Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)

Abstract

The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/g/min ؊1 . We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of ⌬dnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding.Escherichia coli DnaK is a member of the highly conserved heat shock protein 70 (Hsp70) 4 family, and it is involved in a variety of cellular pathways, including protein folding, transport, and degradation (1, 2). As a central player in protein quality control and homeostasis, Hsp70 has also been implicated in the pathogenesis of a variety of diseases (3-5). These observations have led to an interest in understanding how the various activities of Hsp70 are correlated.One of the main roles of DnaK is to enable the folding of nascent or otherwise unfolded proteins (6). In this role, DnaK is thought to limit aggregation and facilitate folding by binding to the hydrophobic regions exposed in these substrates. Briefly, DnaK, like all the Hsp70 family members, consists of a substrate-binding domain (SBD) and a nucleotide-binding domain (NBD) connected by a hydrophobic linker (7-9). The NBD of DnaK is further divided into four subdomains as follows: IA/IIA, which form the base, and IB/IIB, which form the upper walls of the nucleotide binding cleft (Fig. 1A). The binding of ATP to DnaK results in an "open" conformation with low substrate affinity. Upon hydrolysis, the ADP-bound form assumes a "closed" conformation that binds substrate with higher affinity (10 -16). Thus, allosteric communication between the two domains is thought to link nucleotide turnover to substrate binding and release.DnaK alone has a low intrinsic ATPase rate, which facilitates regulation by the important co-chaperones, DnaJ and GrpE. DnaJ specifically stimulates ATP hydrolysis and thus...

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