Article  CAS  Google Scholar 

  9. Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009)

    Article  CAS  Google Scholar 

  10. Hofmann, R. M. & Pickart, C. M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999)

    Article  CAS  Google Scholar 

  11. Yin, L., Krantz, B., Russell, N. S., Deshpande, S. & Wilkinson, K. D. Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation. Biochemistry 39, 10001–10010 (2000)

    Article  CAS  Google Scholar 

  12. James, T. W. et al. Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease. Proc. Natl Acad. Sci. USA 108, 2222–2227 (2011)

    Article  ADS  CAS  Google Scholar 

  13. Capodagli, G. C. et al. Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin. J. Virol. 85, 3621–3630 (2011)

    Article  CAS  Google Scholar 

  14. Akutsu, M., Ye, Y., Virdee, S., Chin, J. W. & Komander, D. Molecular basis for ubiquitin and ISG15 cross-reactivity in viral ovarian tumor domains. Proc. Natl Acad. Sci. USA 108, 2228–2233 (2011)

    Article  ADS  CAS  Google Scholar 

  15. Sato, Y. et al. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80. EMBO J. 28, 2461–2468 (2009)

    Article  CAS  Google Scholar 

  16. Moraes, T. F. et al. Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2–hUbc13. Nature Struct. Biol. 8, 669–673 (2001)

    Article  CAS  Google Scholar 

  17. Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M. & Wolberger, C. Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nature Struct. Mol. Biol. 13, 915–920 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. McKenna, S. et al. An NMR-based model of the ubiquitin-bound human ubiquitin conjugation complex Mms2·Ubc13. The structural basis for lysine 63 chain catalysis. J. Biol. Chem. 278, 13151–13158 (2003)

    Article  CAS  Google Scholar 

  20. Yin, Q. et al. E2 interaction and dimerization in the crystal structure of TRAF6. Nature Struct. Mol. Biol. 16, 658–666 (2009)

    Article  CAS  Google Scholar 

  21. Berndsen, C. E. & Wolberger, C. A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins. Anal. Biochem. 418, 102–110 (2011)

    Article  CAS  Google Scholar 

  22. Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005)

    Article  CAS  Google Scholar 

  23. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271–281 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  30. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

Download references

Acknowledgements

We thank E. Henderson for generating the human/worm OTUB1 clone and C. Berndsen, A. DiBello, A. Datta and M. Bianchet for discussions. GM/CA-CAT has been funded in whole or in part with funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357.

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Authors

Contributions

R.W. and C.W. designed the experiments and R.W. performed all biochemical experiments. Cloning, expression and protein purification were carried out by X.Z., T.W. and R.W. Complexes were prepared for crystallization and crystals were grown by X.Z. and R.W.; R.W. determined the crystal structure with guidance from C.W.; R.W. and C.W. wrote the manuscript.

Corresponding author

Correspondence to Cynthia Wolberger.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-13 with legends and Supplementary Table 1. (PDF 1423 kb)

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Wiener, R., Zhang, X., Wang, T. et al. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483, 618–622 (2012). https://doi.org/10.1038/nature10911

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