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Quantitative dynamics and binding studies of the 20S proteasome by NMR


The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.

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Figure 1: Details of the proteasome structure and methyl assignments.
Figure 2: Details of the assignment strategy.
Figure 3: Quantification of dynamics and structure.
Figure 4: Interaction between the 20S proteasome and the 11S activator.


  1. Pickart, C. M. & Cohen, R. E. Proteasomes and their kin: proteases in the machine age. Nature Rev. Mol. Cell Biol. 5, 177–187 (2004)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Adams, J. The proteasome: a suitable antineoplastic target. Nature Rev. Cancer 4, 349–360 (2004)

    Article  CAS  Google Scholar 

  4. 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 

  5. Glickman, M. H. et al. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615–623 (1998)

    Article  CAS  Google Scholar 

  6. Whitby, F. G. et al. Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 408, 115–120 (2000)

    Article  ADS  CAS  Google Scholar 

  7. 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 

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

    Article  ADS  CAS  Google Scholar 

  9. Unno, M. et al. The structure of the mammalian 20S proteasome at 2.75 Å resolution. Structure 10, 609–618 (2002)

    Article  CAS  Google Scholar 

  10. Kwon, Y. D., Nagy, I., Adams, P. D., Baumeister, W. & Jap, B. K. Crystal structures of the Rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly. J. Mol. Biol. 335, 233–245 (2004)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Mittermaier, A. & Kay, L. E. New tools provide new insights in NMR studies of protein dynamics. Science 312, 224–228 (2006)

    Article  ADS  CAS  Google Scholar 

  13. Tugarinov, V., Hwang, P. M. & Kay, L. E. Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Annu. Rev. Biochem. 73, 107–146 (2004)

    Article  CAS  Google Scholar 

  14. 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 

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

    Article  CAS  Google Scholar 

  16. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. USA 94, 12366–12371 (1997)

    Article  ADS  CAS  Google Scholar 

  17. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl Acad. Sci. USA 95, 13585–13590 (1998)

    Article  ADS  CAS  Google Scholar 

  18. Tugarinov, V. & Kay, L. E. Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J. Am. Chem. Soc. 125, 13868–13878 (2003)

    Article  CAS  Google Scholar 

  19. Sprangers, R., Gribun, A., Hwang, P. M., Houry, W. A. & Kay, L. E. Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release. Proc. Natl Acad. Sci. USA 102, 16678–16683 (2005)

    Article  ADS  CAS  Google Scholar 

  20. Tugarinov, V., Ollerenshaw, J. E. & Kay, L. E. Probing side-chain dynamics in high molecular weight proteins by deuterium NMR spin relaxation: an application to an 82-kDa enzyme. J. Am. Chem. Soc. 127, 8214–8225 (2005)

    Article  CAS  Google Scholar 

  21. Tugarinov, V. & Kay, L. E. Quantitative 13C and 2H NMR relaxation studies of the 723-residue enzyme malate synthase G reveal a dynamic binding interface. Biochemistry 44, 15970–15977 (2005)

    Article  CAS  Google Scholar 

  22. Forster, A., Masters, E. I., Whitby, F. G., Robinson, H. & Hill, C. P. The 1.9 Å structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Mol. Cell 18, 589–599 (2005)

    Article  Google Scholar 

  23. Nederlof, P. M., Wang, H. R. & Baumeister, W. Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro. Proc. Natl Acad. Sci. USA 92, 12060–12064 (1995)

    Article  ADS  CAS  Google Scholar 

  24. Korzhnev, D. M., Kloiber, K., Kanelis, V., Tugarinov, V. & Kay, L. E. Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme. J. Am. Chem. Soc. 126, 3964–3973 (2004)

    Article  CAS  Google Scholar 

  25. Benaroudj, N., Zwickl, P., Seemuller, E., Baumeister, W. & Goldberg, A. L. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol. Cell 11, 69–78 (2003)

    Article  CAS  Google Scholar 

  26. Fiaux, J., Bertelsen, E. B., Horwich, A. L. & Wüthrich, K. NMR analysis of a 900K GroEL GroES complex. Nature 418, 207–211 (2002)

    Article  ADS  CAS  Google Scholar 

  27. 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 

  28. Neri, D., Szyperski, T., Otting, G., Senn, H. & Wüthrich, K. Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28, 7510–7516 (1989)

    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 

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Acknowledgements We thank J. Forman-Kay for discussions and for providing laboratory space, F. Hansen for discussions, J. Rubenstein for electron microscopy images, R. Muhandiram for NMR support and C. Hill for a plasmid of the 11S activator complex. R.S. acknowledges EMBO and the Canadian Institutes of Health Research (CIHR) Training Program in Protein Folding and Disease for fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. Grant support from CIHR and NSERC is acknowledged.

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Correspondence to Lewis E. Kay.

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Supplementary Information

This file contains Supplementary figures S1-S7, Supplementary Notes which include sample preparation, NMR Experiments and measurement of binding constants and additional references. (PDF 2763 kb)

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

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