An unexpected second binding site for polypeptide substrates is essential for Hsp70 chaperone activity

Heat shock proteins of 70 kDa (Hsp70s) are ubiquitous and highly conserved molecular chaperones. They play multiple essential roles in assisting with protein folding and maintaining protein homeostasis. Their chaperone activity has been proposed to require several rounds of binding to and release of polypeptide substrates at the substrate-binding domain (SBD) of Hsp70s. All available structures have revealed a single substrate-binding site in the SBD that binds a single segment of an extended polypeptide of 3?4 residues. However, this well-established single peptide-binding site alone has made it difficult to explain the efficient chaperone activity of Hsp70s. In this study, using purified proteins and site-directed mutagenesis, along with fluorescence polarization and luciferase-refolding assays, we report the unexpected discovery of a second peptide-binding site in Hsp70s. More importantly, the biochemical analyses suggested that this novel binding site, named here P2, is essential for Hsp70 chaperone activity. Furthermore, cross-linking and mutagenesis studies indicated that this second binding site is in the SBD adjacent to the first binding site. Taken together, our results suggest that these two essential binding sites of Hsp70s cooperate in protein folding.