Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of <i>Escherichia coli</i> using Ni‐histidine tag affinity chromatography

Hidaka, Yuji; Park, Heiyoung; Inouye, Masayori

doi:10.1016/s0014-5793(96)01396-8

Cited by 30 publications

(19 citation statements)

References 30 publications

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“…2a. Because it has been demonstrated that EnvZ(C) forms a dimer (20,26), the present result indicates that the region required for the dimer formation resides in the 67 residue of subdomain A. This domain also contains the region required for OmpR interaction, because OmpR was trapped on the resin only in the presence of His-tagged subdomain A (compare lane 4 and lane 5 in Fig.…”

Section: Resultssupporting

confidence: 53%

“…After the sample was washed three times with buffer II [50 mM sodium phosphate buffer (pH 6.0)͞0.3 M NaCl͞5% glycerol] by using ultrafree-MC centrifugal filters (Millipore), proteins bound to Ni-NTA resin were eluted with 0.2 M imidazole/buffer II. The binding experiments between OmpR and either H6-EnvZ(C)⅐wt or H6-EnvZ(C)⅐(223-289) were carried out as described (26). The proteins eluted with 0.2 M imidazole/ buffer II in each binding assay were subjected to SDS͞PAGE (20% gel) and then stained with silver (27).…”

Section: ј-Tatgcaccatcaccatcacca-3јmentioning

confidence: 99%

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Two-domain reconstitution of a functional protein histidine kinase

Park¹,

Saha²,

Inouye³

1998

Proc. Natl. Acad. Sci. U.S.A.

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In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C)⅐(223-289); 67 residues] and subdomain B [EnvZ(C)⅐(290-450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H-243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an ␣/␤-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.

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Section: Resultssupporting

confidence: 53%

Section: ј-Tatgcaccatcaccatcacca-3јmentioning

confidence: 99%

Two-domain reconstitution of a functional protein histidine kinase

Park¹,

Saha²,

Inouye³

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…Most mutations that cluster near or at the site of phosphorylation result in a locked high osmolarity phenotype. Mutations and deletions of the linker region exhibit various low and high osmolarity locked phenotypes and provide evidence that this region is critical for signalling (Park & Inouye 1997). Periplasmic deletions result in a locked high osmolarity phenotype or have no effect (reviewed in Forst & Roberts 1994;Leonardo & Forst 1996).…”

Section: Genetic Analysismentioning

confidence: 99%

“…In contrast, two EnvZ phosphatase mutants cannot complement each other which implies that the kinase and phosphatase activities may be regulated as a dimer to monomer transition, respectively. The K a for EnvZ dimerization has been estimated at 10 5 M ¹1 (Hidaka et al 1997).…”

Section: Catalytic Domainmentioning

confidence: 99%

Signal transduction via the histidyl‐aspartyl phosphorelay

1997

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The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is the predominant mode of signal transduction in bacteria. Adaptation to environmental changes occurs through a sensor histidine protein kinase and a response regulator. The histidine protein kinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein.Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation events that control downstream effectors. Following autophosphorylation at a conserved histidine residue, the histidine kinase serves as a phospho-donor for the response regulator. Once phosphorylated, the response regulator mediates changes in gene expression or cellular locomotion. The EnvZ-OmpR phosphorelay system in Escherichia coli, which monitors external osmolarity and responds by differentially modulating the expression of the OmpF and OmpC major outer membrane porins, will be described as a model system. While histidine kinases were thought to be present only in prokaryotes, they have recently been identified in eukaryotic systems. Here, we review the unique and conserved features of this growing family of signal transducers.

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“…WalK cytoplasmic domains have been purified, and results of in vitro studies show that they are autophosphorylated and phosphorylate their cognate RRs (6,7,17,31,32). Cytoplasmic or truncated forms of HKs have been reported to dimerize in vitro (15). In addition, a solution-state nuclear magnetic resonance (NMR) study (30) described dimerization of the homodimeric core domain of EnvZ, which belongs to the same Pho subfamily of HKs as WalK.…”

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confidence: 99%

Isolation and Characterization of Signermycin B, an Antibiotic That Targets the Dimerization Domain of Histidine Kinase WalK

Watanabe

Igarashi

Okajima

et al. 2012

Antimicrob Agents Chemother

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ABSTRACTThe WalK (histidine kinase)/WalR (response regulator) two-component signal transduction system is a master regulatory system for cell wall metabolism and growth. This system is conserved in low G+C Gram-positive bacteria, includingBacillus subtilis,Staphylococcus aureus,Enterococcus faecalis, andStreptococcus mutans. In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000Streptomycesextracts. The chemical structure (C23H35NO4; molecular weight, 389.5) comprises a tetramic acid moiety and a decalin ring. Signermycin B exhibited antimicrobial activity, with MIC values ranging from 3.13 μg/ml (8 μM) to 6.25 μg/ml (16 μM) against Gram-positive bacteria that possess the WalK/WalR two-component signal transduction system, including the drug-resistant bacteria methicillin-resistantStaphylococcus aureusand vancomycin-resistantEnterococcus faecalis. The half-maximal inhibitory concentrations of signermycin B against WalK in these organisms ranged from 37 to 61 μM. To determine the mechanism of action of signermycin B, surface plasmon resonance response analysis with the two WalK domains ofBacillus subtilisand competition assay with ATP were performed. The results showed that signermycin B binds to the dimerization domain but not the ATP-binding domain of WalK. In the presence of the cross-linker glutaraldehyde, signermycin B did not cause protein aggregation but interfered with the cross-linking of WalK dimers. These results suggest that signermycin B targets the conserved dimerization domain of WalK to inhibit autophosphorylation. InBacillus subtilisandStaphylococcus aureus, signermycin B preferentially controlled the WalR regulon, thereby inhibiting cell division. These phenotypes are consistent with those of cells starved for the WalK/WalR system.

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Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni‐histidine tag affinity chromatography

Cited by 30 publications

References 30 publications

Two-domain reconstitution of a functional protein histidine kinase

Two-domain reconstitution of a functional protein histidine kinase

Signal transduction via the histidyl‐aspartyl phosphorelay

Isolation and Characterization of Signermycin B, an Antibiotic That Targets the Dimerization Domain of Histidine Kinase WalK

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