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:

The proteasome antechamber maintains substrates in an unfolded state

Nature volume 467, pages 868–871 (2010)Cite this article

Subjects

Abstract

Eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins1,2,3,4. Proteolysis occurs in a barrel-shaped 20S core particle, which in Thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and β, arranged α7β7β7α7 (ref. 5). These rings form three interconnected cavities, including a pair of antechambers (formed by α7β7) through which substrates are passed before degradation and a catalytic chamber (β7β7) where the peptide-bond hydrolysis reaction occurs4,5. Although it is clear that substrates must be unfolded to enter through narrow, gated passageways (13 Å in diameter) located on the α-rings1,6,7, the structural and dynamical properties of substrates inside the proteasome antechamber remain unclear. Confinement in the antechamber might be expected to promote folding and thus impede proteolysis. Here we investigate the folding, stability and dynamics of three small protein substrates in the antechamber by methyl transverse-relaxation-optimized NMR spectroscopy8. We show that these substrates interact actively with the antechamber walls and have drastically altered kinetic and equilibrium properties that maintain them in unstructured states so as to be accessible for hydrolysis.

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: Encapsulation of substrates in α7α7.
Figure 2: Folding of proteasome substrates in α7α7.
Figure 3: Interactions between unfolded substrates and the α-ring.
Figure 4: Substrate stability and proteolysis rates are anticorrelated.

Similar content being viewed by others

References

  1. Förster, A. & Hill, C. P. Proteasome degradation: enter the substrate. Trends Cell Biol. 13, 550–553 (2003)

    Article  Google Scholar 

  2. Coux, O., Tanaka, K. & Goldberg, A. L. Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65, 801–847 (1996)

    Article  CAS  Google Scholar 

  3. Baumeister, W., Walz, J., Zühl, F. & Seemüller, E. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380 (1998)

    Article  CAS  Google Scholar 

  4. Marques, A. J., Palanimurugan, R., Matias, A. C., Ramos, P. C. & Dohmen, R. J. Catalytic mechanism and assembly of the proteasome. Chem. Rev. 109, 1509–1536 (2009)

    Article  CAS  Google Scholar 

  5. Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268, 533–539 (1995)

    Article  ADS  Google Scholar 

  6. Liu, C. W., Corboy, M. J., DeMartino, G. N. & Thomas, P. J. Endoproteolytic activity of the proteasome. Science 299, 408–411 (2003)

    Article  ADS  CAS  Google Scholar 

  7. Cheng, Y. Toward an atomic model of the 26S proteasome. Curr. Opin. Struct. Biol. 19, 203–208 (2009)

    Article  Google Scholar 

  8. Tugarinov, V., Hwang, P. M., Ollerenshaw, J. E. & Kay, L. E. Cross-correlated relaxation enhanced 1H-13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J. Am. Chem. Soc. 125, 10420–10428 (2003)

    Article  CAS  Google Scholar 

  9. Sharon, M. et al. 20S proteasomes have the potential to keep substrates in store for continual degradation. J. Biol. Chem. 281, 9569–9575 (2006)

    Article  CAS  Google Scholar 

  10. Hutschenreiter, S., Tinazli, A., Model, K. & Tampé, R. Two-substrate association with the 20S proteasome at single-molecule level. EMBO J. 23, 2488–2497 (2004)

    Article  CAS  Google Scholar 

  11. Ellis, R. J. Protein folding: importance of the Anfinsen cage. Curr. Biol. 13, R881–R883 (2003)

    Article  CAS  Google Scholar 

  12. Zhou, H. X. Protein folding in confined and crowded environments. Arch. Biochem. Biophys. 469, 76–82 (2008)

    Article  CAS  Google Scholar 

  13. Ruschak, A. M. & Kay, L. E. Methyl groups as probes of supra-molecular structure, dynamics and function. J. Biomol. NMR 46, 75–87 (2010)

    Article  CAS  Google Scholar 

  14. Mayor, U. et al. The complete folding pathway of a protein from nanoseconds to microseconds. Nature 421, 863–867 (2003)

    Article  ADS  CAS  Google Scholar 

  15. Neudecker, P. et al. Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy. J. Mol. Biol. 363, 958–976 (2006)

    Article  CAS  Google Scholar 

  16. Jager, M. et al. Understanding the mechanism of beta-sheet folding from a chemical and biological perspective. Biopolymers 90, 751–758 (2008)

    Article  CAS  Google Scholar 

  17. Zwickl, P., Kleinz, J. & Baumeister, W. Critical elements in proteasome assembly. Nature Struct. Biol. 1, 765–770 (1994)

    Article  CAS  Google Scholar 

  18. Sprangers, R. & Kay, L. E. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445, 618–622 (2007)

    Article  CAS  Google Scholar 

  19. Wishart, D. S., Bigam, C. G., Holm, A., Hodges, R. S. & Sykes, B. D. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J. Biomol. NMR 5, 67–81 (1995)

    Article  CAS  Google Scholar 

  20. Fitzkee, N. C. & Rose, G. D. Reassessing random-coil statistics in unfolded proteins. Proc. Natl Acad. Sci. USA 101, 12497–12502 (2004)

    Article  ADS  CAS  Google Scholar 

  21. Jäger, M. et al. Structure-function-folding relationship in a WW domain. Proc. Natl Acad. Sci. USA 103, 10648–10653 (2006)

    Article  ADS  Google Scholar 

  22. Kubelka, J., Hofrichter, J. & Eaton, W. A. The protein folding ‘speed limit’. Curr. Opin. Struct. Biol. 14, 76–88 (2004)

    Article  CAS  Google Scholar 

  23. Jäger, M., Nguyen, H., Crane, J. C., Kelly, J. W. & Gruebele, M. The folding mechanism of a beta-sheet: the WW domain. J. Mol. Biol. 311, 373–393 (2001)

    Article  Google Scholar 

  24. Tugarinov, V., Sprangers, R. & Kay, L. E. Probing side-chain dynamics in the proteasome by relaxation violated coherence transfer NMR spectroscopy. J. Am. Chem. Soc. 129, 1743–1750 (2007)

    Article  CAS  Google Scholar 

  25. Choy, W. Y., Shortle, D. & Kay, L. E. Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of Δ131Δ. J. Am. Chem. Soc. 125, 1748–1758 (2003)

    Article  CAS  Google Scholar 

  26. Battiste, J. L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39, 5355–5365 (2000)

    Article  CAS  Google Scholar 

  27. Clore, G. M., Tang, C. & Iwahara, J. Elucidating transient macromolecular interactions using paramagnetic relaxation enhancement. Curr. Opin. Struct. Biol. 17, 603–616 (2007)

    Article  CAS  Google Scholar 

  28. Kisselev, A. F., Songyang, Z. & Goldberg, A. L. Why does threonine, and not serine, function as the active site nucleophile in proteasomes? J. Biol. Chem. 275, 14831–14837 (2000)

    Article  CAS  Google Scholar 

  29. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    Article  CAS  Google Scholar 

  30. Fersht, A. Structure and Mechanism in Protein Science (Freeman, 1999)

  31. Sprangers, R., Velyvis, A. & Kay, L. E. Solution NMR of supramolecular complexes: providing new insights into function. Nature Methods 4, 697–703 (2007)

    Article  CAS  Google Scholar 

  32. Stollar, E. J. et al. Crystal structures of engrailed homeodomain mutants: implications for stability and dynamics. J. Biol. Chem. 278, 43699–43708 (2003)

    Article  CAS  Google Scholar 

  33. Tugarinov, V. & Kay, L. E. Relaxation rates of degenerate 1H transitions in methyl groups of proteins as reporters of side-chain dynamics. J. Am. Chem. Soc. 128, 7299–7308 (2006)

    Article  CAS  Google Scholar 

  34. Matsunaga, N. &. Nagashima, A. Transport properties of liquid and gaseous D2O over a wide range of temperature and pressure. J. Phys. Chem. 6, 1133–1166 (1977)

Download references

Acknowledgements

The authors would like to thank J. Forman-Kay for providing laboratory space and for discussions, R. Muhandiram for NMR support, H. Lin for help designing protein purification protocols, X. Li and A. Shimmer for assistance with the plate reader, and W. Baumeister for discussions. T.L.R. acknowledges The European Molecular Biology Organization (ALTF 827-2006) and The Canadian Institutes of Health Research (CIHR) for postdoctoral fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. This work was supported by grants from the CIHR and the Natural Sciences and Engineering Research Council of Canada.

Author information

Authors and Affiliations

  1. Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada,

    Amy M. Ruschak, Tomasz L. Religa & Lewis E. Kay

  2. Department of Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany,

    Sarah Breuer & Susanne Witt

Authors
  1. Amy M. Ruschak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomasz L. Religa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarah Breuer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Susanne Witt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lewis E. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.M.R. made samples. T.L.R. helped make plasmids for protein expression and trained A.M.R. to express and purify proteins. A.M.R. and L.E.K. designed experiments, recorded and analysed NMR data, and wrote the manuscript. S.B. and S.W. were involved in preliminary experimental design, and S.W. and T.L.R. commented on the manuscript.

Corresponding author

Correspondence to Lewis E. Kay.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12 with legends, Supplementary Table 1 and additional references. (PDF 4049 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ruschak, A., Religa, T., Breuer, S. et al. The proteasome antechamber maintains substrates in an unfolded state. Nature 467, 868–871 (2010). https://doi.org/10.1038/nature09444

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09444

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