Letter | Published:

Alternative modes of client binding enable functional plasticity of Hsp70

Nature volume 539, pages 448451 (17 November 2016) | Download Citation

Abstract

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation1,2,3,4,5, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins6,7,8,9,10. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid11,12,13,14,15. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70–substrate complexes raise the possibility of additional interaction mechanisms15,16,17,18. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle19. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. Molecular basis for regulation of the heat shock transcription factor sigma32 by the DnaK and DnaJ chaperones. Mol. Cell 32, 347–358 (2008)

  2. 2.

    , & The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell 62, 939–944 (1990)

  3. 3.

    , , , & Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157, 1685–1697 (2014)

  4. 4.

    , De los Rios, P., Christen, P., Lustig, A. & Goloubinoff, P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat. Chem. Biol. 6, 914–920 (2010)

  5. 5.

    , , & DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 12, 4137–4144 (1993)

  6. 6.

    & Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670–684 (2005)

  7. 7.

    , , & Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370, 111–117 (1994)

  8. 8.

    & Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852–1858 (2002)

  9. 9.

    , & Cellular strategies for controlling protein aggregation. Nat. Rev. Mol. Cell Biol. 11, 777–788 (2010)

  10. 10.

    et al. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat. Genet. 23, 425–428 (1999)

  11. 11.

    et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606–1614 (1996)

  12. 12.

    , , & Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507 (1997)

  13. 13.

    et al. The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. Proc. Natl Acad. Sci. USA 91, 10345–10349 (1994)

  14. 14.

    et al. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nat. Struct. Mol. Biol. 20, 900–907 (2013)

  15. 15.

    , , & Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863–874 (2012)

  16. 16.

    , , & Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat. Struct. Mol. Biol. 18, 345–351 (2011)

  17. 17.

    , & Conserved, disordered C terminus of DnaK enhances cellular survival upon stress and DnaK in vitro chaperone activity. J. Biol. Chem. 286, 31821–31829 (2011)

  18. 18.

    , , , & Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding. J. Mol. Biol. 261, 328–333 (1996)

  19. 19.

    et al. Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions. Nat. Struct. Mol. Biol. 18, 150–158 (2011)

  20. 20.

    et al. Direct observation of chaperone-induced changes in a protein folding pathway. Science 318, 1458–1461 (2007)

  21. 21.

    et al. Reshaping of the conformational search of a protein by the chaperone trigger factor. Nature 500, 98–101 (2013)

  22. 22.

    , , , & Stretching DNA with optical tweezers. Biophys. J. 72, 1335–1346 (1997)

  23. 23.

    , , , & Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET. EMBO J. 32, 1639–1649 (2013)

  24. 24.

    et al. Multistep mechanism of substrate binding determines chaperone activity of Hsp70. Nat. Struct. Biol. 7, 586–593 (2000)

  25. 25.

    et al. Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein. Proc. Natl Acad. Sci. USA 111, 13355–13360 (2014)

  26. 26.

    et al. A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32. EMBO J. 15, 607–617 (1996)

  27. 27.

    , , & Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor). Proc. Natl Acad. Sci. USA 91, 3839–3843 (1994)

  28. 28.

    , , & Action of the Hsp70 chaperone system observed with single proteins. Nat. Commun. 6, 6307 (2015)

  29. 29.

    & Hsp90 as a capacitor for morphological evolution. Nature 396, 336–342 (1998)

  30. 30.

    , & Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle. Nat. Struct. Mol. Biol. 20, 929–935 (2013)

  31. 31.

    , , & Reversible thermal transition in GrpE, the nucleotide exchange factor of the DnaK heat-shock system. J. Biol. Chem. 276, 6098–6104 (2001)

  32. 32.

    , , , & The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171. EMBO J. 13, 1687–1695 (1994)

  33. 33.

    et al. GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism. Biochemistry 36, 3417–3422 (1997)

  34. 34.

    , , , & NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone. J. Mol. Biol. 260, 236–250 (1996)

  35. 35.

    , & The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate. Structure 11, 1475–1483 (2003)

  36. 36.

    , , , & Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science 276, 431–435 (1997)

  37. 37.

    Gymnastics of molecular chaperones. Mol. Cell 39, 321–331 (2010)

  38. 38.

    , , & Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc. Natl Acad. Sci. USA 106, 8471–8476 (2009)

Download references

Acknowledgements

Work in the group of S.T. is supported by the Foundation for Fundamental Research on Matter (FOM) and the Netherlands Organization for Scientific Research (NWO). Work in the laboratory of B.B. was supported by research grants from the Deutsche Forschungsgemeinschaft (SFB638 and FOR1805) to G.K. and B.B. The work in the laboratory of M.P.M. was funded by the Deutsche Forschungsgemeinschaft (MA 1278/4-1). We thank M. M. Naqvi for performing control experiments, M. Avellaneda for help with preparing protein structure illustrations, and T. Shimizu, E. Garnett and F. Huber for critical reading of the manuscript.

Author information

Author notes

    • Alireza Mashaghi
    •  & Sergey Bezrukavnikov

    These authors contributed equally to this work.

Affiliations

  1. FOM institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands

    • Alireza Mashaghi
    • , Sergey Bezrukavnikov
    • , David P. Minde
    •  & Sander J. Tans
  2. Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany

    • Anne S. Wentink
    • , Roman Kityk
    • , Beate Zachmann-Brand
    • , Matthias P. Mayer
    • , Günter Kramer
    •  & Bernd Bukau
  3. German Cancer Research Center (DKFZ), Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany

    • Anne S. Wentink
    • , Beate Zachmann-Brand
    • , Günter Kramer
    •  & Bernd Bukau

Authors

  1. Search for Alireza Mashaghi in:

  2. Search for Sergey Bezrukavnikov in:

  3. Search for David P. Minde in:

  4. Search for Anne S. Wentink in:

  5. Search for Roman Kityk in:

  6. Search for Beate Zachmann-Brand in:

  7. Search for Matthias P. Mayer in:

  8. Search for Günter Kramer in:

  9. Search for Bernd Bukau in:

  10. Search for Sander J. Tans in:

Contributions

A.M., S.B., D.M., M.P.M., G.K., B.B. and S.T. conceived and designed the research; B.Z.B. and D.M. designed and purified the MBP protein constructs; A.M. and S.B. performed the optical tweezers experiments; B.Z.B., G.K., A.W. and R.K. purified the DnaK system chaperones and DnaK mutants; A.W. performed the bulk RepE54 assays; R.K. performed the bulk luciferase assays; A.M., S.B., A.W. and S.T. analysed the data; and A.M., S.B., D.M., M.P.M., G.K., B.B. and S.T. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sander J. Tans.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature20137

Further reading

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.