Insights into the effect of the J-domain on the substrate binding domain (SBD) of the 70 kDa heat shock protein, Hsp70, from a chimeric human J-SBD polypeptide

Abstract

DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain, a crucial event for the function that this system has in assisting protein folding. The interaction between Hsp40 and Hsp70 is transient and thus difficult to study, since mixing the binding partners can lead to quick dissociation due to their low affinity, creating a challenge for detailed analysis. As a consequence, knowledge of many important aspects of the mechanism of interaction is still lacking, for instance, the effect that J-domain binding has on Hsp70. In this study, we investigated whether it would be possible to gain understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale for this is that an increase in the proximity between the interacting partners in this engineered chimera will promote the natural interaction and facilitate the characterization of the protein- protein interaction, which is a requirement to gain further understanding of many biological processes. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the SBD alone. J-SBD behaved primarily as a monomer in all conditions tested and exhibited chaperone activity, as shown by aggregation protection and substrate binding assays, which revealed decreased binding to bis-ANS, a probe for hydrophobic patches. Collectively, our results suggest that Hsp40 binding to Hsp70 via the J-domain shifts the Hsp70 equilibrium towards the monomer state to expose hydrophobic sites prone to substrate accommodation.\n\nAbbreviationsBis-ANS (4,4-Dianilino-1,1-Binaphthyl-5,5-Disulfonic Acid; CD, circular dichroism; Hsp, heat shock protein; J-SBD, chimeric polypeptide in which the J-domain of Hsp40 (at the N-terminus) is covalently attached to the substrate binding domain of Hsp70 (at the C-terminus) by a flexible linker; SBD: substrate binding domain of Hsp70.