Interdomain communication in the molecular chaperone DnaK

Background: DnaK is a Hsp70 (heat shock protein) molecular chaperone whose function is controlled in part by the nucleotide exchange factor GrpE. Results: We describe the structures of several complexes formed between GrpE and different DnaK variants. Conclusion: The DnaK-GrpE complex has several conformations, an important factor in regulating DnaK function. Significance: The information obtained explains the nucleotide exchange role of GrpE in much more detail.

Section: Discussionsupporting

confidence: 77%

Section: Dnak-grpe Complex Formation With the Dnak And Grpesupporting

confidence: 73%

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Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Melero

Moro

Pérez-Calvo

et al. 2015

“…Several studies have shown evidence for such communication using intrinsic tryptophan fluorescence or intramolecular suppression of mutations in distinct domains (31)(32)(33)(34)(35). The data presented in Fig.…”

Section: Sse1 Domains Can Function and Interact In Trans-mentioning

confidence: 81%

The Function of the Yeast Molecular Chaperone Sse1 Is Mechanistically Distinct from the Closely Related Hsp70 Family

Shaner

Trott

Goeckeler

et al. 2004

130

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1⌬ yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1⌬ and lethality of sse1⌬sse2⌬ cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1⌬ growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

“…The rate of the conversion was determined at fixed temperatures within the physiologically relevant range during a stepwise increase in temperature from 15 to 48°C. Two different types of measurements were performed: (i) DnaK possesses a single tryptophan residue at position 102, which allows fluorescence spectroscopic monitoring of conformational changes including those accompanying the R 3 T conversion (6,11,26,27) and (ii) fluorescence-labeled ADP allows to monitor the release of the nucleotide, the rate-determining step in ADP/ATP exchange, which underlies the R 3 T conversion (10,17). With both reduced and oxidized GrpE R40C, the rates of the R 3 T conversion of DnaK at 25°C, followed by the decrease in either intrinsic fluorescence of DnaK or fluorescence of MABA-ADP, were similar to the rates that had been measured with wild-type GrpE.…”

Section: Fig 2 Formation Of the Disulfide-linked Grpe R40c Dimermentioning

confidence: 99%

Thermosensor Action of GrpE

Grimshaw

Jelesarov

Siegenthaler

et al. 2003

Self Cite

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...