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.

A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE

Abstract

The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP–controlled release of protein substrates. The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE. We find that GrpE interacts stably with the amino–terminal ATPase domain of DnaK. Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding. Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK. Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity. Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE–like proteins may be a general feature of this class of chaperone.

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.

References

  1. Gething, M.-J. & Sambrook, J. Protein folding in the cell. Nature 355, 33–45 (1992).

    Article  CAS  Google Scholar 

  2. Hartl, F.U., Martin, J. & Neupert, W. Protein folding in the cell: the role of molecular chaperones Hsp70 and Hsp60. A. Rev. Biophys. biomol. Struct. 21, 293–322 (1992).

    Article  CAS  Google Scholar 

  3. Schröder, H., Langer, T., Hartl, F.U. & Bukau, B. DnaK, DnaJ, GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 12, 4137–4144 (1993).

    Article  Google Scholar 

  4. Skowyra, D., Georgopoulos, C. & Zylicz, M. 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).

    Article  CAS  Google Scholar 

  5. Flynn, G.C., Chappell, T.G. & Rothman, J.E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 245, 385–390 (1989).

    Article  CAS  Google Scholar 

  6. Flaherty, K.M., Deluca-Flaherty, C. & McKay, D.B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346, 623–628 (1990).

    Article  CAS  Google Scholar 

  7. Flaherty, K.M., McKay, D.B. Kabsch W. & Holmes, K.C. Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70K heat shock cognate protein. Proc. natn. Acad. Sci. U.S.A. 88, 5041–5045 (1991).

    Article  CAS  Google Scholar 

  8. Bork, P., Sander, C. & Valencia, A., An TPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc. natn. Acad. Sci. U.S.A. 89, 7290–7294 (1992).

    Article  CAS  Google Scholar 

  9. Holmes, K.C., Sander, C. & Valencia, A. A new ATP-binding fold in actin, hexokinase and Hsc70. Trends Cell Biol. 3, 53–59 (1993).

    Article  CAS  Google Scholar 

  10. McCarty, J.S. & Walker, G.C. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc. natn. Acad. Sci. U.S.A. 88, 9513–9517 (1991).

    Article  CAS  Google Scholar 

  11. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C. & Zylicz, M. Escherichia coli. DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc. natn. Acad. Sci. U.S.A. 88, 2874–2878 (1991).

    Article  CAS  Google Scholar 

  12. Langer, T. et al. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 356, 683–689 (1992).

    Article  CAS  Google Scholar 

  13. Zylicz, M., Ang, D. & Georgopoulos, C. The grpE protein of Escherichia coli. Purification and properties. J. biol. Chem. 262, 17437–17442(1987).

    CAS  PubMed  Google Scholar 

  14. Georgopoulos, C., Ang, D., Liberek, K. & Zylicz, M. in: Stress proteins in biology and medicine (eds R. Morimoto, A. Tissieres & C. Georgopoulos) 191–222 (Cold Spring Harbor Press, New York, 1990).

    Google Scholar 

  15. Gross, C.A., Straus, D.B. & Erickson, J.W. in: Stress proteins in biology and medicine (eds R. Morimoto, A. Tissieres & C. Georgopoulos) 167–190 (Cold Spring Harbor Press, New York, 1990).

    Google Scholar 

  16. Yochem, J., Uchida, H. Sunshine, M. Saito, H. Georgopoulos, C. & Feiss, M. Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli. and bacteriophage lambda DNA replication. Molec. Gen. Genet. 164, 9–14 (1978).

    Article  CAS  Google Scholar 

  17. Georgopoulos, C. A new bacterial gene (groPC) which affects lambda DNA replication. Molec. Gen. Genet. 151, 35–39 (1977).

    Article  CAS  Google Scholar 

  18. Johnson, C., Chandrasekhar, G.N. & Georgopoulos, C. Escherichia coli. dnaK and grpE heat shock proteins interact both in vivo and in vitro. J. Bact. 171, 1590–1596 (1989).

    Article  CAS  Google Scholar 

  19. Miyazaki,T., Tanaka, S. Fujita, H. & Itikawa, H. DNA sequence analysis of the dnaK gene of Escherichia coli B and of two dnaK genes carrying the temperature-sensitive mutations dnaK7(Ts) and dnaK756(Ts). J. Bact. 174, 3715–3722 (1992).

    Article  CAS  Google Scholar 

  20. Casadaban, M.J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli. using bacteriophage lambda and Mu. J. molec. Biol. 104, 541–555 (1976).

    Article  CAS  Google Scholar 

  21. Stüber, D., Matile, H. & Garotta, G. in: Immunological Methods (eds I. Levkovits & B. Pernis) 121–152 (Academic Press, Orlando, 1990).

    Book  Google Scholar 

  22. Stüber, D. & Bujard, H. Transcription from efficient promotors can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1, 1399–1404 (1982).

    Article  Google Scholar 

  23. Gamer, J., Bujard, H. & Bukau, B. Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32. Cell 69, 833–842 (1992).

    Article  CAS  Google Scholar 

  24. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  25. Harlow, E. & Lane, D. Antibodies: A Laboratory Manual. (Cold Spring Harbor Press, New York, 1988).

    Google Scholar 

  26. Schmid, F.X. in: Protein Structure: A Practical Approach (ed. IE. Creighton) 283 (IRL, Oxford, 1989).

    Google Scholar 

  27. Bukau, B. & Walker, G. Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli. mutants lacking the DnaK chaperone. EMBO J. 9, 4027–4036 (1990).

    Article  CAS  Google Scholar 

  28. Miller, J.H. Experiments in molecular genetics. (Cold Spring Harbor Press, New York, 1972).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Buchberger, A., Schröder, H., Büttner, M. et al. A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. Nat Struct Mol Biol 1, 95–101 (1994). https://doi.org/10.1038/nsb0294-95

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0294-95

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