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Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments

Nature Structural & Molecular Biology volume 15, pages 641–650 (2008)Cite this article

Abstract

The ring-forming AAA+ chaperone ClpB cooperates with the DnaK chaperone system to reactivate aggregated proteins. With the assistance of DnaK, ClpB extracts unfolded polypeptides from aggregates via substrate threading through its central channel. Here we analyze the processing of mixed aggregates consisting of protein fusions of misfolded and native domains. ClpB–DnaK reactivated all aggregated fusion proteins with similar efficiency, without unfolding native domains, demonstrating that partial threading of the misfolded moiety is sufficient to solubilize aggregates. Reactivation by ClpB–DnaK occurred even when two stably folded domains flanked the aggregated moiety, indicating threading of internal substrate segments. In contrast with the related AAA+ chaperone ClpC, ClpB lacks a robust unfolding activity, enabling it to sense the conformational state of substrates. ClpB rings are highly unstable, which may facilitate dissociation from trapped substrates during threading.

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Figure 1: Mixed protein aggregates, harboring misfolded and native domains, are efficiently processed by KJE–ClpB.
Figure 2: Partial threading of the misfolded luciferase moiety is sufficient for solubilization of mixed protein aggregates.
Figure 3: Exchange of the labile moiety reveals the same principles of KJE–ClpB-mediated disaggregation of mixed protein aggregates.
Figure 4: The folding state of aggregated luciferase-DHFR influences both ClpB threading mode and luciferase reactivation.
Figure 5: Partial substrate threading mode is operative during ClpB-mediated protein disaggregation of an authentic thermolabile E. coli protein.
Figure 6: ClpB hexamers are dynamic assemblies.
Figure 7: Solubilization of mixed protein aggregates by peptidase-cooperating ClpC–MecA.

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Acknowledgements

We thank P. Tessarz for critical reading of the manuscript and E. Vierling (Department of Biochemistry and molecular biophysics, University of Arizona, USA) for providing 16.6 sHsp antibodies. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bu617/17-1) to B.B. and A.M., the Fond der Chemischen Industrie to B.B. and a Heisenberg Fellowship of the DFG to A.M.

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  1. Institut für Biologie-Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany.

Authors and Affiliations

  1. Zentrum für Molekulare Biologie Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Universität Heidelberg, Im Neuenheimer Feld 282, Heidelberg, D-69120, Germany

    Tobias Haslberger, Agnieszka Zdanowicz, Ingo Brand, Axel Mogk & Bernd Bukau

  2. European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Agnieszka Zdanowicz

  3. Department of Biochemistry, La Trobe University, Melbourne, Victoria, 3086, Australia

    Ingo Brand

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Contributions

T.H., A.Z., A.M. and B.B conceived and designed experiments. T.H., A.Z. and I.B. performed experiments; T.H., A.Z., I.B., J.K., K.T., A.M. and B.B. analyzed the data; J.K. and K.T. assisted in experimental design; A.M. and B.B. wrote the manuscript.

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Correspondence to Axel Mogk or Bernd Bukau.

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Haslberger, T., Zdanowicz, A., Brand, I. et al. Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments. Nat Struct Mol Biol 15, 641–650 (2008). https://doi.org/10.1038/nsmb.1425

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