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 analyted mutants of Dna K, an Hsp70 homolog, altered in key residues of its substrate binding domai n. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered cl osing device involving independent action of an alpha-helical lid and an ar ch, and a superimposed allosteric mechanism of ATP-controlled opening of th e 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 ch anges in the alpha-helical lid and beta-domain that cause opening of the su bstrate binding cavity, The affinity for substrates, as defined by this mec hanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis media ted locking-in of substrates and chaperone activity of DnaK.