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Multistep mechanism of substrate binding determines chaperone activity of Hsp70


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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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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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).

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