DnaK, an Hsp70 molecular chaperone, processes its substrates in an ATP-driven cycle, which is controlled by the cochaperones DnaJ and GrpE. The kinetic analysis of substrate binding and release has as yet been limited to fluorescence-labeled peptides. Here, we report a comprehensive kinetic analysis of the chaperone action with protein substrates. The kinetic partitioning of the (ATP⅐DnaK)⅐substrate complexes between dissociation and conversion into stable (ADP⅐DnaK)⅐substrate complexes is determined by DnaJ. In the case of substrates that allow the formation of ternary (ATP⅐DnaK)⅐substrate⅐DnaJ complexes, the cis-effect of DnaJ markedly accelerates ATP hydrolysis. This triage mechanism efficiently selects from the (ATP⅐DnaK)⅐substrate complexes those to be processed in the chaperone cycle; at 45°C, the fraction of protein complexes fed into the cycle is 20 times higher than that of peptide complexes. The thermosensor effect of the ADP/ATP exchange factor GrpE retards the release of substrate from the cycle at higher temperatures; the fraction of total DnaK in stable (ADP⅐DnaK)⅐substrate complexes is 2 times higher at 45°C than at 25°C. Monitoring the cellular situation by DnaJ as nonnative protein sensor and GrpE as thermosensor thus directly adapts the operational mode of the DnaK system to heat shock conditions. Molecular chaperones of the 70-kDa heat shock protein (Hsp70) 2 family assist folding and refolding of nascent and stress-denatured proteins, assembly and disassembly of protein complexes, and translocation of polypeptide chains across membranes. All of these activities appear to rely on the transient interaction of Hsp70 with short hydrophobic segments of their protein substrates. The cycle of substrate binding and release is driven by the hydrolysis of ATP. DnaK, an Hsp70 homolog in Escherichia coli, consists of a 44-kDa NH 2 -terminal ATPase domain (1, 2) and a 25-kDa COOH-terminal substratebinding domain (3). Hydrolysis of DnaK-bound ATP and ADP/ ATP exchange control the functional properties of the substrate-binding domain (4). ATP-liganded DnaK (T-state DnaK) exhibits low affinity for substrates and fast rates of binding and release, whereas ADP-liganded (R-state) DnaK is characterized by high substrate affinity and slow kinetics (5, 6). DnaK acts in concert with two co-chaperones (Fig. 1). DnaJ, an Hsp40 homolog, stimulates the hydrolysis of ATP and thus promotes the formation of high affinity (ADP⅐DnaK)⅐substrate complexes, whereas GrpE facilitates the exchange of ADP for ATP and thus triggers the release of substrate from the cycle (7-9).Fluorescence-labeled peptides have allowed the kinetic analysis of the formation of DnaK⅐peptide complexes (6). With protein substrates, however, no kinetic data have been reported to date, DnaK⅐protein complexes having solely been examined by size exclusion chromatography (5), by nondenaturing gel electrophoresis (2), and by measuring fluorescence anisotropy under steady-state conditions (10).A major question in the field of molecular chaperones is how targe...

Temperature directly controls functional properties of the DnaK/DnaJ/GrpE chaperone system. The rate of the high to low affinity conversion of DnaK shows a non-Arrhenius temperature dependence and above ϳ40°C even decreases. In the same temperature range, the ADP/ATP exchange factor GrpE undergoes an extensive, fully reversible thermal transition (Grimshaw, J. P. A., Jelesarov, I., Schö nfeld, H. J., and Christen, P. (2001) J. Biol. Chem. 276, 6098 -6104). To show that this transition underlies the thermal regulation of the chaperone system, we introduced an intersubunit disulfide bond into the paired long helices of the GrpE dimer. The transition was absent in disulfide-linked GrpE R40C but was restored by reduction. With disulfide-stabilized GrpE, the rate of ADP/ATP exchange and conversion of DnaK from its ADP-liganded high affinity R state to the ATP-liganded low affinity T state continuously increased with increasing temperature. With reduced GrpE R40C, the conversion became slower at temperatures >40°C, as observed with wild-type GrpE. Thus, the long helix pair in the GrpE dimer acts as a thermosensor that, by decreasing its ADP/ATP exchange activity, induces a shift of the DnaK⅐substrate complexes toward the high affinity R state and in this way adapts the DnaK/DnaJ/GrpE system to heat shock conditions. Cells respond to an increase in temperature by increased synthesis of heat shock proteins (Hsps).1 Molecular chaperone systems of the Hsp70 family prevent the formation of protein aggregates and facilitate the folding of nascent polypeptide chains and denatured proteins (for comprehensive reviews, see Refs. 1 and 2). DnaK, an Hsp70 homolog of Escherichia coli, binds peptides and segments of denatured proteins in extended conformation (3, 4) and cooperates with two cohort heat shock proteins: DnaJ, an Hsp40 homolog, and GrpE (5). The DnaK/ DnaJ/GrpE chaperone system has been extensively studied in vitro at ambient temperatures (6 -12). DnaK alternates between two states, the ATP-liganded low affinity T state with fast binding and release of the substrate and the ADP-liganded high affinity R state with slow kinetics. A substrate is first bound by T state DnaK, which is then converted to the high affinity R state through DnaJ-triggered hydrolysis of DnaKbound ATP. With the assistance of GrpE, which serves as an ADP/ATP exchange factor, DnaK is reconverted from the R state into the low affinity T state, releasing the substrate.Heat shock proteins, by definition, are induced by a heat shock, i.e. a transient increase in temperature enhances the expression level of chaperones and co-chaperones. The transcription of the genes of DnaK and its co-chaperones DnaJ and GrpE is controlled by the initiation factor 32 of RNA polymerase (for a recent review, see Ref. 13). Recently, we have investigated the direct effect of elevated temperatures on the isolated DnaK/DnaJ/GrpE chaperone system. GrpE, which is an elongated homodimer both in solution (14, 15) and in crystalline form (Fig. 1 and Ref. 16), has been found to und...

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Osm1 and Frd1 are soluble fumarate reductases from yeast that are critical for allowing survival under anaerobic conditions. Although they maintain redox balance during anaerobiosis, the underlying mechanism is not understood. Here, we report the crystal structure of a eukaryotic soluble fumarate reductase, which is unique among soluble fumarate reductases as it lacks a heme domain. Structural and enzymatic analyses indicate that Osm1 has a specific binding pocket for flavin molecules, including FAD, FMN, and riboflavin, catalyzing their oxidation while reducing fumarate to succinate. Moreover, ER-resident Osm1 can transfer electrons from the Ero1 FAD cofactor to fumarate either by free FAD or by a direct interaction, allowing de novo disulfide bond formation in the absence of oxygen. We conclude that soluble eukaryotic fumarate reductases can maintain an oxidizing environment under anaerobic conditions, either by oxidizing cellular flavin cofactors or by a direct interaction with flavoenzymes such as Ero1.

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...