Binding of a Small Molecule at a Protein–Protein Interface Regulates the Chaperone Activity of Hsp70–Hsp40

Abstract

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.Heat shock protein 70 (Hsp70) is a member of a ubiquitously expressed family of molecular chaperones that are involved in protein homeostasis. In its role as a mediator of protein fate, this chaperone has been linked to multiple tasks, including roles in de novo protein folding, subcellular trafficking, protein disaggregation, proteasome-mediated degradation, and autophagy (1-6). In addition, Hsp70 has been linked to numerous diseases, especially cancer and disorders of protein folding (7). Thus, there is interest in better understanding the biology of Hsp70 in order to test its potential as a therapeutic target (8).To accomplish its various chaperone functions, Hsp70 physically interacts with the exposed hydrophobic residues of polypeptides via its C-terminal substrate-binding domain (SBD). NIH-PA Author ManuscriptHydrolysis of ATP in the adjacent, N-terminal nucleotide-binding domain (NBD) propagates an allosteric change to the SBD, resulting in an approximately 10-fold enhancement in substrate affinity (9-12). These findings suggest an important role for the nucleotide state in controlling the interactions of Hsp70 with misfolded substrates. Consistent with the proposed importance of nucleotide turnover, a family of essential cochaperones, the Hsp40s, is known to tightly regulate the ATPase rate of Hsp70. These cochaperones are defined by the presence of a conserved, 60 amino acid J-domain. Interaction of the J-domain with the NBD of a Hsp70 stimulates its ATP hydrolysis and favors tight association with bound substrates. For example, the J-domain containing co-chaperone, DnaJ, stimulates the nucleotide hydrolysis rate o...

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