Specificity of DnaK-peptide Binding

Gragerov, Alexander; Zeng, Li; Zhao, Xiaoliang; Burkholder, William F.; Gottesman, Max E.

doi:10.1006/jmbi.1994.1043

Cited by 239 publications

(222 citation statements)

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“…Nucleotide binding and hydrolysis are important steps of the action Hsp70/DnaK proteins. In vitro studies have shown that Hsp70/DnaK proteins bind both denatured proteins and some short peptides, and release these substrates in response to the addition of ATP [47,48]. Hsp70/DnaK proteins have a weak ATPase activity and a peptide binding activity with a mechanism of coupling these two activities such that the addition of ATP induces peptide release [5,49].…”

Section: Secondary Structure Of Bldnakmentioning

confidence: 99%

“…The open/closed state of the Hsp70/ DnaK substrate-binding domain, which is mediated by the repositioning of a α-helical lid over the substrate-binding pocket, is governed by the nucleotide occupancy and status in the ATPase domain [5,50]. The conformational change induced by ATP is the switch that releases the bound peptides and denatured proteins in vivo [47,48]. Accordingly, our results confi rm that the secondary structures of Hsp70/ DnaK proteins are insensitive to change in the presence of nucleotide.…”

Section: Secondary Structure Of Bldnakmentioning

confidence: 99%

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A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

et al. 2009

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The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purifi ed to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purifi ed BlDnaK was 40°C in the presence of 100 mM KCl. The purifi ed BlDnaK had a V max of 32.5 nmol Pi/min and a K M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg 2+ and K + ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.

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Section: Secondary Structure Of Bldnakmentioning

confidence: 99%

Section: Secondary Structure Of Bldnakmentioning

confidence: 99%

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

et al. 2009

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“…(A DnaK-binding motif contains a hydrophobic core flanked by positively charged residues [21]. Leucine residues within the hydrophobic core are particularly important, because the substrate-binding site of DnaK contains a leucine-pocket [19].)…”

Section: Discussionmentioning

confidence: 99%

The insect antimicrobial peptide, -pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

CHESNOKOVA¹

2004

FEBS Letters

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Recent reports have indicated that insect antimicrobial peptides kill bacteria by inhibiting the molecular chaperone DnaK. It was proposed that the antimicrobial peptide, all-L L -pyrrhocoricin (L L -PYR), binds to two sites on DnaK, the conventional substratebinding site and the multi-helical C-terminal lid, and that inhibition of DnaK comes about from the lid mode of binding. In this report, we show using two different assays that L L -PYR binds to and stimulates the ATPase activity of both wild-type and a lidless variant of DnaK. Our study shows that L L -PYR interacts with DnaK much like the all-L L NR (NRLLLTG) peptide, which is known to bind in the conventional substrate-binding site of DnaK. L L -PYR antimicrobial activity is thus a consequence of the competitive inhibition of bacterial DnaK.

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“…Steady state levels can be found in [5,6]; they vary from approximately 4000 at T 13.5 to approximately 6000 at 37 ± C. DnaK is a chaperone and it has a high affinity for hydrophobic residues [7]; as these signal a possible misfold, s 32 controls the expression of DnaK by binding to the RNAp. One expects at most a few hundred s 32 in the cell, a number which is dynamical adjustable because of the short in vivo half-life of s 32 (0.7 min at 42 ± C and 15 min at 22 ± C [8,9]).…”

Section: (Received 29 July 1999)mentioning

confidence: 99%

“…The key inhibitory control mechanism is the association of DnaK to s 32 . DnaK binds to unfolded protein residues [7], and the amount of DnaK-s 32 association thereby monitors cellular consequences of a shift in temperature.…”

Section: (Received 29 July 1999)mentioning

confidence: 99%

Thermodynamics of Heat-Shock Response

Arnvig¹,

Pedersen²,

Sneppen

2000

Phys. Rev. Lett.

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Production of heat-shock proteins is induced when a living cell is exposed to a rise in temperature. The heat-shock response of protein DnaK synthesis in E.coli for temperature shifts T ! T 1 DT and T ! T 2 DT is measured as a function of the initial temperature T . We observe a reversed heat shock at low T. The magnitude of the shock increases when one increases the distance to the temperature T 0 ഠ 23 ± C, thereby mimicking the nonmonotonous stability of proteins at low temperature. This suggests that stability related to hot as well as cold unfolding of proteins is directly implemented in the biological control of protein folding.PACS numbers: 87.14. Ee, Chaperones direct protein folding in the living cell by binding to unfolded or misfolded proteins. The expression level of many of these catalysts of protein folding changes in response to environmental changes. In particular, when any living cell is exposed to a temperature shock the production of these evolutionary conserved proteins is transiently increased [1]. The heat-shock (HS) response in E.coli involves about 40 widely dispersed genes and is mediated through the s 32 protein [2,3]. The s 32 binds to RNA polymerase (RNAp), where it displaces the s 70 subunit and thereby changes RNAp's affinity to a number of promoters. This induces production of the heat-shock proteins. If the gene for s 32 is removed from the E.coli genome, the HS is suppressed [2,4] and also the cell cannot grow above 20 ± C.The HS is fast. In some cases it can be detected by a changed synthesis rate of, e.g., the chaperone protein DnaK, about a minute after the temperature shift. Given that the DnaK protein in itself takes about 45 seconds to synthesize, the observed fast change in DnaK production must be very close to the physical mechanism that triggers the response. We will argue for a mechanism that does not demand an additional synthesis of s 32 and thus postulate that a changed synthesis of s 32 only plays a role in the latter stages of the HS. To quantify the physical mechanism we measure the dependence of HS with initial temperature and find that the magnitude of the shock is inversely proportional to the folding stability of a typical globular protein.This paper measures the expression of protein DnaK. Steady state levels can be found in [5,6]; they vary from approximately 4000 at T 13.5 to approximately 6000 at 37 ± C. DnaK is a chaperone and it has a high affinity for hydrophobic residues [7]; as these signal a possible misfold, s 32 controls the expression of DnaK by binding to the RNAp. One expects at most a few hundred s 32 in the cell, a number which is dynamical adjustable because of the short in vivo half-life of s 32 (0.7 min at 42 ± C and 15 min at 22 ± C [8,9]). The lifetime s 32 is known to increase transiently under the HS.The measurement was on an E.coli K12 strain grown on an A 1 B medium with a 3 H labeled amino acid as described in [10]. After the temperature shift we extracted samples of the culture at subsequent times. Each sample was exposed to radioact...

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Specificity of DnaK-peptide Binding

Cited by 239 publications

References 0 publications

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

The insect antimicrobial peptide, -pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

Thermodynamics of Heat-Shock Response

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