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:

An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains

This article has been updated

Abstract

Eight different types of ubiquitin linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are mainly unknown. We here reveal how the human ovarian tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin. A crystal structure of the extended catalytic domain reveals an unpredicted ankyrin repeat domain that precedes an A20-like catalytic core. NMR analysis identifies the ankyrin domain as a new ubiquitin-binding fold, which we have termed AnkUBD, and DUB assays in vitro and in vivo show that this domain is crucial for TRABID efficiency and linkage specificity. Our data are consistent with AnkUBD functioning as an enzymatic S1′ ubiquitin-binding site, which orients a ubiquitin chain so that Lys29 and Lys33 linkages are cleaved preferentially.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Structure and specificity of an extended TRABID OTU domain.
Figure 2: TRABID contains two ankyrin repeats with roles in ubiquitin binding.
Figure 3: A conserved hydrophobic surface on AnkUBD binds ubiquitin.
Figure 4: AnkUBD binds the ubiquitin hydrophobic patch.
Figure 5: Analysis of TRABID DUB activity.
Figure 6: Role of the NZF domains in cleaving longer ubiquitin chains.
Figure 7: In vivo DUB assay NZF and AnkUBD are essential for TRABID puncta.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

Change history

  • 29 February 2012

    In the version of Supplementary Figure 5b originally posted online, three images were incorrect: those for DMSO K27only Ub, MG132 K27only Ub and MG132 K29only Ub. The correct figure is now shown. The conclusions from this figure remain unchanged. These errors have been corrected in this file as of 29 February 2012.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series. EMBO Rep. 9, 536–542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 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 

  4. Dammer, E.B. et al. Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease. J. Biol. Chem. 286, 10457–10465 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang, M., Cheng, D., Peng, J. & Pickart, C.M. Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase. EMBO J. 25, 1710–1719 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hay-Koren, A., Caspi, M., Zilberberg, A. & Rosin-Arbesfeld, R. The EDD E3 ubiquitin ligase ubiquitinates and up-regulates β-catenin. Mol. Biol. Cell 22, 399–411 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chastagner, P., Israël, A. & Brou, C. Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep. 7, 1147–1153 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Al-Hakim, A.K. et al. Control of AMPK-related kinases by USP9X and atypical lysine(29)/lysine(33)-linked polyubiquitin chains. Biochem. J. 411, 249–260 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Johnson, E.S., Ma, P.C., Ota, I.M. & Varshavsky, A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Koegl, M. et al. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Virdee, S., Ye, Y., Nguyen, D.P., Komander, D. & Chin, J.W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750–757 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. El Oualid, F. et al. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Edn Engl. 49, 10149–10153 (2010).

    Article  CAS  Google Scholar 

  13. Kumar, K.S., Spasser, L., Erlich, L.A., Bavikar, S.N. & Brik, A. Total chemical synthesis of di-ubiquitin chains. Angew. Chem. Int. Edn Engl. 49, 9126–9131 (2010).

    Article  CAS  Google Scholar 

  14. Virdee, S. et al. Traceless and site-specific ubiquitination of recombinant proteins. J. Am. Chem. Soc. 133, 10708–10711 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cooper, E.M. et al. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1. EMBO J. 28, 621–631 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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 

  21. Bremm, A., Freund, S.M. & 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nanao, M.H. et al. Crystal structure of human otubain 2. EMBO Rep. 5, 783–788 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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 

  24. Lin, S.C. et al. Molecular basis for the unique deubiquitinating activity of the NF-κB inhibitor A20. J. Mol. Biol. 376, 526–540 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. 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  PubMed  PubMed Central  Google Scholar 

  26. 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  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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  PubMed  PubMed Central  Google Scholar 

  28. 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  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dikic, I., Wakatsuki, S. & Walters, K.J. Ubiquitin-binding domains—from structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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 

  31. Kayagaki, N. et al. DUBA: a deubiquitinase that regulates type I interferon production. Science 318, 1628–1632 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Li, S. et al. Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6. J. Biol. Chem. 285, 4291–4297 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Uchiyama, K. et al. VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo. J. Cell Biol. 159, 855–866 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ernst, R., Mueller, B., Ploegh, H.L. & Schlieker, C. The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER. Mol. Cell 36, 28–38 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 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 

  36. Evans, P.C. et al. Isolation and characterization of two novel A20-like proteins. Biochem. J. 357, 617–623 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li, J., Mahajan, A. & Tsai, M.D. Ankyrin repeat: a unique motif mediating protein-protein interactions. Biochemistry 45, 15168–15178 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  40. de Vries, S.J., van Dijk, M. & Bonvin, A.M. The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5, 883–897 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Mosavi, L.K., Cammett, T.J., Desrosiers, D.C. & Peng, Z.Y. The ankyrin repeat as molecular architecture for protein recognition. Protein Sci. 13, 1435–1448 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sato, Y., Yoshikawa, A., Yamashita, M., Yamagata, A. & Fukai, S. Structural basis for specific recognition of lysine 63-linked polyubiquitin chains by NZF domains of TAB2 and TAB3. EMBO J. 28, 3903–3909 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kulathu, Y., Akutsu, M., Bremm, A., Hofmann, K. & Komander, D. Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain. Nat. Struct. Mol. Biol. 16, 1328–1330 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Bosanac, I. et al. Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling. Mol. Cell 40, 548–557 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Reyes-Turcu, F.E., Shanks, J.R., Komander, D. & Wilkinson, K.D. Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T. J. Biol. Chem. 283, 19581–19592 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ohshima, R. et al. Putative tumor suppressor EDD interacts with and up-regulates APC. Genes Cells 12, 1339–1345 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Tanaka, N. et al. Structural basis for recognition of 2′,5′-linked oligoadenylates by human ribonuclease L. EMBO J. 23, 3929–3938 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Pape, T. & Schneider, T.R. HKL2map: a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).

    Article  CAS  Google Scholar 

  49. Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D Biol. Crystallogr. 59, 2023–2030 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  52. 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 

  53. 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 

Download references

Acknowledgements

We would like to thank R. Williams, M. Allen, M. Fiedler, N. Soler, S. Freund, C. Johnson, S. McLaughlin, Y. Kulathu, Y. Ye, A. Bremm, M. Busch (Medical Research Council Laboratory of Molecular Biology), D.S. Hameed (Netherlands Cancer Institute) and K. Hofmann (Miltenyi Biotec) for reagents, help with experiments and discussions. Crystallographic data were collected at the European Synchrotron Radiation Facility beamline ID23-2. J.D.F.L. was supported by an Association for International Cancer Research grant (no. 07-0040 to M.B.). D.K. is an European Molecular Biology Organization (EMBO) Young Investigator. This work was supported by the Medical Research Council (MC_US_A024_0059 to M.B., MC_US_A024_0056 to D.K., and MC_US_A024_0051 to J.W.C.).

Author information

Authors and Affiliations

Authors

Contributions

D.K., M.B. and J.D.F.L. designed the research. J.D.F.L., D.K., J.M., T.E.T.M., T.J.R. and M.A. conducted the experiments. F.E., H.O., S.V. and J.W.C. contributed reagents. D.K. wrote the manuscript, with help from all authors.

Corresponding author

Correspondence to David Komander.

Ethics declarations

Competing interests

H.O. and F.E. are cofounders of UbiQ Bio BV.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Methods (PDF 3212 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Licchesi, J., Mieszczanek, J., Mevissen, T. et al. An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains. Nat Struct Mol Biol 19, 62–71 (2012). https://doi.org/10.1038/nsmb.2169

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2169

This article is cited by

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