Stimulus perception in bacterial signal-transducing histidine kinases.

Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.     

Differential localization of two histidine kinases controlling bacterial cell differentiation

The bacterium C. crescentus coordinates cellular differentiation and cell cycle progression via a network of signal transduction proteins. Here, we demonstrate that the antagonistic DivJ and PleC histidine kinases that regulate polar differentiation are differentially localized as a function of the cell cycle. The DivJ kinase localizes to the stalked pole in response to a signal at the G1-to-S transition, while the PleC kinase is localized to the flagellar pole in swarmer and predivisional cells but is dispersed throughout the cell in the stalked cell. PleC, which is required for DivJ localization, may provide the cue at the G1-to-S transition that directs the polar positioning of DivJ. The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle.     

A bacterial inhibitor of animal protein kinases

Cellular extracts ofEscherichia coli severely impair the protein-phosphorylating activity in vitro of animal enzymes of both the casein-kinase type and the histone-kinase type. This blockade is due to a protein, probably dimeric, acting as a noncompetitive inhibitor, which has been purified to near-homogeneity from the soluble fraction of bacterial cells by chromatographic procedures.     

Mammalian histidine kinases

Protein phosphorylation is one of the most ubiquitous and important types of post-translational modification for the regulation of cell function. The importance of two-component histidine kinases in bacteria, fungi and plants has long been recognised. In mammals, the regulatory roles of serine/threonine and tyrosine kinases have attracted most attention. However, the existence of histidine kinases in mammalian cells has been known for many years, although little is still understood about their biological roles by comparison with the hydroxyamino acid kinases. In addition, with the exception of NDP kinase, other mammalian histidine kinases remain to be identified and characterised. NDP kinase is a multifunctional enzyme that appears to act as a protein histidine kinase and as such, to regulate the activation of some G-proteins. Histone H4 histidine kinase activity has been shown to correlate with cellular proliferation and there is evidence that it is an oncodevelopmental marker in liver. This review mainly concentrates on describing recent research on these two types of histidine kinase. Developments in methods for the detection and assay of histidine kinases, including mass spectrometric methods for the detection of phosphohistidines in proteins and in-gel kinase assays for histone H4 histidine kinases, are described. Little is known about inhibitors of mammalian histidine kinases, although there is much interest in two-component histidine kinase inhibitors as potential antibiotics. The inhibition of a histone H4 histidine kinase by genistein is described and that of two-component histidine kinase inhibitors of structurally-related mammalian protein kinases. In addition, recent findings concerning mammalian protein histidine phosphatases are briefly described.     

Mammalian protein histidine kinases

The existence of protein kinases, known as histidine kinases, which phosphorylate their substrates on histidine residues has been well documented in bacteria and also in lower eukaryotes such as yeast and plants. Their biological roles in cellular signalling pathways within these organisms have also been well characterised. The evidence for the existence of such enzymes in mammalian cells is much less well established and little has been determined about their cellular functions. The aim of the current review is to present a summary of what is known about mammalian histidine kinases. In addition, by consideration of the chemistry of phosphohistidine, what is currently known of some mammalian histidine kinases and the way in which they act in bacteria and other eukaryotes, a general role for mammalian histidine kinases is proposed. A histidine kinase phosphorylates a substrate protein, by virtue of the relatively high free energy of hydrolysis of phosphohistidine the phosphate group is easily transferred to either a small molecule or another protein with which the phosphorylated substrate protein specifically interacts. This allows a signalling process to occur, which may be downregulated by the action of phosphatases. Given the known importance of protein phosphorylation to the regulation of almost all aspects of cellular function, the investigation of the largely unexplored area of histidine phosphorylation in mammalian cells is likely to provide a greater understanding of cellular action and possibly provide a new set of therapeutic drug targets.     

The HWE histidine kinases, a new family of bacterial two-component sensor kinases with potentially diverse roles in environmental signaling

Two-component signal transduction pathways play a major role in the response of bacteria to external cues. These pathways are initiated by large collection of histidine kinases (HKs) containing a sensor domain that perceives the environmental signal followed by an HK domain that triggers a histidine-aspartate phosphorelay. Previous phylogenetic analyses identified 11 major families of two-component HKs by comparing signature motifs within the HK domain. Here we describe a new family with homology to Agrobacterium tumefaciens BphP2, an HK first discovered by the presence of a phytochrome sensor domain involved in light perception. Members of this sensor HK family differ from most others by the absence of a recognizable F box and the presence of several uniquely conserved residues, including a histidine in the N box and a tryptophan-X-glutamic acid sequence in the G1 box, which we have used to define the family (HWE). At least 81 members were identified in a variety of alpha- and gamma-proteobacteria, with a significant enrichment in the Rhizobiaceae family. Several representatives were shown to have HK activity in vitro, supporting their proposed participation in phosphorelays. One or more domains related to signal transduction were evident N-terminal to the HK domain, including chemotactic methyltransferase domains, suggesting that this family has multiple roles in environmental signaling. The discovery of the HWE family further extends the diversity within the HK superfamily and expands the importance of two-component signaling in bacteria.     

Hybrid histidine kinases in pathogenic fungi

Histidine kinases (HK) sense and transduce via phosphorylation events many intra‐ and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N‐terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape‐function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.     

Signal transduction in photoreceptor histidine kinases

Two‐component systems (TCS) constitute the predominant means by which prokaryotes read out and adapt to their environment. Canonical TCSs comprise a sensor histidine kinase (SHK), usually a transmembrane receptor, and a response regulator (RR). In signal‐dependent manner, the SHK autophosphorylates and in turn transfers the phosphoryl group to the RR which then elicits downstream responses, often in form of altered gene expression. SHKs also catalyze the hydrolysis of the phospho‐RR, hence, tightly adjusting the overall degree of RR phosphorylation. Photoreceptor histidine kinases are a subset of mostly soluble, cytosolic SHKs that sense light in the near‐ultraviolet to near‐infrared spectral range. Owing to their experimental tractability, photoreceptor histidine kinases serve as paradigms and provide unusually detailed molecular insight into signal detection, decoding, and regulation of SHK activity. The synthesis of recent results on receptors with light‐oxygen‐voltage, bacteriophytochrome and microbial rhodopsin sensor units identifies recurring, joint signaling strategies. Light signals are initially absorbed by the sensor module and converted into subtle rearrangements of α helices, mostly through pivoting and rotation. These conformational transitions propagate through parallel coiled‐coil linkers to the effector unit as changes in left‐handed superhelical winding. Within the effector, subtle conformations are triggered that modulate the solvent accessibility of residues engaged in the kinase and phosphatase activities. Taken together, a consistent view of the entire trajectory from signal detection to regulation of output emerges. The underlying allosteric mechanisms could widely apply to TCS signaling in general.     

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.     

Mechanism of regulation of receptor histidine kinases

Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.     

New class of bacterial membrane oxidoreductases

A new class of bacterial multisubunit membrane-bound electron-transfer complexes has been identified based on biochemical and bioinformatic data. It contains subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. The complex was purified from the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus, and putative operons for similar complexes were identified in a wide range of bacteria. In most cases, the presence of the new complex is anticorrelated with the cytochrome bc or bf electron-transfer complex, suggesting that it replaces it functionally. This appears to be a widespread yet previously unrecognized protein complex involved in energy metabolism in bacteria.     

Determinants of homodimerization specificity in histidine kinases

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.     

Targeting a highly conserved domain in bacterial histidine kinases to generate inhibitors with broad spectrum activity

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Three (and more) component regulatory systems – auxiliary regulators of bacterial histidine kinases

Two‐component signal transduction (TCST) is the most prevalent mechanism employed by microbes to sense and respond to environmental changes. It is characterized by the signal‐induced transfer of phosphate from a sensor histidine kinase (HK) to a response regulator (RR), resulting in a cellular response. An emerging theme in the field of TCST signalling is the discovery of auxiliary factors, distinct from the HK and RR, which are capable of influencing phosphotransfer. One group of TCST auxiliary proteins accomplishes this task by acting on HKs. Auxiliary regulators of HKs are widespread and have been identified in all cellular compartments, where they can influence HK activity through interactions with the sensing, transmembrane or enzymatic domains of the HK. The effects of an auxiliary regulator are controlled by its regulated expression, modification and/or through ligand binding. Ultimately, auxiliary regulators can connect a given TCST system to other regulatory networks in the cell or result in regulation of the TCST system in response to an expanded range of stimuli. The studies highlighted in this review draw attention to an emerging view of bacterial TCST systems as core signalling units upon which auxiliary factors act.     

Characterization of sporulation histidine kinases of Bacillus anthracis

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

Repurposing Hsp90 inhibitors as antibiotics targeting histidine kinases

To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.     

Atypical modes of bacterial histidine kinase signaling

The environment of a cell has a profound influence on its physiology, development and evolution. Accordingly, the capacity to sense and respond to physical and chemical signals in the environment is an important feature of cellular biology. In bacteria, environmental sensory perception is often regulated by two‐component signal transduction systems (TCSTs). Canonical TCST entails signal‐induced autophosphorylation of a sensor histidine kinase (HK) followed by phosphoryl transfer to a cognate response regulator (RR) protein, which may affect gene expression at multiple levels. Recent studies provide evidence for systems that do not adhere to this archetypal TCST signaling model. We present selected examples of atypical modes of signal transduction including inactivation of HK activity via homo‐ and hetero oligomerization, and cross‐phosphorylation between HKs. These examples highlight mechanisms bacteria use to integrate environmental signals to control complex adaptive processes.     

Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases

An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae. In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation. Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation. Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine. Hence, the isolated enzyme is a protein histidine kinase. A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg. The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone. Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively. The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine. This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.