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Mechanics of Hsp70 chaperones enables differential interaction with client proteins

Nature Structural & Molecular Biology volume 18, pages 345–351 (2011)Cite this article

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Abstract

Hsp70 chaperones interact with a wide spectrum of substrates ranging from unfolded to natively folded and aggregated proteins. Structural evidence suggests that bound substrates are entirely enclosed in a β-sheet cavity covered by a helical lid, which requires structural rearrangements including lid opening to allow substrate access. We analyzed the mechanics of the lid movement of bacterial DnaK by disulfide fixation of lid elements to the β-sheet and by electron paramagnetic resonance spectroscopy using spin labels in the lid and β-sheet. Our results indicate that the lid-forming helix B adopts at least three conformational states and, notably, does not close over bound proteins, implying that DnaK does not only bind to extended peptide stretches of protein substrates but can also accommodate regions with substantial tertiary structure. This flexible binding mechanism provides a basis for the broad spectrum of substrate conformers of Hsp70s.

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Figure 1: Structure of the DnaK substrate-binding domain.
Figure 2: DnaK variants show altered interaction with substrates.
Figure 3: Only reduced DnaK-HA is capable of refolding luciferase.
Figure 4: Effects of disulfide bond formation on interdomain communication.
Figure 5: EPR spectra of a spin pair in the SBD of DnaK in the presence of nucleotide and substrate.
Figure 6: The distal part of helix B can be cross-linked to bound substrate.
Figure 7: Cartoon representation of the SBD of DnaK illustrating possible lid movements.

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Acknowledgements

We are grateful to F. Rodriguez for providing RepE, C. Altenbach for providing the fitting software and T. Ruppert for mass spectrometry. We thank S. R. Scholz for critical reading of the manuscript and S. Miller for help with figure preparation. We thank CellNetworks for providing the FTIR spectrometer. This work was supported by the Deutsche Forschungsgemeinschaft (SFB638 and MA 1278/4-1 to M.P.M.) and a Kekulé fellowship of the Fonds der Chemischen Industrie to R.S.

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  1. Annette H Erbse

    Present address: Present address: Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA.,

  2. Rainer Schlecht and Annette H Erbse: These authors contributed equally to this work.

Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ–ZMBH Allianz, Im Neuenheimer Feld 282, Heidelberg, Germany

    Rainer Schlecht, Annette H Erbse, Bernd Bukau & Matthias P Mayer

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Contributions

R.S. designed, performed and interpreted the experiments in Figures 2,3,4 and Figure 6, Table 1, Supplementary Figures 1,4,5 and 9 and Supplementary Table 1. A.H.E. designed, performed and interpreted the experiments in Figure 5 and Supplementary Figures 6–8. B.B. designed the experiments and supervised R.S. and A.H.E. M.P.M. designed all mutant proteins; designed, performed and interpreted experiments in Supplementary Figure 2; was involved in the design and interpretation of all experiments; and supervised R.S. and A.H.E. All authors prepared figures and wrote the manuscript.

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Correspondence to Bernd Bukau or Matthias P Mayer.

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Supplementary Figures 1–9 and Supplementary Table 1 (PDF 1780 kb)

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Schlecht, R., Erbse, A., Bukau, B. et al. Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat Struct Mol Biol 18, 345–351 (2011). https://doi.org/10.1038/nsmb.2006

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