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Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces

Nature Structural & Molecular Biology volume 19, pages 1347–1355 (2012)Cite this article

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Abstract

Bacteria, fungi and plants rescue aggregated proteins using a powerful bichaperone system composed of an Hsp70 chaperone and an Hsp100 AAA+ disaggregase. In Escherichia coli, the Hsp70 chaperone DnaK binds aggregates and targets the disaggregase ClpB to the substrate. ClpB hexamers use ATP to thread substrate polypeptides through the central pore, driving disaggregation. How ClpB finds DnaK and regulates threading remains unclear. To dissect the disaggregation mechanism, we separated these steps using primarily chimeric ClpB-ClpV constructs that directly recognize alternative substrates, thereby obviating DnaK involvement. We show that ClpB has low intrinsic disaggregation activity that is normally repressed by the ClpB middle (M) domain. In the presence of aggregate, DnaK directly binds M-domain motif 2, increasing ClpB ATPase activity to unleash high ClpB threading power. Our results uncover a new function for Hsp70: the coupling of substrate targeting to AAA+ chaperone activation at aggregate surfaces.

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Figure 1: ClpVB binds to VipA–VipB tubules but exhibits low disassembly activity.
Figure 2: The M domain acts as a negative regulator of ClpVB unfolding activity.
Figure 3: Hsp70 chaperones stimulate the unfolding activity of hybrid Hsp100 proteins in a species-specific manner.
Figure 4: Stabilizing the interaction of the ClpB M-domain motif 2 with AAA-1 prevents activation by Hsp70.
Figure 5: Activated ClpVB has increased ATPase activity and is sensitive to incorporation of ATPase-deficient subunits.
Figure 6: DnaK interacts with ClpB M-domain motif 2.
Figure 7: DnaK preferentially interacts with the activated state of ClpB.
Figure 8: M-domain motif 2 couples DnaK interaction and ClpB activity control.

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Acknowledgements

We thank H. Zentgraf for initial EM experiments; J. Krijnse-Locker and I. Haußer-Siller from the EM core facility of CellNetworks, University of Heidelberg for support; and L. Guilbride for critical editing of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (BU617-17) to B.B. and A.M. F.S. and E.K. were supported by the Hartmut-Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS). Y.O. was supported by a Humboldt fellowship.

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  1. Fabian Seyffer and Eva Kummer: These authors contributed equally to this work.

Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany

    Fabian Seyffer, Eva Kummer, Yuki Oguchi, Juliane Winkler, Mohit Kumar, Regina Zahn, Victor Sourjik, Bernd Bukau & Axel Mogk

  2. Deutsche Krebsforschungszentrum (DKFZ), Heidelberg, Germany

    Fabian Seyffer, Eva Kummer, Yuki Oguchi, Juliane Winkler, Regina Zahn, Bernd Bukau & Axel Mogk

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Contributions

F.S., E.K., Y.O., J.W., M.K., V.S., B.B. and A.M. conceived and designed experiments. F.S., E.K., Y.O., J.W., M.K. and R.Z. performed experiments. F.S., E.K., Y.O., J.W., M.K., V.S., B.B. and A.M. analyzed the data. V.S., B.B. and A.M. wrote the manuscript.

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Correspondence to Bernd Bukau or Axel Mogk.

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Seyffer, F., Kummer, E., Oguchi, Y. et al. Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces. Nat Struct Mol Biol 19, 1347–1355 (2012). https://doi.org/10.1038/nsmb.2442

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