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.

  • Letter
  • Published:

Ubiquitin chain conformation regulates recognition and activity of interacting proteins

Nature volume 492, pages 266–270 (2012)Cite this article

Subjects

Abstract

Mechanisms of protein recognition have been extensively studied for single-domain proteins1, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins2,3. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended ‘open’ and more compact ‘closed’ conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48–diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.

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: Ubiquitin chain conformations and ensemble FRET measurements.
Figure 2: Single-molecule FRET of K48NC (a), K63NC (b) and M1NC (c).
Figure 3: Ubiquitin-interacting protein binding to Lys 63- and Met 1-linked chains.
Figure 4: DUB interaction with Lys48-linked chains.

Similar content being viewed by others

References

  1. Lo Conte, L., Chothia, C. & Janin, J. The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285, 2177–2198 (1999)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Iwai, K. Linear polyubiquitin chains: a new modifier involved in NFκB activation and chronic inflammation, including dermatitis. Cell Cycle 10, 3095–3104 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Cook, W. J., Jeffrey, L. C., Carson, M., Chen, Z. & Pickart, C. M. Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). J. Biol. Chem. 267, 16467–16471 (1992)

    CAS  PubMed  Google Scholar 

  9. Ryabov, Y. & Fushman, D. Interdomain mobility in di-ubiquitin revealed by NMR. Proteins 63, 787–796 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Hirano, T. et al. Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution. J. Biol. Chem. 286, 37496–37502 (2011)

    Article  CAS  Google Scholar 

  12. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Rohaim, A., Kawasaki, M., Kato, R., Dikic, I. & Wakatsuki, S. Structure of a compact conformation of linear diubiquitin. Acta Crystallogr. D 68, 102–108 (2012)

    Article  CAS  Google Scholar 

  15. Orte, A., Clarke, R., Balasubramanian, S. & Klenerman, D. Determination of the fraction and stoichiometry of femtomolar levels of biomolecular complexes in an excess of monomer using single-molecule, two-color coincidence detection. Anal. Chem. 78, 7707–7715 (2006)

    Article  CAS  Google Scholar 

  16. Fraser, J. S. et al. Hidden alternative structures of proline isomerase essential for catalysis. Nature 462, 669–673 (2009)

    Article  ADS  CAS  Google Scholar 

  17. Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008)

    Article  CAS  Google Scholar 

  18. Rahighi, S. et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell 136, 1098–1109 (2009)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  20. McCullough, J. et al. Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery. Curr. Biol. 16, 160–165 (2006)

    Article  CAS  Google Scholar 

  21. Ye, Y. et al. Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21. EMBO Rep. 12, 350–357 (2011)

    Article  CAS  Google Scholar 

  22. Wiener, R., Zhang, X., Wang, T. & Wolberger, C. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483, 618–622 (2012)

    Article  ADS  CAS  Google Scholar 

  23. Juang, Y.-C. et al. OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function. Mol. Cell 45, 384–397 (2012)

    Article  CAS  Google Scholar 

  24. Boehr, D. D., Nussinov, R. & Wright, P. E. The role of dynamic conformational ensembles in biomolecular recognition. Nature Chem. Biol. 5, 789–796 (2009)

    Article  CAS  Google Scholar 

  25. Eddins, M. J., Varadan, R., Fushman, D., Pickart, C. M. & Wolberger, C. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH. J. Mol. Biol. 367, 204–211 (2007)

    Article  CAS  Google Scholar 

  26. Schaefer, J. B. & Morgan, D. O. Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes. J. Biol. Chem. 286, 45186–45196 (2011)

    Article  CAS  Google Scholar 

  27. Thrower, J. S., Hoffman, L., Rechsteiner, M. & Pickart, C. M. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000)

    Article  CAS  Google Scholar 

  28. Orte, A., Clarke, R. W. & Klenerman, D. Fluorescence coincidence spectroscopy for single-molecule fluorescence resonance energy-transfer measurements. Anal. Chem. 80, 8389–8397 (2008)

    Article  CAS  Google Scholar 

  29. Clarke, R. W., Orte, A. & Klenerman, D. Optimized threshold selection for single-molecule two-color fluorescence coincidence spectroscopy. Anal. Chem. 79, 2771–2777 (2007)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank members of the Komander, Jackson and Klenerman laboratories, R. Williams, S. Freund, C. Johnson, S. McLaughlin and A. Fersht for discussions. Work in the Komander laboratory is supported by the Medical Research Council (U105192732) and the EMBO Young Investigator Program. G.B. and S.I. were supported by the BBSRC, the Newton Trust and an EMBO YIP small grant to D.Ko. Work in the Klenerman laboratory is supported by EPSRC.

Author information

Authors and Affiliations

  1. Division of Protein and Nucleic Acids Chemistry, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK,

    Yu Ye & David Komander

  2. Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK,

    Georg Blaser, Mathew H. Horrocks, Shehu Ibrahim, Alexander A. Zhukov, David Klenerman & Sophie E. Jackson

  3. Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071 Granada, Spain,

    Maria J. Ruedas-Rama & Angel Orte

Authors
  1. Yu Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Georg Blaser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mathew H. Horrocks
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria J. Ruedas-Rama
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shehu Ibrahim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexander A. Zhukov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Angel Orte
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David Klenerman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sophie E. Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. David Komander
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.Y., G.B. and M.H.H. designed and performed the experiments, including single-molecule measurements, and analysed the data. Y.Y. and G.B. generated all proteins used in this study. Y.Y. performed kinetic experiments. M.H.H. and S.I. built the PAX instrument and A.A.Z. programmed the control for PAX measurements. S.I. performed single molecule experiments and contributed to data analysis. M.J.R.-R. and A.O. performed lifetime measurements. D.Kl., S.E.J. and D.Ko. directed the research and analysed the results. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to David Klenerman, Sophie E. Jackson or David Komander.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13, Supplementary Methods and Supplementary References. (PDF 3875 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ye, Y., Blaser, G., Horrocks, M. et al. Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266–270 (2012). https://doi.org/10.1038/nature11722

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11722

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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