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Substrate recognition by the AAA+ chaperone ClpB

Abstract

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

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Figure 1: Identification and characterization of ClpB-interacting peptides.
Figure 2: Preloading of ClpB-B1/2A with peptide B1 prevents further substrate association.
Figure 3: Peptides bind to the first AAA domain of ClpB.
Figure 4: Identification of a potential substrate-binding site.
Figure 5: ClpB pore mutants exhibit reduced chaperone activities and their ATPase activities are stimulated less by peptides and substrates.
Figure 6: Reduced binding of ClpB pore mutants to peptides and substrates in vitro.
Figure 7: The substrate interactions of ClpB pore mutants are affected in vivo.
Figure 8: Direct interaction between the ClpB pore site and substrates revealed by crosslinking.

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Acknowledgements

We thank D. Dougan, K. Turgay and E. Weber-Ban for discussions and critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Leibnizprogramm and Bu617/14-1) and the Fond der Chemischen Industrie to B.B. and A.M.

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Correspondence to Bernd Bukau or Axel Mogk.

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Supplementary information

Supplementary Fig. 1

The peptide-induced oligomerization of ClpB-B1/2A is ATP-dependent. (PDF 250 kb)

Supplementary Fig. 2

ClpB pore mutants exhibit no oligomerization and structural defects. (PDF 1034 kb)

Supplementary Fig. 3

ClpB interacts with a short 14-mer oligopeptide. (PDF 14 kb)

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Schlieker, C., Weibezahn, J., Patzelt, H. et al. Substrate recognition by the AAA+ chaperone ClpB. Nat Struct Mol Biol 11, 607–615 (2004). https://doi.org/10.1038/nsmb787

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