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.

Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation

Abstract

Polyubiquitin chains target protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 to allow efficient substrate degradation. Despite Rpn11's essential function during substrate processing, its detailed structural and biochemical characterization has been hindered by difficulties in purifying the isolated enzyme. Here we report the 2.0-Å crystal structures of Zn2+-free and Zn2+-bound Saccharomyces cerevisiae Rpn11 in an MPN-domain heterodimer with Rpn8. The Rpn11-Rpn8 interaction occurs via two distinct interfaces that may be conserved in related MPN-domain complexes. Our structural and mutational studies reveal that Rpn11 lacks a conserved surface to bind the ubiquitin Ile44 patch, does not interact with the moiety on the proximal side of the scissile isopeptide bond and exhibits no linkage specificity for ubiquitin cleavage. These findings explain how Rpn11 functions as a promiscuous deubiquitinase for cotranslocational substrate deubiquitination during proteasomal degradation.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Rpn11 and Rpn8 form a heterodimer through two distinct interfaces.
Figure 2: Rpn11 is missing a conserved binding site for the Ile44 patch of ubiquitin.
Figure 3: Missing proximal contacts allow Rpn11 cleavage promiscuity.
Figure 4: The Ins-1 loop of Rpn11 acts as a flap to fold over the ubiquitin C terminus.
Figure 5: Model for Rpn11-mediated deubiquitination.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).

    CAS  Article  Google Scholar 

  2. Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    CAS  Article  Google Scholar 

  3. Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  5. Saeki, Y. et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J. 28, 359–371 (2009).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  7. Matyskiela, M.E. & Martin, A. Design principles of a universal protein degradation machine. J. Mol. Biol. 425, 199–213 (2013).

    CAS  Article  Google Scholar 

  8. Saeki, Y. & Tanaka, K. Assembly and function of the proteasome. Methods Mol. Biol. 832, 315–337 (2012).

    CAS  Article  Google Scholar 

  9. Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386, 463–471 (1997).

    CAS  Article  Google Scholar 

  10. Groll, M. et al. A gated channel into the proteasome core particle. Nat. Struct. Biol. 7, 1062–1067 (2000).

    CAS  Article  Google Scholar 

  11. Glickman, M.H., Rubin, D.M., Fried, V.A. & Finley, D. The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol. Cell. Biol. 18, 3149–3162 (1998).

    CAS  Article  Google Scholar 

  12. Smith, D.M. et al. Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's a ring opens the gate for substrate entry. Mol. Cell 27, 731–744 (2007).

    CAS  Article  Google Scholar 

  13. Beckwith, R., Estrin, E., Worden, E.J. & Martin, A. Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase. Nat. Struct. Mol. Biol. 20, 1164–1172 (2013).

    CAS  Article  Google Scholar 

  14. Erales, J., Hoyt, M.A., Troll, F. & Coffino, P. Functional asymmetries of proteasome translocase pore. J. Biol. Chem. 287, 18535–18543 (2012).

    CAS  Article  Google Scholar 

  15. Zhang, F. et al. Mechanism of substrate unfolding and translocation by the regulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol. Cell 34, 485–496 (2009).

    CAS  Article  Google Scholar 

  16. Peth, A., Nathan, J.A. & Goldberg, A.L. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome. J. Biol. Chem. 288, 29215–29222 (2013).

    CAS  Article  Google Scholar 

  17. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002).

    CAS  Article  Google Scholar 

  18. Yao, T. & Cohen, R.E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002).

    CAS  Article  Google Scholar 

  19. Inobe, T., Fishbain, S., Prakash, S. & Matouschek, A. Defining the geometry of the two-component proteasome degron. Nat. Chem. Biol. 7, 161–167 (2011).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  21. Beck, F. et al. Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl. Acad. Sci. USA 109, 14870–14875 (2012).

    CAS  Article  Google Scholar 

  22. Matyskiela, M.E., Lander, G.C. & Martin, A. Conformational switching of the 26S proteasome enables substrate degradation. Nat. Struct. Mol. Biol. 20, 781–788 (2013).

    CAS  Article  Google Scholar 

  23. Lander, G.C. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012).

    CAS  Article  Google Scholar 

  24. 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).

    CAS  Article  Google Scholar 

  25. Estrin, E., Lopez-Blanco, J.R., Chacon, P. & Martin, A. Formation of an intricate helical bundle dictates the assembly of the 26S proteasome lid. Structure 21, 1624–1635 (2013).

    CAS  Article  Google Scholar 

  26. Echalier, A. et al. Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1. Proc. Natl. Acad. Sci. USA 110, 1273–1278 (2013).

    CAS  Article  Google Scholar 

  27. Sanches, M., Alves, B.S., Zanchin, N.I. & Guimaraes, B.G. The crystal structure of the human Mov34 MPN domain reveals a metal-free dimer. J. Mol. Biol. 370, 846–855 (2007).

    CAS  Article  Google Scholar 

  28. Cooper, E.M., Boeke, J.D. & Cohen, R.E. Specificity of the BRISC deubiquitinating enzyme is not due to selective binding to Lys63-linked polyubiquitin. J. Biol. Chem. 285, 10344–10352 (2010).

    CAS  Article  Google Scholar 

  29. Hofmann, K. & Bucher, P. The PCI domain: a common theme in three multiprotein complexes. Trends Biochem. Sci. 23, 204–205 (1998).

    CAS  Article  Google Scholar 

  30. Wang, B. et al. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316, 1194–1198 (2007).

    CAS  Article  Google Scholar 

  31. Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007).

    CAS  Article  Google Scholar 

  32. Davies, C.W., Paul, L.N., Kim, M.I. & Das, C. Structural and thermodynamic comparison of the catalytic domain of AMSH and AMSH-LP: nearly identical fold but different stability. J. Mol. Biol. 413, 416–429 (2011).

    CAS  Article  Google Scholar 

  33. Ye, Y. et al. Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266–270 (2012).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  35. Ciechanover, A., Elias, S., Heller, H. & Hershko, A. “Covalent affinity” purification of ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542 (1982).

    CAS  PubMed  Google Scholar 

  36. Dong, K.C. et al. Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19, 1053–1063 (2011).

    CAS  Article  Google Scholar 

  37. Sem, D.S. & McNeeley, P.A. Application of fluorescence polarization to the steady-state enzyme kinetic analysis of calpain II. FEBS Lett. 443, 17–19 (1999).

    CAS  Article  Google Scholar 

  38. Adams, P.D., Mustyakimov, M., Afonine, P.V. & Langan, P. Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules. Acta Crystallogr. D Biol. Crystallogr. 65, 567–573 (2009).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

We thank the members of the Martin laboratory for helpful discussions and D. Morgan (University of California, San Francisco) for providing expression constructs for E1 and E2 enzymes. We are also grateful to M. Herzik (University of California, Berkeley) and A. Lyubimov (Stanford University) for help with crystallography data collection and processing. E.J.W. acknowledges support from the US National Science Foundation Graduate Research Fellowship. This research was funded in part by the Searle Scholars Program (A.M.), start-up funds from the University of California, Berkeley Department of Molecular and Cell Biology (A.M.) and the US National Science Foundation CAREER Program (NSF-MCB-1150288 to A.M.).

Author information

Authors and Affiliations

Authors

Contributions

E.J.W. and A.M. designed experiments; E.J.W. and C.P. expressed, purified and characterized protein variants; and E.J.W. performed X-ray crystallographic studies. E.J.W. and A.M. prepared the manuscript.

Corresponding author

Correspondence to Andreas Martin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Electron density of the Rpn11 catalytic active site in the apo and Zn2+-bound states.

(a) The 2|Fo|-|Fc| electron density map of the Zn2+-bound active site is shown in grey and contoured at 1.5σ. Depicted in cyan is the |Fo|-|Fc| difference electron density map contoured at 5σ, calculated from the final coordinates after three rounds of ADP and reciprocal space XYZ refinement with the Zn2+ ion and the catalytic water omitted. (b) 2|Fo|-|Fc| electron density map of the Rpn11 active site in the apo state, contoured at 1.5σ. Also included is the corresponding |Fo|-|Fc| difference electron density map contoured at 5σ, which does not show any peaks at this contouring level.

Supplementary Figure 2 Specific interface residues facilitate Rpn11–Rpn8 heterodimer formation.

Close-up view of interactions between Rpn11 (green) and Rpn8 (blue, surface representation) at interface 2. Rpn8 Gly170 packs against Rpn11 Leu34, Leu35, and Leu38 (shown in stick representation). For the interaction of the corresponding residue Rpn11 Met212 with Rpn8 see Figure 1e.

Supplementary Figure 3 Alignment of the hydrophobic dimerization interfaces of Rpn11 and related MPN-containing proteins.

Residues of the hydrophobic interfaces 1 and 2 predicted to contribute >0.45 kcal/mol of binding energy between Rpn11 and Rpn8 are colored red. Residues predicted to be at the same position in other proteins are colored red if hydrophobic.

Supplementary Figure 4 Alignment of the hydrophobic dimerization interfaces of Rpn8 and related MPN-containing proteins.

Residues of the hydrophobic interfaces 1 and 2 predicted to contribute >0.45 kcal/mol of binding energy between Rpn11 and Rpn8 are colored red. Residues predicted to be at the same position in other proteins are colored red if hydrophobic.

Supplementary Figure 5 Structure-based alignment of Rpn11 and AMSH-LP.

Supplementary Figure 6 Example gels for the Lys48-linked diubiquitin cleavage assay of Rpn11.

Shown are the gels for one Michealis-Menten experiment analyzing the 30-minute time courses of Lys48-linked di-ubiquitin cleavage by wild-type Rpn11-Rpn8 (5 μM) at substrate concentrations between 15 and 500 μM. Bands indicated by asterisks are due to contaminating proteins that co-purify at low abundance with Rpn11 and Rpn8 heterodimers. Covalently linked ClpX hexamer was used for normalization of staining and enzyme concentrations across different gels, which were all processed in parallel.

Supplementary Figure 7 The N terminus and Ins-2 region of Rpn11 contact Rpn2.

Crystal structures of the Rpn11-Rpn8 dimer and Rpn2 (ref. 1) (PDB ID: 4ADY), with Rpn11 in green, Rpn8 in blue and Rpn2 in orange, are docked into the segmented EM 3D-reconstruction of the substrate-bound 26S proteasome2 (EMDB ID: EMD-5669). The Ins-1 loop of Rpn11 is colored gold. The N-terminal residue of Rpn11 as well as the residues flanking its unstructured Ins-2 region are colored red and labeled. Extra electron density not accounted for by the crystal structures of Rpn11-Rpn8 and Rpn2, and presumably corresponding to the Ins-2 region of Rpn11, is circled.

Supplementary Figure 8 Fluorescence-based assays for Rpn11 ubiquitin binding and cleavage.

(a) Example time-based polarization measurements of the cleavage of 5 and 10 μM Ub-Lys-TAMRA by 1.25 μM Rpn11-Rpn8. (b) Example single-turnover kinetics measurement for the cleavage of 100 nM Ub-Lys-TAMRA by 450 μM Rpn11-Rpn8. The data are fit by a single exponential, with a calculated kcat = 0.95 min−1. (c) Michaelis-Menten curve for Ub-Lys-TAMRA cleavage by Rpn11, where kcat was constrained to the experimentally determined value in (b). Limitations in substrate solubility precluded using Ub-Lys-TAMRA at concentrations higher than KM, so measurement of a complete curve was not possible. The estimated KM for Ub-Lys-TAMRA cleavage by Rpn11 is 20 μM. (d) Tryptophan fluorescence-based assay of K48-linked di-ubiquitin binding to Rpn11V80A. Tryptophan fluorescence of 5 μM Rpn11-Rpn8 heterodimers was measured in the presence of Lys48-linked di-ubiquitin at concentrations between 0 and 500 μM, as discussed in the methods. Triplicate fluorescence measurements were averaged and fit to a simple binding curve with a KD of 67 μM.

Supplementary information

Supplementary Figures

Supplementary Figures 1–8 (PDF 12936 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Worden, E., Padovani, C. & Martin, A. Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation. Nat Struct Mol Biol 21, 220–227 (2014). https://doi.org/10.1038/nsmb.2771

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2771

Further reading

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