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 structures of HslU and the ATP-dependent protease HslU–HslV

Nature volume 403, pages 800–805 (2000)Cite this article

Abstract

The degradation of cytoplasmic proteins is an ATP-dependent process1. Substrates are targeted to a single soluble protease, the 26S proteasome2,3, in eukaryotes and to a number of unrelated proteases in prokaryotes4,5. A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU)6,7,8 in Escherichia coli. Its protease component HslV shares ∼20% sequence similarity6 and a conserved fold9 with 20S proteasome β-subunits. HslU is a member of the Hsp100 (Clp) family of ATPases. Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV–the first structure of a complete set of components of an ATP-dependent protease. HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components. Instead, there is conformational flexibility and domain motion in HslU and a localized order–disorder transition in HslV. Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states. They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein10,11, the prototype of an AAA-ATPase. A third, mostly α-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases.

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: Summary of the three crystal forms (a–c) that were used for structure determination.
Figure 2: Comparison of HsIU and NSF main chains.
Figure 3: HsIV–HsIU cyrstal complex.
Figure 4: Representation of the nucleotide-binding site in HslU.
Figure 5: View of HslU along the sixfold axis (indicated by a circle).

Similar content being viewed by others

References

  1. Goldberg, A. L. & St. John, A. C. Intracellular protein degradation in mammalian and bacterial cells: part 2. Annu. Rev. Biochem. 45, 747–803 (1976).

    Article  CAS  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61, 761 –807 (1992).

    Article  CAS  Google Scholar 

  3. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  Google Scholar 

  4. Maurizi, M. R. Proteases and protein degradation in Escherichia coli. Experientia 48, 178–201 (1992).

    Article  CAS  Google Scholar 

  5. Gottesman, S., Wickner, S. & Maurizi, M. R. Protein quality control: triage by chaperones and proteases. Genes Dev. 11, 815– 823 (1997).

    Article  CAS  Google Scholar 

  6. Chuang, S. E., Burland, V., Plunkett, G., Daniels, D. L. & Blattner, F. R. Sequence analysis of four new heat shock genes constituting the hslu and hslv operons in Escherichia coli. Gene 134, 1–6 (1993).

    Article  CAS  Google Scholar 

  7. Rohrwild, M. et al. HslV-HslU: a novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc. Natl Acad. Sci. USA 93, 5808–5813 ( 1996).

    Article  CAS  ADS  Google Scholar 

  8. Missiakis, D., Schwager, F., Betton, J. -M., Georgopoulos, C. & Raina, S. Identification and characterizaton of HslV HslU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. EMBO J. 15, 6899–6909 (1996).

    Article  Google Scholar 

  9. Bochtler, M., Ditzel, L., Groll, M. & Huber, R. Crystal structure of heat shock locus V (HslV) from Escherichia coli. Proc. Natl Acad. Sci. USA 94, 6070–6074 (1997).

    Article  CAS  ADS  Google Scholar 

  10. Lenzen, C. U., Steinmann, D., Whiteheart, S. W. & Weis, W. I. Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell 94, 525– 536 (1998).

    Article  CAS  Google Scholar 

  11. Yu, R. C., Hanson, P. I., Jahn, R. & Brünger, A. T. Structure of the ATP-dependent oligomerization domain of the N-ethylmaleimide sensitive factor complexed with ATP. Nature Struct. Biol. 5, 803–811 (1998).

    Article  CAS  Google Scholar 

  12. Huang, H. -C. & Goldberg, A. L. Proteolytic activity of the ATP-dependent protease HslVU can be uncoupled from ATP-hydrolysis. J. Biol. Chem. 272, 21364–21372 ( 1997).

    Article  CAS  Google Scholar 

  13. Neuwald, A. F., Aravind, L., Spouge, J. L. & Koonin, E. V. AAA+: a class of chaperone-like ATPases associated with the assembly, operation and disassembly of protein complexes. Genome Res. 9, 27– 43 (1999).

    CAS  PubMed  Google Scholar 

  14. Feng, H. P. & Gierasch, L. M. Molecular chaperones: clamps for the Clps? Curr. Biol. 8, 464– 467 (1998).

    Article  Google Scholar 

  15. Traut, T. W. The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide binding sites. Eur. J. Biochem. 222, 9–19 ( 1994).

    Article  CAS  Google Scholar 

  16. Saraste, M., Sibbald, P. R. & Wittinghofer, A. The P-loop—a common motif in ATP—and GTP-binding proteins. Trends Biochem. 15, 430– 434 (1990).

    Article  Google Scholar 

  17. Smith, C. A. & Rayment, I. Active site comparisons highlight structural similarities between myosin and other P-loop proteins. Biophys. J. 70, 1590–1602 (1996).

    Article  CAS  ADS  Google Scholar 

  18. Rohrwild, M. et al. The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome. Nature Struct. Biol 4, 133–139 (1997).

    Article  CAS  Google Scholar 

  19. Schirmer, E. C., Glover, J. R., Singer, M. A. & Lindquist, S. Hsp100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21, 289–296 (1996).

    Article  CAS  Google Scholar 

  20. Karata, K., Inagawa, T., Wilkinson, A. J., Tatsuta, T. & Ogura, T. Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases. J. Biol. Chem. 274, 26225–26232 (1999).

    Article  CAS  Google Scholar 

  21. Levchenko, I., Smith, C. K., Walsh, N. P., Sauer, R. T. & Baker, T. A. PDZ-like domains mediate binding specificity in the Clp/Hsp100 family of chaperone. Cell 91, 939–947 (1997).

    Article  CAS  Google Scholar 

  22. Smith, C. K., Baker, T. A. & Sauer, R. T. Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc. Natl Acad. Sci. 96, 6678–6682 (1999).

    Article  CAS  ADS  Google Scholar 

  23. Weber-Ban, E. U., Reid, B. G., Miranker, A. D. & Horwich, A. L. Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90–93 ( 1999).

    Article  CAS  ADS  Google Scholar 

  24. 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 

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

    Article  CAS  ADS  Google Scholar 

  26. Knowlton, J. R. et al. Structure of the proteasome activator REGα (PA28α). Nature 390, 639–643 (1997).

    Article  CAS  ADS  Google Scholar 

  27. Glickman, M. H. et al. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and elF3. Cell 94, 615–623 (1998).

    Article  CAS  Google Scholar 

  28. Project, C. C. C. The CCP4 suite: programs for protein crystallography. Acta Cryst. D 50, 760–763 ( 1994).

    Article  Google Scholar 

  29. Jones, A. T. A graphics model building and refinement system for macromolecules. J. Appl. Crystallogr. 11, 268–272 (1978).

    Article  CAS  Google Scholar 

  30. Brünger, A. T. et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Cryst. D 54, 905–921 (1998).

    Article  Google Scholar 

Download references

Acknowledgements

We thank P. Zwickl for 14C-labelled casein, for his help with the protein degradation assay and for essential discussions; S. Grazulis for sharing PDB-scripts M. Boicu for DNA sequencing; and R. Engh for critically reading the manuscript. The financial support of the Deutsche Forschungsgemeinschaft and of the Human Frontier Science Program is acknowledged.

Author information

Author notes

  1. Matthias Bochtler, Claudia Hartmann, Gleb P. Bourenkov and Hans D. Bartunik: These authors contributed equally to this work

Authors and Affiliations

  1. Max-Planck-Institut für Biochemie , Am Klopferspitz 18a, Martinsried , D-82152, Planegg, Germany

    Matthias Bochtler, Claudia Hartmann, Hyun Kyu Song & Robert Huber

  2. Arbeitsgruppen für strukturelle Molekularbiologie der Max-Planck-Gesellschaft, Notkestr. 85 , Hamburg, D-22603, Germany

    Gleb P. Bourenkov & Hans D. Bartunik

Authors
  1. Matthias Bochtler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudia Hartmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hyun Kyu Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gleb P. Bourenkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans D. Bartunik
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Robert Huber
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthias Bochtler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bochtler, M., Hartmann, C., Song, H. et al. The structures of HslU and the ATP-dependent protease HslU–HslV . Nature 403, 800–805 (2000). https://doi.org/10.1038/35001629

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35001629

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