Multistep mechanism of substrate binding determines chaperone activity of Hsp70

Abstract

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 alpha-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a beta-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 alpha-helical lid and beta-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.