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.

Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

Abstract

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Mpa1–234 hexamer has three 75 Å–long coiled coils that are needed for Pup recognition.
Figure 2: Full-length Pup in the context of hexameric Mpa1–234.
Figure 3: The Pup helical region is essential for proteasomal degradation, which supports the idea that Mpa uses a binding-induced folding recognition mechanism.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

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

    Article  CAS  Google Scholar 

  2. Lupas, A. et al. Eubacterial proteasomes. Mol. Biol. Rep. 24, 125–131 (1997).

    Article  CAS  Google Scholar 

  3. Lin, G. et al. Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity. Mol. Microbiol. 59, 1405–1416 (2006).

    Article  CAS  Google Scholar 

  4. Hu, G. et al. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate. Mol. Microbiol. 59, 1417–1428 (2006).

    Article  CAS  Google Scholar 

  5. Wang, T. et al. Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa. Structure 17, 1377–1385 (2009).

    Article  CAS  Google Scholar 

  6. Gandotra, S., Schnappinger, D., Monteleone, M., Hillen, W. & Ehrt, S. In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice. Nat. Med. 13, 1515–1520 (2007).

    Article  CAS  Google Scholar 

  7. Voges, D., Zwickl, P. & Baumeister, W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068 (1999).

    Article  CAS  Google Scholar 

  8. Nathan, C. et al. A philosophy of anti-infectives as a guide in the search for new drugs for tuberculosis. Tuberculosis (Edinb.) 88 Suppl 1, S25–S33 (2008).

    Article  CAS  Google Scholar 

  9. Darwin, K.H., Lin, G., Chen, Z., Li, H. & Nathan, C.F. Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue. Mol. Microbiol. 55, 561–571 (2005).

    Article  CAS  Google Scholar 

  10. Zhang, F. et al. Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol. Cell 34, 473–484 (2009).

    Article  Google Scholar 

  11. Djuranovic, S. et al. Structure and activity of the N-terminal substrate recognition domains in proteasomal ATPases. Mol. Cell 34, 580–590 (2009).

    Article  CAS  Google Scholar 

  12. Pearce, M.J., Mintseris, J., Ferreyra, J., Gygi, S.P. & Darwin, K.H. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis. Science 322, 1104–1107 (2008).

    Article  CAS  Google Scholar 

  13. Burns, K.E., Liu, W.T., Boshoff, H.I., Dorrestein, P.C. & Barry, C.E. III. Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein. J. Biol. Chem. 284, 3069–3075 (2009).

    Article  CAS  Google Scholar 

  14. Sutter, M., Damberger, F.F., Imkamp, F., Allain, F.H. & Weber-Ban, E. Prokaryotic ubiquitin-like protein (Pup) is coupled to substrates via the side chain of its C-terminal glutamate. J. Am. Chem. Soc. 132, 5610–5612 (2010).

    Article  CAS  Google Scholar 

  15. Striebel, F., Hunkeler, M., Summer, H. & Weber-Ban, E. The mycobacterial Mpa-proteasome unfolds and degrades pupylated substrates by engaging Pup's N-terminus. EMBO J. 29, 1262–1271 (2010).

    Article  CAS  Google Scholar 

  16. Burns, K.E., Pearce, M.J. & Darwin, K.H. Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates. J. Bacteriol. 192, 2933–2935 (2010).

    Article  CAS  Google Scholar 

  17. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968).

    Article  CAS  Google Scholar 

  18. Zhou, F.X., Merianos, H.J., Brunger, A.T. & Engelman, D.M. Polar residues drive association of polyleucine transmembrane helices. Proc. Natl. Acad. Sci. USA 98, 2250–2255 (2001).

    Article  CAS  Google Scholar 

  19. Meindl-Beinker, N.M., Lundin, C., Nilsson, I., White, S.H. & von Heijne, G. Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly. EMBO Rep. 7, 1111–1116 (2006).

    Article  CAS  Google Scholar 

  20. Sutter, M., Striebel, F., Damberger, F.F., Allain, F.H. & Weber-Ban, E. A distinct structural region of the prokaryotic ubiquitin-like protein (Pup) is recognized by the N-terminal domain of the proteasomal ATPase Mpa. FEBS Lett. 583, 3151–3157 (2009).

    Article  CAS  Google Scholar 

  21. Chen, X. et al. Prokaryotic ubiquitin-like protein pup is intrinsically disordered. J. Mol. Biol. 392, 208–217 (2009).

    Article  CAS  Google Scholar 

  22. Martin, A., Baker, T.A. & Sauer, R.T. Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nat. Struct. Mol. Biol. 15, 1147–1151 (2008).

    Article  CAS  Google Scholar 

  23. Burns, K.E. et al. “Depupylation” of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates. Mol. Cell 39, 821–827 (2010).

    Article  CAS  Google Scholar 

  24. Darwin, K.H. & Hofmann, K. SAMPyling proteins in archaea. Trends Biochem. Sci. 35, 348–351 (2010).

    Article  CAS  Google Scholar 

  25. Humbard, M.A. et al. Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii. Nature 463, 54–60 (2010).

    Article  CAS  Google Scholar 

  26. Dyson, H.J. & Wright, P.E. Coupling of folding and binding for unstructured proteins. Curr. Opin. Struct. Biol. 12, 54–60 (2002).

    Article  CAS  Google Scholar 

  27. Shoemaker, B.A., Portman, J.J. & Wolynes, P.G. Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc. Natl. Acad. Sci. USA 97, 8868–8873 (2000).

    Article  CAS  Google Scholar 

  28. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. & Pease, L.R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    Article  CAS  Google Scholar 

  29. Hatfull, G.F. & Jacobs, J.W.R. Molecular Genetics of Mycobacteria (ASM, Washington, DC, 2000).

  30. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  31. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  32. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  34. Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K. & Noble, M. The CCP4 molecular-graphics project. Acta Crystallogr. D Biol. Crystallogr. 58, 1955–1957 (2002).

    Article  Google Scholar 

Download references

Acknowledgements

We thank K. Burns for reviewing this manuscript and C. Nathan for advice and encouragement. X-ray diffraction data for this study were collected at beamlines X25 and X29 of the National Synchrotron Light Source. Financial support was principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the US National Institutes of Health (NIH). This work was supported by NIH grant AI070285 and Brookhaven National Laboratory LDRD grant 10-016 to H.L. and by NIH grant HL092774 to K.H.D. K.H.D. was also supported by a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Diseases award.

Author information

Authors and Affiliations

Authors

Contributions

T.W. performed protein purification, crystallization and structure determination; K.H.D. performed mutagenesis and in vivo degradation assays; T.W., K.H.D. and H.L. designed experiments and wrote the manuscript.

Corresponding authors

Correspondence to K Heran Darwin or Huilin Li.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 2054 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wang, T., Darwin, K. & Li, H. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation. Nat Struct Mol Biol 17, 1352–1357 (2010). https://doi.org/10.1038/nsmb.1918

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1918

This article is cited by

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