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Multistep mechanism of substrate binding determines chaperone activity of Hsp70

Nature Structural Biology volume 7, pages 586–593 (2000)Cite this article

Abstract

The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding. We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an α-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a β-structured subdomain. Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the α-helical lid and β-domain that cause opening of the substrate binding cavity. The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK.

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Figure 1: Structure of DnaK wild type and mutant proteins.
Figure 2: Characterization of the DnaK(2–538) mutant protein.
Figure 3: Peptide binding to wild type and mutant DnaK.
Figure 4: ATPase stimulation correlates with substrate affinity.
Figure 5: Refolding of chemically denatured luciferase.
Figure 6: Model of the interaction of the Hsp70 chaperones with substrates.

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References

  1. Buchberger, A. et al. Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication. J. Biol. Chem. 270, 16903–16910 (1995).

    Article  CAS  Google Scholar 

  2. Schmid, D., Baici, A., Gehring, H. & Christen, P. Kinetics of molecular chaperone action. Science 263, 971–973 (1994).

    Article  CAS  Google Scholar 

  3. Bukau, B. & Horwich, A.L. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351–366 (1998).

    Article  CAS  Google Scholar 

  4. Karzai, A.W. & McMacken, R. A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271, 11236–11246 (1996).

    Article  CAS  Google Scholar 

  5. Laufen, T. et al. Mechanism of regulation of Hsp70 chaperones by DnaJ co-chaperones. Proc. Natl. Acad. Sci. USA 96, 5452–5457 (1999).

    Article  CAS  Google Scholar 

  6. Misselwitz, B., Staeck, O. & Rapoport, T.A. J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. Mol. Cell 2, 593–603 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Morshauser, R.C. et al. High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70. J. Mol. Biol. 289, 1387–1403 (1999).

    Article  CAS  Google Scholar 

  9. Wang, H. et al. NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone–protein interaction. Biochemistry 37, 7929–7940 (1998).

    Article  CAS  Google Scholar 

  10. Pellecchia, M. et al. Structural insights into substrate binding by the molecular chaperone DnaK. Nature Struct. Biol. 7, 298–303 (2000).

    Article  CAS  Google Scholar 

  11. Matlack, K.E.S., Misselwitz, B., Plath, K. & Rapoport, T.A. BiP acts as a molecular ratchet during posttranslational transport of prepro-α factor across the ER membrane. Cell 97, 553–564 (1999).

    Article  CAS  Google Scholar 

  12. Vriend, G. A molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990).

    Article  CAS  Google Scholar 

  13. McCarty, J.S. et al. Regulatory region C of the E. coli heat shock transcription factor, α32, constitutes a DnaK binding site and is conserved among eubacteria. J. Mol. Biol. 256, 829–837 (1996).

    Article  CAS  Google Scholar 

  14. 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 

  15. Takeda, S. & McKay, D.B. Kinetics of peptide binding to the bovine 70 kDa heat shock cognate protein, a molecular chaperone. Biochemistry 35, 4636–4644 (1996).

    Article  CAS  Google Scholar 

  16. Greene, L.E., Zinner, R., Naficy, S. & Eisenberg, E. Effect of nucleotide on the binding of peptides to 70-kDa heat shock protein. J. Biol. Chem. 270, 2967–2973 (1995).

    Article  CAS  Google Scholar 

  17. Pierpaoli, E.V., Gisler, S.M. & Christen, P. Sequence-specific rates of interaction of target peptides with the molecular chaperones DnaK and DnaJ. Biochemistry 37, 16741–16748 (1998).

    Article  CAS  Google Scholar 

  18. Rüdiger, S., Germcroth, L., Schneider-Mergener, J. & Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507 (1997).

    Article  Google Scholar 

  19. Bukau, B. & Walker, G.C. ΔdnaK mutants of Escherichia coli have defects in chromosomal segregation and plasmid maintenance at normal growth temperatures. J. Bacteriol. 171, 6030–6038 (1989).

    Article  CAS  Google Scholar 

  20. Spence, J., Cegielska, A. & Georgopoulos, C. Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation. J. Bacteriol. 172, 7157–7166 (1990).

    Article  CAS  Google Scholar 

  21. Ungewickell, E., Ungewickell, H. & Holstein, S.E.H. Functional interaction of the auxilin J domain with the nucleotide- and substrate-binding modules of Hsc70. J. Biol. Chem. 272, 19594–19600 (1997).

    Article  CAS  Google Scholar 

  22. Gamer, J. et al. A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the E. coli heat shock transcription factor σ32. EMBO J. 15, 607–617 (1996).

    Article  CAS  Google Scholar 

  23. 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 

  24. Rüdiger, S. Buchberger, A. & Bukau, B. Interaction of Hsp70 chaperones with substrates. Nature Struct. Biol. 4, 342–349 (1997).

    Article  Google Scholar 

  25. Stenberg, G. & Fersht, A.R. Folding of barnase in the presence of the molecular chaperone SecB. J. Mol. Biol. 274, 268–275 (1997).

    Article  CAS  Google Scholar 

  26. Fekkes, P., den Blaauwen, T. & Driessen, A.J.M. Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB. Biochemistry 34, 10078–10085 (1995).

    Article  CAS  Google Scholar 

  27. Kunkel, T.A., Bebenek, K. & McClary, J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 204, 125–139 (1991).

    Article  CAS  Google Scholar 

  28. Buchberger, A., Schröder, H., Büttner, M., Valencia, A. & Bukau, B. A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. Nature Struct. Biol. 1, 95–101 (1994).

    Article  CAS  Google Scholar 

  29. Mayer, M.P., Laufen, T., Paal, K., McCarty, J.S. & Bukau, B. Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance. J. Mol. Biol. 289, 1131–1144 (1999).

    Article  CAS  Google Scholar 

  30. Theyssen, H., Schuster, H.-P., Bukau, B. & Reinstein, J. The second step of ATP binding to DnaK induces peptide release. J. Mol. Biol. 263, 657–670 (1996).

    Article  CAS  Google Scholar 

  31. McCarty, J.S., Buchberger, A., Reinstein, J. & Bukau, B. The role of ATP in the functional cycle of the DnaK chaperone system. J. Mol. Biol. 249, 126–137 (1995).

    Article  CAS  Google Scholar 

  32. Szabo, A. 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).

    Article  CAS  Google Scholar 

  33. Kraulis, J. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

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Acknowledgements

We thank B. Krauβ for technical assistance and W. Haehnel and R. Loyal for peptide synthesis. This work was supported by grants from the DFG and the Fonds der Chemie to B.B.

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Author notes

  1. Hartwig Schröder

    Present address: Central Research Facility, A30, BASF AG, D-67056, Ludwigshafen, Germany

Authors and Affiliations

  1. Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str.7, D-Freiburg, 79104, Germany

    Matthias P. Mayer, Hartwig Schröder, Stefan Rüdiger, Klaus Paal, Thomas Laufen & Bernd Bukau

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Correspondence to Bernd Bukau.

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Mayer, M., Schröder, H., Rüdiger, S. et al. Multistep mechanism of substrate binding determines chaperone activity of Hsp70. Nat Struct Mol Biol 7, 586–593 (2000). https://doi.org/10.1038/76819

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