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:

Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains

Nature Structural & Molecular Biology volume 16, pages 883–889 (2009)Cite this article

Abstract

Ubiquitin (denoted Ub) receptor proteins as a group must contain a diverse set of binding specificities to distinguish the many forms of polyubiquitin (polyUb) signals. Previous studies suggested that the large class of ubiquitin-associated (UBA) domains contains members with intrinsic specificity for Lys63-linked polyUb or Lys48-linked polyUb, thus explaining how UBA-containing proteins can mediate diverse signaling events. Here we show that previously observed Lys63-polyUb selectivity in UBA domains is the result of an artifact in which the dimeric fusion partner, glutathione S-transferase (GST), positions two UBAs for higher affinity, avid interactions with Lys63-polyUb, but not with Lys48-polyUb. Freed from GST, these UBAs are either nonselective or prefer Lys48-polyUb. Accordingly, NMR experiments reveal no Lys63-polyUb–specific binding epitopes for these UBAs. We reexamine previous conclusions based on GST-UBAs and present an alternative model for how UBAs achieve a diverse range of linkage specificities.

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: GST-Ede1 UBA is selective for Lys63-linked polyUb.
Figure 2: CSP mapping of the interactions of Ede1 UBA with monoUb, Lys63-polyUb and Lys48-polyUb reveals no linkage-specific mode of interaction.
Figure 3: Free Ede1-UBA is not linkage selective.
Figure 4: The bivalency of GST-Ede1 UBA preferentially promotes binding to Lys63-polyUb over Lys48-polyUb.
Figure 5: HR23A-UBA1 is a Lys48-selective UBA domain.
Figure 6: Oligomeric UBA proteins may achieve Lys63 selectivity through linkage-specific avidity.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Pickart, C.M. & Eddins, M.J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).

    Article  CAS  Google Scholar 

  4. Sun, L. & Chen, Z.J. The novel functions of ubiquitination in signaling. Curr. Opin. Cell Biol. 16, 119–126 (2004).

    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. Hicke, L. & Dunn, R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172 (2003).

    Article  CAS  Google Scholar 

  7. Shilatifard, A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annu. Rev. Biochem. 75, 243–269 (2006).

    Article  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. Raasi, S. & Pickart, C.M. Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains. J. Biol. Chem. 278, 8951–8959 (2003).

    Article  CAS  Google Scholar 

  10. Raasi, S., Orlov, I., Fleming, K.G. & Pickart, C.M. Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A. J. Mol. Biol. 341, 1367–1379 (2004).

    Article  CAS  Google Scholar 

  11. Trempe, J.F. et al. Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain. EMBO J. 24, 3178–3189 (2005).

    Article  CAS  Google Scholar 

  12. Swanson, K.A., Hicke, L. & Radhakrishnan, I. Structural basis for monoubiquitin recognition by the Ede1 UBA domain. J. Mol. Biol. 358, 713–724 (2006).

    Article  CAS  Google Scholar 

  13. 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  Google Scholar 

  14. Haririnia, A., D'Onofrio, M. & Fushman, D. Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a. J. Mol. Biol. 368, 753–766 (2007).

    Article  CAS  Google Scholar 

  15. Scheibner, K.A., Zhang, Z. & Cole, P.A. Merging fluorescence resonance energy transfer and expressed protein ligation to analyze protein-protein interactions. Anal. Biochem. 317, 226–232 (2003).

    Article  CAS  Google Scholar 

  16. Zhang, D., Raasi, S. & Fushman, D. Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains. J. Mol. Biol. 377, 162–180 (2008).

    Article  CAS  Google Scholar 

  17. Sims, J.J. & Cohen, R.E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80. Mol. Cell 33, 775–783 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Kaplan, W. et al. Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag. Protein Sci. 6, 399–406 (1997).

    Article  CAS  Google Scholar 

  20. Mueller, T.D. & Feigon, J. Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions. J. Mol. Biol. 319, 1243–1255 (2002).

    Article  CAS  Google Scholar 

  21. Bayrer, J.R., Zhang, W. & Weiss, M.A. Dimerization of doublesex is mediated by a cryptic ubiquitin-associated domain fold: implications for sex-specific gene regulation. J. Biol. Chem. 280, 32989–32996 (2005).

    Article  CAS  Google Scholar 

  22. Kozlov, G. et al. Structural basis for UBA-mediated dimerization of c-Cbl ubiquitin ligase. J. Biol. Chem. 282, 27547–27555 (2007).

    Article  CAS  Google Scholar 

  23. Prag, G. et al. Mechanism of ubiquitin recognition by the CUE domain of Vps9p. Cell 113, 609–620 (2003).

    Article  CAS  Google Scholar 

  24. Peschard, P. et al. Structural basis for ubiquitin-mediated dimerization and activation of the ubiquitin protein ligase Cbl-b. Mol. Cell 27, 474–485 (2007).

    Article  CAS  Google Scholar 

  25. Lowe, E.D. et al. Structures of the Dsk2 UBL and UBA domains and their complex. Acta Crystallogr. D Biol. Crystallogr. 62, 177–188 (2006).

    Article  Google Scholar 

  26. Sasaki, T., Funakoshi, M., Endicott, J.A. & Kobayashi, H. Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain. Biochem. Biophys. Res. Commun. 336, 530–535 (2005).

    Article  CAS  Google Scholar 

  27. Matiuhin, Y. et al. Extraproteasomal Rpn10 restricts access of the polyubiquitin-binding protein Dsk2 to proteasome. Mol. Cell 32, 415–425 (2008).

    Article  CAS  Google Scholar 

  28. 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 

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

    Article  CAS  Google Scholar 

  30. Kim, I. & Rao, H. What's Ub chain linkage got to do with it? Sci. STKE 2006, pe18 (2006).

    PubMed  Google Scholar 

  31. Friedlander, R., Jarosch, E., Urban, J., Volkwein, C. & Sommer, T. A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat. Cell Biol. 2, 379–384 (2000).

    Article  CAS  Google Scholar 

  32. 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 

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

    Article  CAS  Google Scholar 

  34. Lo, Y.C. et al. Structural basis for recognition of diubiquitins by NEMO. Mol. Cell 33, 602–615 (2009).

    Article  CAS  Google Scholar 

  35. Broemer, M. & Meier, P. Ubiquitin-mediated regulation of apoptosis. Trends Cell Biol. 19, 130–140 (2009).

    Article  CAS  Google Scholar 

  36. Gyrd-Hansen, M. et al. IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-κB as well as cell survival and oncogenesis. Nat. Cell Biol. 10, 1309–1317 (2008).

    Article  CAS  Google Scholar 

  37. Seibenhener, M.L., Geetha, T. & Wooten, M.W. Sequestosome 1/p62–more than just a scaffold. FEBS Lett. 581, 175–179 (2007).

    Article  CAS  Google Scholar 

  38. Bjørkøy, G., Lamark, T. & Johansen, T. p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy 2, 138–139 (2006).

    Article  Google Scholar 

  39. Tan, J.M., Wong, E.S., Dawson, V.L., Dawson, T.M. & Lim, K.L. Lysine 63-linked polyubiquitin potentially partners with p62 to promote the clearance of protein inclusions by autophagy. Autophagy 4, 251–253 (2007).

    Article  Google Scholar 

  40. Seibenhener, M.L. et al. Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol. Cell. Biol. 24, 8055–8068 (2004).

    Article  CAS  Google Scholar 

  41. Bjørkøy, G. et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 171, 603–614 (2005).

    Article  Google Scholar 

  42. Tan, J.M. et al. Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Hum. Mol. Genet. 17, 431–439 (2008).

    Article  CAS  Google Scholar 

  43. Olzmann, J.A. & Chin, L.S. Parkin-mediated K63-linked polyubiquitination: a signal for targeting misfolded proteins to the aggresome-autophagy pathway. Autophagy 4, 85–87 (2008).

    Article  CAS  Google Scholar 

  44. Kirkin, V. et al. A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol. Cell 33, 505–516 (2009).

    Article  CAS  Google Scholar 

  45. Aguilar, R.C., Watson, H.A. & Wendland, B. The yeast Epsin Ent1 is recruited to membranes through multiple independent interactions. J. Biol. Chem. 278, 10737–10743 (2003).

    Article  CAS  Google Scholar 

  46. Gagny, B. et al. A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis. J. Cell Sci. 113, 3309–3319 (2000).

    CAS  PubMed  Google Scholar 

  47. Maldonado-Báez, L. et al. Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis. Mol. Biol. Cell 19, 2936–2948 (2008).

    Article  Google Scholar 

  48. Wilkinson, K.D. Quantitative analysis of protein-protein interactions. Methods Mol. Biol. 261, 15–32 (2004).

    CAS  Google Scholar 

  49. Keller, R. The Computer Aided Resonance Assignment Tutorial (CANTINA, Goldau, Switzerland, 2004).

    Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Cole (Johns Hopkins School of Medicine) for materials and advice for fluorescent labeling by expressed protein ligation. Work in this study was supported in part by the US National Istitutes of Health (NIH) grant GM065334 (to D.F.) and NIH Roadmap grant RR020839.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA

    Joshua J Sims & Robert E Cohen

  2. Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA

    Aydin Haririnia, Bryan C Dickinson & David Fushman

  3. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA

    Robert E Cohen

Authors
  1. Joshua J Sims
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aydin Haririnia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bryan C Dickinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Fushman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Robert E Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.J.S. and R.E.C. conceived of the study; J.J.S., D.F. and R.E.C. designed the experiments; J.J.S., R.E.C., A.H. and B.C.D. prepared the proteins; J.J.S. performed all the cloning and binding assays, and interpreted the data with R.E.C.; A.H. and B.C.D. performed the NMR experiments and analyzed the data with D.F.; J.J.S., D.F. and R.E.C. wrote the paper.

Corresponding author

Correspondence to Robert E Cohen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Methods (PDF 579 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sims, J., Haririnia, A., Dickinson, B. et al. Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains. Nat Struct Mol Biol 16, 883–889 (2009). https://doi.org/10.1038/nsmb.1637

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1637

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