The Importance of Having Thermosensor Control in the DnaK Chaperone System

Abstract

In addition to the 32 -mediated heat shock response, the DnaK/DnaJ/GrpE molecular chaperone system of Escherichia coli directly adapts to elevated temperatures by sequestering a higher fraction of substrate. This immediate heat shock response is due to the differential temperature dependence of the activity of DnaJ, which stimulates the hydrolysis of DnaK-bound ATP, and the activity of GrpE, which facilitates ADP/ATP exchange and converts DnaK from its high-affinity ADPliganded state into its low-affinity ATP-liganded state. GrpE acts as thermosensor with its ADP/ATP exchange activity decreasing above 40°C. To assess the importance of this reversible thermal adaptation for the chaperone action of the DnaK/DnaJ/GrpE system during heat shock, we used glucose-6-phosphate dehydrogenase and luciferase as substrates. We compared the performance of wild-type GrpE as a component of the chaperone system with that of GrpE R40C. In this mutant, the thermosensing helices are stabilized with an intersubunit disulfide bond and its nucleotide exchange activity thus increases continuously with increasing temperature. Wild-type GrpE with intact thermosensor proved superior to GrpE R40C with desensitized thermosensor. The chaperone system with wild-type GrpE yielded not only a higher fraction of refolding-competent protein at the end of a heat shock but also protected luciferase more efficiently against inactivation during heat shock. Consistent with their differential thermal behavior, the protective effects of wild-type GrpE and GrpE R40C diverged more and more with increasing temperature. Thus, the direct thermal adaptation of the DnaK chaperone system by thermosensing GrpE is essential for efficient chaperone action during heat shock.Molecular chaperones of the 70-kDa heat shock protein (Hsp70) 1 family participate in many cellular processes, including the folding, membrane translocation, and degradation of proteins (1). The chaperones recognize and interact with hydrophobic peptide segments, which are exposed by nascent polypeptide chains during synthesis and by misfolded proteins during stress, in particular heat shock. The irreversible formation of protein aggregates thus is reduced, increasing the yield of properly folded and refolded native protein. Hsp70 bind and release their substrates in an ATP-driven cycle (2, 3). The ATPase activity resides in the NH 2 -terminal domain (4, 5) and modulates the substrate binding properties of the COOH-terminal peptide-binding domain (6). The ATP-liganded T state is characterized by low affinity for substrates and fast rates of binding and release, whereas the ADP-liganded R state shows high affinity for substrates with slow kinetics (7,8). DnaK, an Hsp70 homolog in Escherichia coli, acts in concert with its co-chaperones DnaJ, an Hsp40 homolog, and GrpE (2, 9, 10). DnaJ stimulates the hydrolysis of DnaK-bound ATP and converts T-state DnaK into its high-affinity R state (Fig. 1). GrpE facilitates ADP/ATP exchange and reconverts DnaK into the low-affinity T state. In the presence of A...

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters inShigella

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Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch

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The NH2-terminal Domain of the Chloroplast GrpE Homolog CGE1 Is Required for Dimerization and Cochaperone Function in Vivo

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