Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne

Nature Structural & Molecular Biology volume 17, pages 939–947 (2010)Cite this article

Subjects

Abstract

Ubiquitin is a versatile cellular signaling molecule that can form polymers of eight different linkages, and individual linkage types have been associated with distinct cellular functions. Though little is currently known about Lys11-linked ubiquitin chains, recent data indicate that they may be as abundant as Lys48 linkages and may be involved in vital cellular processes. Here we report the generation of Lys11-linked polyubiquitin in vitro, for which the Lys11-specific E2 enzyme UBE2S was fused to a ubiquitin binding domain. Crystallographic and NMR analyses of Lys11-linked diubiquitin reveal that Lys11-linked chains adopt compact conformations in which Ile44 is solvent exposed. Furthermore, we identify the OTU family deubiquitinase Cezanne as the first deubiquitinase with Lys11-linkage preference. Our data highlight the intrinsic specificity of the ubiquitin system that extends to Lys11-linked chains and emphasize that differentially linked polyubiquitin chains must be regarded as independent post-translational modifications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: UBE2S is a Lys11-specific E2 enzyme.
Figure 2: Assembly of Lys11-linked diubiquitin.
Figure 3: Assembly of Lys11-linked tetraubiquitin.
Figure 4: NMR solution studies of Lys11-linked diubiquitin.
Figure 5: Crystal structure of Lys11-linked diubiquitin.
Figure 6: Hydrolysis of Lys11 linkages by DUBs.
Figure 7: Cezanne cleaves Lys11-linkages preferentially.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Komander, D. The emerging complexity of protein ubiquitination. Biochem. Soc. Trans. 37, 937–953 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Chen, Z.J. & Sun, L.J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Dye, B.T. & Schulman, B.A. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 36, 131–150 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. 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 

  8. 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  PubMed  Google Scholar 

  9. Chen, Z. & Pickart, C.M. A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. J. Biol. Chem. 265, 21835–21842 (1990).

    CAS  PubMed  Google Scholar 

  10. Baboshina, O.V. & Haas, A.L. Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5. J. Biol. Chem. 271, 2823–2831 (1996).

    Article  CAS  PubMed  Google Scholar 

  11. Jin, L., Williamson, A., Banerjee, S., Philipp, I. & Rape, M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133, 653–665 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Williamson, A. et al. Identification of a physiological E2 module for the human anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 106, 18213–18218 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Garnett, M.J. et al. UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit. Nat. Cell Biol. 11, 1363–1369 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wu, T. et al. UBE2S drives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 107, 1355–1360 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rodrigo-Brenni, M.C. & Morgan, D.O. Sequential E2s drive polyubiquitin chain assembly on APC targets. Cell 130, 127–139 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Alexandru, G. et al. UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover. Cell 134, 804–816 (2008).

    Article  CAS  PubMed  PubMed Central  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. Nat. Struct. Mol. Biol. 13, 915–920 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Reyes-Turcu, F.E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. McCullough, J., Clague, M.J. & Urbe, S. AMSH is an endosome-associated ubiquitin isopeptidase. J. Cell Biol. 166, 487–492 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Varadan, R. et al. Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling. J. Biol. Chem. 279, 7055–7063 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Varadan, R., Walker, O., Pickart, C. & Fushman, D. Structural properties of polyubiquitin chains in solution. J. Mol. Biol. 324, 637–647 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Tenno, T. et al. Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains. Genes Cells 9, 865–875 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Zhang, N. et al. Structure of the s5a:k48-linked diubiquitin complex and its interactions with rpn13. Mol. Cell 35, 280–290 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Varadan, R., Assfalg, M., Raasi, S., Pickart, C. & Fushman, D. Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. Mol. Cell 18, 687–698 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Raasi, S., Varadan, R., Fushman, D. & Pickart, C.M. Diverse polyubiquitin interaction properties of ubiquitin-associated domains. Nat. Struct. Mol. Biol. 12, 708–714 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Komander, D., Clague, M.J. & Urbe, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Komander, D. et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Komander, D. et al. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol. Cell 29, 451–464 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Tran, H., Hamada, F., Schwarz-Romond, T. & Bienz, M. Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains. Genes Dev. 22, 528–542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Komander, D. & Barford, D. Structure of the A20 OTU domain and mechanistic insights into deubiquitination. Biochem. J. 409, 77–85 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Winborn, B.J. et al. The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains. J. Biol. Chem. 283, 26436–26443 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hymowitz, S.G. & Wertz, I.E. A20: from ubiquitin editing to tumour suppression. Nat. Rev. Cancer 10, 332–341 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Skaug, B., Jiang, X. & Chen, Z.J. The role of ubiquitin in NF-κB regulatory pathways. Annu. Rev. Biochem. 78, 769–796 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Enesa, K. et al. NF-κB suppression by the deubiquitinating enzyme Cezanne: a novel negative feedback loop in pro-inflammatory signaling. J. Biol. Chem. 283, 7036–7045 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Fushman, D. & Walker, O. Exploring the linkage dependence of polyubiquitin conformations using molecular modeling. J. Mol. Biol. 395, 803–814 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. van Wijk, S.J. et al. A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system. Mol. Syst. Biol. 5, 295 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Markson, G. et al. Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network. Genome Res. 19, 1905–1911 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Xia, Z.P. et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 461, 114–119 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Nijman, S.M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Neidhardt, F.C., Bloch, P.L. & Smith, D.F. Culture medium for enterobacteria. J. Bacteriol. 119, 736–747 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Pickart, C.M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Mori, S., Abeygunawardana, C., Johnson, M.O. & van Zijl, P.C. Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. J. Magn. Reson. B. 108, 94–98 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Hajduk, P.J. et al. NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein. J. Med. Chem. 40, 3144–3150 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  PubMed  Google Scholar 

  46. Vagin, A. & Teplyakov, A. An approach to multi-copy search in molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 56, 1622–1624 (2000).

    Article  CAS  PubMed  Google Scholar 

  47. Vijay-Kumar, S., Bugg, C.E. & Cook, W.J. Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531–544 (1987).

    Article  CAS  PubMed  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. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank E. Stephens, S. Peak-Chew and F. Begum for MS analysis, M. Allen for help with isotope labeling, T. Nicholson (ENZO LifeSciences) for providing ubiquitin mutants, P. Cohen (Univ. of Dundee), S. Urbe, M. Clague (Univ. of Liverpool), K.D. Wilkinson (Emory Univ.), S. Todi (Univ. Of Michigan) and B. Kessler (Oxford Univ.) for constructs, T. Tenno (Kobe Univ.) and M. Shirakawa (Kyoto Univ.) for kindly providing NMR data for Lys48- and Lys63-linked diubiquitin and members of the Komander lab, especially Y. Kulathu, Y. Ye, M. Akutsu, S. Virdee and A. Eslambolchi for providing reagents and many discussions. A.B. is a MRC Career Development Fellow.

Author information

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Anja Bremm, Stefan M V Freund & David Komander

Authors
  1. Anja Bremm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan M V Freund
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Komander
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.B. designed, performed and analyzed all experiments; S.M.V.F. performed solution measurements and analyzed the data; D.K. designed research, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to David Komander.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1, Supplementary Discussion, Supplementary Methods and Supplementary Data (PDF 5381 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bremm, A., Freund, S. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat Struct Mol Biol 17, 939–947 (2010). https://doi.org/10.1038/nsmb.1873

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1873

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing