Comparison of the Pseudomonas aeruginosa and Escherichia coli PhoQ sensor domains: evidence for distinct mechanisms of signal detection

The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.     

Determination of the physiological dimer interface of the PhoQ sensor domain

PhoQ is the transmembrane sensor kinase of the phoPQ two-component system, which detects and responds to divalent cations and antimicrobial peptides and can trigger bacterial virulence. Despite their ubiquity and importance in bacterial signaling, the structure and molecular mechanism of the sensor kinases is not fully understood. Frequently, signals are transmitted from a periplasmic domain in these proteins to the cytoplasmic kinase domains via an extended dimeric interface, and the PhoQ protein would appear to follow this paradigm. However, the isolated truncated periplasmic domain of PhoQ dimerizes poorly, so it has been difficult to distinguish the relevant interface in crystal structures of the PhoQ periplasmic domain. Thus, to determine the arrangement of the periplasmic domains of Escherichia coli PhoQ in the physiological homodimer, disulfide-scanning mutagenesis was used. Single cysteine substitutions were introduced along the N-terminal helix of the periplasmic region, and the degree of cross-linking in each protein variant was determined by Western blotting and immunodetection. The results were subjected to periodicity analysis to generate a profile that provides information concerning the C^β distances between corresponding residues at the interface. This profile, together with a rigid-body search procedure, side-chain placement, and energy minimization, was used to build a model of the dimer arrangement. The final model proved to be highly compatible with one of the PhoQ crystal structures, 3BQ8, indicating that 3BQ8 is representative of the physiological arrangement. The model of the periplasmic region is also compatible with a full-length PhoQ protein in which a four-helix bundle forms in the membrane. The membrane four-helix bundle has been proposed for other sensor kinases and is thought to have a role in the mechanism of signal transduction; our model supports the idea that signaling through a membrane four-helix bundle is a widespread mechanism in the transmembrane sensor kinases.     

Crystal structure of a functional dimer of the PhoQ sensor domain

The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.     

The connector SafA interacts with the multi-sensing domain of PhoQ in Escherichia coli

Sensor histidine kinases of two-component signal transduction systems (TCSs) respond to various environmental signals and transduce the external stimuli across the cell membrane to their cognate response regulators. Recently, membrane proteins that modulate sensory systems have been discovered. Among such proteins is SafA, which activates the PhoQ/PhoP TCS by direct interaction with the sensor PhoQ. SafA is directly induced by the EvgS/EvgA TCS, thus connecting the two TCSs, EvgS/EvgA and PhoQ/PhoP. We investigated how SafA interacted with PhoQ. Bacterial two-hybrid and reporter assays revealed that the C-terminal region (41-65 aa) of SafA activated PhoQ at the periplasm. Adding synthetic SafA(41-65) peptide to the cell culture also activated PhoQ/PhoP. Furthermore, direct interaction between SafA(41-65) and the sensor domain of PhoQ was observed by means of surface plasmon resonance. NMR spectroscopy of (15) N-labelled PhoQ sensor domain confirmed that SafA and Mg(2+) provoked a different conformational change of PhoQ. Site-directed mutagenesis studies revealed that R53, within SafA(41-65), was important for the activation of PhoQ, and D179 of the PhoQ sensor domain was required for its activation by SafA. SafA activated PhoQ by a different mechanism from cationic antimicrobial peptides and acidic pH, and independent of divalent cations and MgrB.     

Mutational analysis of the Escherichia coli PhoQ sensor kinase: differences with the Salmonella enterica serovar Typhimurium PhoQ protein and in the mechanism of Mg2+ and Ca2+ sensing

The PhoP-PhoQ two-component system plays a role in Mg2+ homeostasis and/or the virulence properties of a number of bacterial species. A Salmonella enterica serovar Typhimurium PhoQ sensor kinase mutant, in which the threonine at residue 48 in the periplasmic sensor domain is changed to an isoleucine, was shown previously to result in elevated expression of PhoP-activated genes and to affect mouse virulence, epithelial cell invasion, and sensitivity to macrophage killing. We characterized a complete set of proteins having amino acid substitutions at position 48 in the closely related Escherichia coli PhoQ protein. Numerous mutant proteins having amino acid substitutions with side chains of various sizes and characters displayed signaling phenotypes similar to that of the wild-type protein, indicating that interactions mediated by the wild-type threonine side chain are not required for normal protein function. Changes to amino acids with aromatic side chains had little impact on signaling in response to extracellular Mg2+ but resulted in reduced sensitivity to extracellular Ca2+, suggesting that the mechanisms of signal transduction in response to these two divalent cations are different. Surprisingly, the Ile48 protein displayed a defective phenotype rather than the hyperactive phenotype seen with the S. enterica serovar Typhimurium protein. We also describe a mutant PhoQ protein lacking the extracellular sensor domain with a defect in the ability to activate PhoP. The defect does not appear to be due to reduced autokinase activity but rather appears to be due to an effect on the stability of the aspartyl-phosphate bond of phospho-PhoP.     

Characterization of the catalytic activities of the PhoQ histidine protein kinase of Salmonella enterica serovar Typhimurium

Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2) or Mn2+ was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.     

Block by extracellular divalent cations of Drosophila big brain channels expressed in Xenopus oocytes

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain (bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca(2+) and Ba(2+) but not by Mg(2+) in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu(274)) and aspartate (Asp(253)) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu(71)) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu(71) to Asp introduced a new sensitivity to block by extracellular Mg(2+); substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca(2+), suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination.     

The phosphatase activity is the target for Mg2+ regulation of the sensor protein PhoQ in Salmonella

The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Environmental deprivation of Mg(2+) activates the PhoP/PhoQ signal transduction cascade, which results in an increased expression of genes necessary for survival inside the host. It was previously demonstrated that the interaction of Mg(2+) with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down-regulation of PhoP-activated genes. We have now examined the regulatory effect of Mg(2+) on the putative activities of the membrane-bound PhoQ. We demonstrated that Mg(2+) promotes a phospho-PhoP phosphatase activity in the sensor protein. This activity depends on the intactness of the conserved His-277, suggesting that the phosphatase active site overlaps the H box. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, because Mg(2+) did not stimulate the release of inorganic phosphate from phospho-PhoP in a fusion protein that lacks this sensing domain. These findings reveal that the sensor PhoQ harbors a phospho-PhoP phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg(2+)-triggered regulation of the PhoP/PhoQ system.     

The PhoQ histidine kinases of Salmonella and Pseudomonas spp. are structurally and functionally different: evidence that pH and antimicrobial peptide sensing contribute to mammalian pathogenesis

The PhoQ sensor kinase is essential for Salmonella typhimurium virulence for animals, and a homologue exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST-PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST-PhoQ has a periplasmic Per-ARNT-Sim domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA-PhoQ). Here, we demonstrate that PA-PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ-null mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST-PhoQ and PA-PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA-PhoQ binding metal in a specific ligand-binding pocket. PA-PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA-PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.     

Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.

Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca(+2)-dependent exocytotic and Ca(+2)- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible seven-amino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 A crystal structure of synaptotagmin III, residues 295-566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca(+2)-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca(+2)-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.     

Extracellular calcium-sensing receptors in fishes.

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.     

Extracellular calcium-sensing receptors in fishes

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.     

Relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 cell

A study was made on the correlation between the degree of membrane fusion and surface tension increase of phosphatidic acid membranes caused by divalent cations. Membrane fusion was followed by the Tb3+/dipicolinic acid assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of small unilamellar lipid vesicles. The surface tension and surface potential of monolayers made of the same lipids as used in the fusion experiments were measured as a function of divalent cation concentration. It was found that the 'threshold' concentration to induce massive vesicle membrane fusion was the same for Ca2+ and Mg2+, and that the surface tension increase in the monolayer, induced by changing divalent cation concentration from zero to a concentration which corresponds to its threshold value, inducing vesicle membrane fusion, was approximately the same: 6.3 dyn/cm for both Ca2+ and Mg2+. Both the divalent cation's threshold concentrations as well as the surface tension change corresponding to the threshold concentration for the phosphatidic acid membrane were smaller than those for the phosphatidylserine membrane. The different fusion capability of these divalent cations for phosphatidic acid and phosphatidylserine membranes is discussed in terms of the different ion binding capabilities of these ions to the membranes.     

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.     

Fibroblasts contracting collagen matrices form transient plasma membrane passages through which the cells take up fluorescein isothiocyanate-dextran and Ca2+.

When fibroblasts contract collagen matrices, the cells activate a Ca(2+)-dependent cyclic AMP signaling pathway. We have found that contraction also stimulates uptake of fluorescein isothiocyanate-dextran molecules from the medium. Our results indicate that fluorescein isothiocyanate-dextran enters directly into the cell cytoplasm through 3- to 5-nm plasma membrane passages. These passages, which reseal in less than 5 s in the presence of divalent cations, also are likely sites of Ca2+ uptake during contraction and the first step in contraction-activated cyclic AMP signaling. The formation of plasma membrane passages during fibroblast contraction may reflect a general cellular response to rapid mechanical changes.     

Identification of an internal cavity in the PhoQ sensor domain for PhoQ activity and SafA-mediated control

The PhoQ/PhoP two-component signal transduction system is conserved in various Gram-negative bacteria and is often involved in the expression of virulence in pathogens. The small inner membrane protein SafA activates PhoQ in Escherichia coli independently from other known signals that control PhoQ activity. We have previously shown that SafA directly interacts with the sensor domain of the periplasmic region of PhoQ (PhoQ-SD) for activation, and that a D179R mutation in PhoQ-SD attenuates PhoQ activation by SafA. In this study, structural comparison of wild-type PhoQ-SD and D179R revealed a difference in the cavity (SD (sensory domain) pocket) found in the central core of this domain. This was the only structural difference between the two proteins. Site-directed mutagenesis of the residues surrounding the SD pocket has supported the SD pocket as a site involved in PhoQ activity. Furthermore, the SD pocket has also been shown to be involved in SafA-mediated PhoQ control.