Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.13.3 (histidine kinase)
2,405 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Local anesthetics are known to reduce the level of OmpF and increase the synthesis of OmpC in the outer membrane of Escherichia coli K-12. It has been shown that the anesthetics procaine and phenethyl alcohol (PEA) act at the transcriptional level for ompF and ompC and that in the case of procaine, its action is dependent on EnvZ, the membrane-bound signal transducer required for ompF and ompC expression. In an effort to further understand how anesthetics regulate ompF and ompC expression, we have analyzed the DNA binding properties of OmpR (the transcriptional activator protein for ompF and ompC genes) from cells treated with procaine or PEA. Treatment of a wild-type cell with either anesthetic converted OmpR from a low-affinity DNA binding form to a high-affinity DNA binding form. The change in DNA binding affinity was correlated with alterations in outer membrane porin profiles and could occur in the absence of protein synthesis. A strain lacking EnvZ was unable to respond to procaine to produce either the shift in the OmpR DNA binding property or cause any change in the outer membrane porin profile. PEA treatment was also dependent on EnvZ for the alteration in the OmpR DNA binding property, but it could induce ompC expression in the absence of EnvZ. Further studies suggest that the amino-terminal region of EnvZ is responsible for the procaine signalling. Our results indicate that procaine and PEA regulate ompF and ompC expression by modifying the DNA binding properties of OmpR through EnvZ signal transduction.
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PMID:Procaine, a local anesthetic, signals through the EnvZ receptor to change the DNA binding affinity of the transcriptional activator protein OmpR. 171 43

The Tar-EnvZ hybrid molecule (Taz1) is an inner membrane transducer that activates OmpR, a transcriptional activator for porin gene expression (ompC), in response to an aspartic acid signal. Signal transduction by Taz1 most likely involves a phosphorylated Taz1 intermediate that donates its phosphate to OmpR. Phosphorylated OmpR has already been implicated in transcriptional activation of porin genes. Using a cell-free system containing Taz1-enriched membrane fractions, we have examined the phosphorylation properties of Taz1 and the stimulatory effects of divalent and monovalent ions. Highest activation of Taz1 phosphorylation was observed with CaCl2, and its stimulation could be observed with as low as 60 microM of CaCl2. Phosphorylated Taz1 could readily donate its phosphate group to OmpR in the presence of calcium. CaCl2 was also able to enhance phosphorylation of intact membrane-bound EnvZ and a cytoplasmic fragment of EnvZ lacking the receptor and transmembrane domains. These results indicate that the site for CaCl2 stimulation is within the cytoplasmic region of EnvZ and probably involves an enhanced rate of EnvZ phosphorylation.
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PMID:Ca2(+)-enhanced phosphorylation of a chimeric protein kinase involved with bacterial signal transduction. 185 Apr 14

The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.
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PMID:Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate. 247 47

EnvZ is a membrane-bound histidine kinase that functions as an osmotic sensor capable of phosphorylating the regulator protein OmpR in Escherichia coli. To characterize the site of phosphorylation biochemically, we overexpressed a 36-kDa truncated EnvZ protein (Glu-106 to Gly-450) that formed inclusion bodies in the cell. After solubilization, the inclusion body form of EnvZ was cleaved into two major fragments with molecular weights of 25,000 and 10,000. The 25-kDa fragment, EnvZc, was purified and found to exist as a dimer. N-terminal sequence analysis established that cleavage had occurred at Arg-214, indicating that EnvZc contained most of the cytoplasmic domain of EnvZ. After labeling EnvZc with [gamma-32P]ATP, the protein was proteolytically digested, and the resulting peptides were separated by reverse phase chromatography using high performance liquid chromatography. One major radioactive peptide containing greater than 90% of the recovered peptide-associated radioactivity was isolated. Amino acid analysis of this purified peptide indicated that the composition was consistent with a peptide that contained His-243. The amino acid sequence of this peptide was determined to be MAGVSHDLRTP (residues 238-248). These results indicate that His-243 is the major site of phosphorylation on EnvZ and represents the first biochemical characterization of the site of phosphorylation of a membrane histidine kinase of the two-component regulatory family of molecules in bacteria.
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PMID:Identification of the site of phosphorylation on the osmosensor, EnvZ, of Escherichia coli. 813 3

The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an alpha-helical pattern of exposed and buried positions spanning residues 270-309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinase-activating state. Each of the lock-on disulfides cross-links the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membrane-bound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.
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PMID:Cysteine and disulfide scanning reveals a regulatory alpha-helix in the cytoplasmic domain of the aspartate receptor. 940 66

Escherichia coli responds rapidly to K+-limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating Kdp-ATPase. This process is controlled by the membrane-bound histidine kinase KdpD and the response regulator KdpE. Here, it is demonstrated that replacements of the native Cys residues at positions 409, 852, and 874 influence distinct activities of KdpD, whereas replacements of Cys residues at positions 32, 256, and 402 have no effect. Replacements of Cys409 in KdpD reveal that transmembrane domain I is important for perception and/or propagation of the stimulus. When Cys409 is replaced with Ala, kdpFABC expression becomes constitutive regardless of the external stimuli. In contrast, when Cys409 is replaced with Val or Tyr, induction of kdpFABC expression in response to different stimuli is drastically reduced. KdpD with Ser at position 409 supports levels of kdpFABC expression comparable to those seen in wild-type. Since neither the kinase nor phosphatase activity of these proteins is affected, it is proposed that different amino acid side-chains at position 409 alter the switch between the inactive and active forms of the kinase. When Cys852 or Cys874 is replaced with Ala or Ser, kinase activity is reduced to 10% of the wild-type level. However, kinetic studies reveal that the apparent ATP binding affinity is not affected. Surprisingly, introduction of Cys852 and Cys874 into a KdpD protein devoid of Cys residues leads to full recovery of the kinase activity. Labeling studies support the idea that a disulfide bridge forms between these two residues.
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PMID:Effect of cysteine replacements on the properties of the turgor sensor KdpD of Escherichia coli. 967 24

The synthesis of the membrane-bound [NiFe]hydrogenase of Rhodobacter capsulatus (HupSL) is regulated negatively by the protein histidine kinase, HupT, and positively by the response regulator, HupR. It is demonstrated in this work that HupT and HupR are partners in a two-component signal transduction system. The binding of HupR protein to the hupS promoter regulatory region (phupS ) was studied using gel retardation and footprinting assays. HupR protected a 50 bp region localized upstream from the binding site of the histone-like integration host factor (IHF) regulator. HupR, which belongs to the NtrC subfamily, binds to an enhancer site (TTG-N5-CAA) localized at -162/-152 nt. However, the enhancer-binding HupR protein does not require the RpoN sigma factor for transcriptional activation, as is the case for NtrC from enteric bacteria, but functions with sigma70-RNA polymerase, as is the case for R. capsulatus NtrC. Besides, unlike NtrC from Escherichia coli, HupR activates transcription in the unphosphorylated form and becomes inactive by phosphorylation. This was demonstrated by replacing the putative phosphorylation site (D54) of the HupR protein with various amino acids or by deleting it using site-directed mutagenesis. Strains expressing mutated hupR genes showed high hydrogenase activities even in the absence of H2, indicating that hupSL transcription is activated by the binding of unphosphorylated HupR protein. Strains producing mutated HupRD54 proteins were derepressed for hupSL expression as were HupT- mutants. It is shown that the phosphorylated form of HupT was able to transfer phosphate to wild-type HupR protein but not to mutated D54 HupR proteins. Thus, it is concluded that HupT and HupR are the partners of a two-component regulatory system that regulates hupSL gene transcription.
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PMID:The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two-component HupT/HupR system. 1059 24

Many bacteria are motile and use a conserved class of transmembrane sensory receptor to regulate cellular taxis toward an optimal living environment. These conserved receptors are typically stimulated by extracellular signals, but also undergo adaptation via covalent modification at specific sites on their cytoplasmic domains. The function of the cytoplasmic domain is to integrate the extracellular and adaptive signals, and to use this integrated information to regulate an associated histidine kinase. The kinase, in turn, triggers a cytoplasmic phosphorylation pathway of the two-component class. The high-resolution structure of a receptor cytoplasmic domain has recently been determined by crystallographic methods and is largely consistent with a structural model independently generated by chemical studies of the domain in the full-length, membrane-bound receptor. These results represent an important step toward a mechanistic understanding of receptor-to-kinase information transfer.
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PMID:Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors. 1098 36

The membrane-bound histidine kinase KdpD is a putative turgor sensor that regulates, together with the response regulator KdpE, the expression of the kdpFABC operon coding for the high affinity K(+)-uptake system KdpFABC of Escherichia coli. To elucidate the nature of the primary stimulus for KdpD, we developed an in vitro assay based on right-side-out membrane vesicles. Conditions were varied inside and outside of the vesicles, and KdpD autophosphorylation activity was tested. It was shown that an increase of the ionic strength inside the vesicles was accompanied by an increase of the autophosphorylation activity of KdpD with ATP. However, K(+) at concentrations higher than 1 mm inhibited KdpD autophosphorylation activity. This K(+)-specific effect was not observed with KdpD-Arg-511 --> Gln, a KdpD derivative, which causes K(+)-independent kdpFABC expression. When the osmolality outside the vesicles was increased, autophosphorylation activity of KdpD was stimulated, whereby salts were more effective than sugars. Treatment of the vesicles with amphipathic compounds did not affect KdpD autophosphorylation activity. Based on these results it is proposed that changes of intracellular parameters elicited by K(+) limitation or osmotic upshock directly influence KdpD autophosphorylation activity, whereby K(+) has an inhibitory and ionic strength a stimulatory effect.
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PMID:K+ and ionic strength directly influence the autophosphorylation activity of the putative turgor sensor KdpD of Escherichia coli. 1101 46

Bacterial two-component regulatory systems control the expression of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA sequences and to alter gene transcription. UhpB is the HK protein which controls production of the sugar phosphate transporter UhpT. Elevated expression of full-length UhpB or of a soluble hybrid protein, GST-Bc, which is glutathione S-transferase (GST) fused to the cytoplasmic C-terminal portion of UhpB, results in complete blockage of uhpT expression in a uhp(+) strain. This dominant-negative interference could result from the ability of GST-Bc to bind and sequester the RR UhpA and to accelerate its dephosphorylation. The portion of GST-Bc responsible for the interference phenotype was localized using truncation, linker insertion, and point mutations to the region between residues 293 and 366 flanking His-313, the putative site of autophosphorylation. Point mutations which allow GST-Bc to activate uhpT expression or which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the phosphoryl transfer domain of EnvZ, which forms half of an interdimer four-helix bundle and is responsible for dimerization of its cytoplasmic domain. The expression of GST fusion proteins carrying the corresponding portions of EnvZ strongly interfered with the activation of porin gene expression by OmpR. The GST-Bc protein accelerated dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA to GST-Bc was observed in the presence of the metal chelator EDTA and depended on the presence of His-313. Phosphate transfer from P-UhpA to the liberated phosphoryl transfer domain also occurred. Taken together, these results indicate that the phosphoryl transfer-dimerization domain of UhpB participates in the specific binding of UhpA, in the control of autokinase activity, and in the dephosphorylation of P-UhpA.
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PMID:The phosphoryl transfer domain of UhpB interacts with the response regulator UhpA. 1132 44


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