Thermosensor Action of GrpE

Grimshaw, John P.A.; Jelesarov, Ilian; Siegenthaler, Rahel K.; Christen, Philipp

doi:10.1074/jbc.m300924200

Cited by 55 publications

(36 citation statements)

References 41 publications

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“…One of the roles of DnaK is to bind these substrates, protecting them from aggregation (3,28,29). Following a return to normal temperature, DnaK also participates in active refolding (30,31). Similar to what was observed in the in vitro luciferase refolding experiments, the ATPase activity of DnaK appears to be required during heat shock, because active site mutations that abolish nucleotide turnover are unable to rescue heat shock (24 -26).…”

supporting

confidence: 58%

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

Chang

Thompson

Ung

et al. 2010

Journal of Biological Chemistry

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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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confidence: 58%

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

Chang

Thompson

Ung

et al. 2010

Journal of Biological Chemistry

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“…Small heat shock proteins are often expressed in stress situations and assist in protein stability, while the DSBA protein catalyzes disulfide bond formation during folding of secreted proteins (47). The GrpE protein prevents in E. coli, together with DnaK and DnaJ, the aggregation of stress-denatured proteins in response to hyperosmotic and heat shock stress (20).…”

Section: Discussionmentioning

confidence: 99%

Exposure to Solute Stress Affects Genome-Wide Expression but Not the Polycyclic Aromatic Hydrocarbon-Degrading Activity of Sphingomonas sp. Strain LH128 in Biofilms

Fida

Breugelmans

Lavigne

et al. 2012

Appl Environ Microbiol

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e Members of the genus Sphingomonas are important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degrading Sphingomonas sp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.

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“…The concentrations of DnaJ and WT GrpE were determined with ⑀ 277 ϭ 18,100 M Ϫ1 cm Ϫ1 and ⑀ 279 ϭ 2,720 M Ϫ1 cm Ϫ1 , respectively. Mutant GrpE R40C was expressed and purified as described previously (15). Its concentration was determined by amino acid analysis.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…Stabilization of the pair of the long NH 2 -terminal helices in the GrpE dimer with an engineered disulfide bond (R40C) (Fig. 2) abolishes the thermal transition in GrpE and reduces the deviation of the ADP/ATP exchange activity from an Arrhenius temperature dependence, indicating that the long helix pair acts as the primary thermosensor of the chaperone system (15,16).In E. coli, DnaK and its co-chaperones are constitutively expressed as well as induced by heat shock or other cellular stress. This familiar heat shock response is mediated by 32 , which directs RNA polymerase to transcribe the particular set of heat shock genes.…”

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The Importance of Having Thermosensor Control in the DnaK Chaperone System

Siegenthaler

Christen

2005

Journal of Biological Chemistry

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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...

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Thermosensor Action of GrpE

Cited by 55 publications

References 41 publications

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

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

Exposure to Solute Stress Affects Genome-Wide Expression but Not the Polycyclic Aromatic Hydrocarbon-Degrading Activity of Sphingomonas sp. Strain LH128 in Biofilms

The Importance of Having Thermosensor Control in the DnaK Chaperone System

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