The 70kD heat shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP, and a substrate-binding domain (SBD) that binds extended polypeptides. NBD and SBD interact little when in ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact Hsp70 from Escherichia coli in an ATP-bound state at 1.96 Å resolution. NBD-ATP adopts a unique conformation, forming extensive interfaces with a radically changed SBD that has its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes together with our biochemical tests provide a long-sought structural explanation for allosteric coupling in Hsp70 activity.

Background: Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the folding activity seen in Hsp70s. Results: In contrast to Hsp70s, Hsp110s exhibit distinct peptide substrate binding properties. Conclusion:The peptide substrate binding properties determine the chaperone activity differences between Hsp70s and Hsp110s. Significance: Our studies shed light on the molecular mechanism of the chaperone activities of Hsp70s and Hsp110s.

Background: Ubiquitous and conserved Hsp70 heat shock proteins play key roles in protein folding. Results: DnaK, a model Hsp70, forms a specific dimer, and mutations on the dimer interfaces compromise both chaperone activity and Hsp40 interaction. Conclusion: Dimerization of DnaK is required for the efficient interaction with Hsp40. Significance: Our studies shed light on the molecular mechanism of the Hsp70 chaperone machinery.

The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaK’s in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.

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.