Modeling Chemotaxis Reveals the Role of Reversed Phosphotransfer and a Bi-Functional Kinase-Phosphatase

Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY₆, aided by its specific phosphatase, the bifunctional kinase CheA₃, acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB₂ connects the cytoplasmic cluster kinase CheA₃ with the polar localised kinase CheA₂, and allows CheA₃-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA₃ to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.     

Specificity of localization and phosphotransfer in the CheA proteins of Rhodobacter sphaeroides

Specificity of protein–protein interactions plays a vital role in signal transduction. The chemosensory pathway of Rhodobacter sphaeroides comprises multiple homologues of chemotaxis proteins characterized in organisms such as Escherichia coli. Three CheA homologues are essential for chemotaxis in R. sphaeroides under laboratory conditions. These CheAs are differentially localized to two chemosensory clusters, one at the cell pole and one in the cytoplasm. The polar CheA, CheA₂, has the same domain structure as E. coli CheA and can phosphorylate all R. sphaeroides chemotaxis response regulators. CheA₃ and CheA₄ independently localize to the cytoplasmic cluster; each protein has a subset of the CheA domains, with CheA₃ phosphorylating CheA₄ together making a functional CheA protein. Interestingly, CheA₃‐P can only phosphorylate two response regulators, CheY₆ and CheB₂. R. sphaeroides CheAs exhibit two interesting differences in specificity: (i) the response regulators that they phosphorylate and (ii) the chemosensory cluster to which they localize. Using a domain‐swapping approach we investigated the role of the P1 and P5 CheA domains in determining these specificities. We show that the P1 domain is sufficient to determine which response regulators will be phosphorylated in vitro while the P5 domain is sufficient to localize the CheAs to a specific chemosensory cluster.     

Phosphate Flow between Hybrid Histidine Kinases CheA3 and CheS3 Controls Rhodospirillum centenum Cyst Formation

Genomic and genetic analyses have demonstrated that many species contain multiple chemotaxis-like signal transduction cascades that likely control processes other than chemotaxis. The Che₃ signal transduction cascade from Rhodospirillum centenum is one such example that regulates development of dormant cysts. This Che-like cascade contains two hybrid response regulator-histidine kinases, CheA₃ and CheS₃, and a single-domain response regulator CheY₃. We demonstrate that cheS₃ is epistatic to cheA₃ and that only CheS₃∼P can phosphorylate CheY₃. We further show that CheA₃ derepresses cyst formation by phosphorylating a CheS₃ receiver domain. These results demonstrate that the flow of phosphate as defined by the paradigm E. coli chemotaxis cascade does not necessarily hold true for non-chemotactic Che-like signal transduction cascades.     

Spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity

Signal transduction proteins are often multi‐domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two‐component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so‐called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross‐talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.     

Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi

In the model organism Escherichia coli, the coupling protein CheW, which bridges the chemoreceptors and histidine kinase CheA, is essential for chemotaxis. Unlike the situation in E. coli, Borrelia burgdorferi, the causative agent of Lyme disease, has three cheW homologues (cheW₁, cheW₂ and cheW₃). Here, a comprehensive approach is utilized to investigate the roles of the three cheWs in chemotaxis of B. burgdorferi. First, genetic studies indicated that both the cheW₁ and cheW₃ genes are essential for chemotaxis, as the mutants had altered swimming behaviours and were non‐chemotactic. Second, immunofluorescence and cryo‐electron tomography studies suggested that both CheW₁ and CheW₃ are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW₁ and cheW₃, cheW₂ is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW₁ and CheW₃ interact with CheA₂ whereas CheW₂ binds to CheA₁. Collectively, our results indicate that CheW₁ and CheW₃ are incorporated into one chemosensory pathway that is essential for B. burgdorferi chemotaxis. Although many bacteria have more than one homologue of CheW, to our knowledge, this report provides the first experimental evidence that two CheW proteins coexist in one chemosensory pathway and that both are essential for chemotaxis.     

Structural Basis for the Localization of the Chemotaxis Phosphatase CheZ by CheAS

CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.During bacterial chemotaxis, transmembrane receptors regulate the activity of the chemotaxis-specific histidine autokinase CheA with the aid of a coupling protein, CheW. CheA acts to phosphorylate the response regulator CheY and the response regulator domain of the methylesterase CheB. Phosphorylated CheY (CheY-P) binds to the “switch complex” in the flagellar motor to regulate the sense of rotation of the motor. CheZ acts as a CheY phosphate phosphatase.Maddock and Shapiro (4) showed that the chemotaxis receptors tend to be clustered and often located at polar ends of bacterial cells. This localization of receptors is in large part dependent on the presence of CheA and CheW, and the clusters that form in wild-type cells contain receptors, CheA, CheW, CheY, and CheZ (8). These clusters are essential for proper communication among receptors and other members of the signal transduction complex.In Escherichia coli and many related bacteria, a naturally occurring short form of CheA (CheA_S) (7) interacts with CheZ, enhances the rate of dephosphorylation of CheY-P (5, 10), and is responsible for the localization of CheZ to the polar assemblies of receptors, CheA, and CheW (1). Having the kinase and the phosphatase colocalized generates more uniform CheY levels within the bacterial cell (9).In order to understand the structural basis of the CheA_S-CheZ interaction, we examined a CheA fragment containing residues 98 to 150 (CheA_98-150). This fragment begins at the alternative site of translation initiation for CheA_S and extends into the linker region joining the histidine phosphotransfer domain to the CheY-binding domain. This fragment includes residues that correspond to the C terminus of the fourth helix and the complete fifth helix of the intact histidine phosphotransfer domain, also known as the P1 domain. Figure ​Figure11 depicts the binding of CheA_98-150 to CheZ, detected by changes in the fluorescence of the tryptophan residues of CheZ. The data points represent the fluorescence intensities from the complex, plotted against CheA_98-150 concentrations. Assay results were collected in triplicate, and the data points indicate the mean values. The fluorescence intensity (excitation wavelength, 295 nm; emission wavelength, 340 nm) was monitored after each addition and corrected for the blank buffer. The solid lines represent least-squares fits to a two-state binding model. As shown in Fig. ​Fig.1,1, CheA_98-150 binds to CheZ with a dissociation constant in the nanomolar range, with a stoichiometry of one CheA_98-150 molecule per CheZ dimer.Open in a separate windowFIG. 1.Intrinsic tryptophan fluorescence detection of the interactions of wild-type CheZ (A) and CheZ_65-139 (B) with CheA_98-150. Wild-type CheZ and CheZ_65-139 (both present at 1.0 μM) were titrated with CheA_98-150 to produce saturation binding curves at 25°C. The dissociation constants fit to values between 10 and 30 nM but are too strong to be determined accurately at these CheZ concentrations. arb units, arbitrary units.Figure ​Figure22 shows the ¹H-¹⁵N correlation spectrum for CheA_98-150. The resonances were mostly resolved and sharp, with a limited dispersion of chemical shifts along the ¹H dimension, suggesting a high degree of backbone mobility (2). Complete backbone assignments for the nonproline residues in CheA_98-150 were made using standard HNCACB and CBCA(CO)NH methods (6). We have assigned Glu100 through His154 (a residue of the His₆ tag). Although the nuclear magnetic resonance (NMR) spectra and ¹⁵N relaxation properties (3) suggest that this fragment has no stable structure under these conditions, the central region (Asp112 to Glu133) exhibits positive (¹H-)¹⁵N nuclear Overhauser effects and large, positive (up to 2.6 ppm) Cα secondary shifts (11), consistent with a partially rigid, helical structure.Open in a separate windowFIG. 2.¹H-¹⁵N heteronuclear single-quantum coherence spectra of 100 mM ¹⁵N-labeled CheA_98-150 in the absence (black) and presence (red) of 200 mM unlabeled CheZ. Amino acid residues disappearing (black) or displaying significant chemical-shift perturbations (red) are identified.To establish which residues of CheA_98-150 are involved in CheZ binding, we titrated ¹⁵N-labeled CheA_98-150 with unlabeled wild-type CheZ. The spectra were collected using a mixture of 50 mM sodium phosphate buffer, pH 6.8, 1 mM EDTA, and 2 mM dithiothreitol at 25°C. The ¹H-¹⁵N resonances from Asp112 to Glu133, the fifth helix of the N-terminal domain in CheA, weakened as CheZ was added, and these peaks disappeared with the addition of two molar equivalents of CheZ subunits. A new set of resonances, albeit somewhat broadened, appeared near Asp112 to Glu133, suggesting that these residues are at the periphery of the binding region and retain sufficient mobility in the bound state to be observed. New resonances for these residues from the bound state strengthened, whereas resonances from the free state weakened, as the CheZ concentration increased. This pattern of resonance changes is characteristic of an interaction that is in the slow-exchange regime on the NMR time scale. The disappearance of the CheA resonances at a 1:2 ratio with CheZ again indicates that the binding stoichiometry is two CheZ monomers per CheA_98-150 molecule, and the bound complex in the protonated form is likely too large (∼100 kDa) or elongated to be observed.To examine the CheA-CheZ interaction in more detail, we assigned the amide resonances from CheA_98-150 in a complex with a shortened form of CheZ comprising residues 65 to 139 (CheZ_65-139). We chose to focus on this central region of CheZ because it gave sharper CheA resonances in the complex but still bound CheA_98-150 very strongly. The ¹³C backbone chemical shifts of the bound form of CheA_98-150 support a fully helical structure (12) for the region of Asp 112 to Glu133.Results from chemical shift perturbation experiments with CheZ_65-139 indicate that the CheA_98-150-binding site in CheZ is the helix bundle tip, where several aromatic residues cluster (data not shown). Several extreme-upfield methyl proton resonances of CheZ_65-139 shifted downfield upon binding with CheA_98-150, indicating a reorganization of aliphatic methyl groups and aromatic rings in CheZ. Many CheZ_65-139 resonances shifted upon binding, strongly indicating that CheA_S induces global structural changes that propagate from the binding site toward the central, CheY-binding region. This possibility is consistent with the observed perturbations of histidine side chain resonances of CheZ_65-139 at the other end of the helix bundle (data not shown). The imidazole rings from the four histidines in a CheZ_65-139 dimer are situated 25 to 30 Å from the helix bundle tip. The resonances from the imidazole nitrogen atoms and carbon-bound protons detected by ¹H-¹⁵N correlation spectra are clearly affected by the binding of CheA_98-150.We identified the region from Asp112 to Glu133 in CheA_98-150 as being responsible for CheZ binding. This region corresponds to the fifth helix in the intact P1 domain of CheA. NMR data indicate that this region is still mildly helical in CheA_98-150, although it lacks folding cooperativity. The helical content is enhanced by CheZ binding. A model of the complex with one CheA and two CheZ molecules was built (Fig. ​(Fig.3).3). The CheA_S helix formed by residues 112 to 133 is shown bound to an opening formed by the two helical hairpins in a CheZ dimer. A space-filling model (not shown) indicates that there is not enough room to accommodate the CheA helix, suggesting that near the hairpin turn region the four-helix bundle of CheZ expands upon the binding of CheA_S, resulting in structural changes remote from the binding area. This assumption is consistent with the extensive peak movements observed in the CheZ_65-139 spectrum upon the binding of CheA_S. These binding-induced structural changes near the middle of the CheZ helical bundle are likely to be responsible for enhanced CheY-P-binding affinity and/or catalysis of phosphate hydrolysis, leading to increased CheY-P phosphatase activity.Open in a separate windowFIG. 3.Model of the CheZ-CheA_S interaction. The model shows residues 65 to 139 of CheZ as a helical ribbon (Protein Data Bank identification number 1KMI). The cluster of aromatic residues in CheZ is shown in magenta, and the helical CheA residues 112 to 133 are shown end-on with blue and red side chains. The CheA helix (residues 112 to 133) was taken from the known structure of the Htp domain of CheA (Protein Data Bank identification number 1I5N) and manually docked with CheZ to maximize hydrophobic contact.     

Rewiring the specificity of two-component signal transduction systems

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.     

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.     

Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.     

Plant two-component systems: principles,functions, complexity and cross talk

Two-component systems have emerged as important sensing/response mechanisms in higher plants. They are composed of hybrid histidine kinases, histidine-containing phosphotransfer domain proteins and response regulators that are biochemically linked by His-to-Asp phosphorelay. In plants two-component systems play a major role in cytokinin perception and signalling and contribute to ethylene signal transduction and osmosensing. Furthermore, developmental processes like megagametogenesis in Arabidopsis thaliana and flowering promotion in rice (Oryza sativa) involve elements of two-component systems. Two-component-like elements also function as components of the Arabidopsis circadian clock. Because of the molecular mode of signalling, plant two-component systems also appear to serve as intensive cross talk and signal integration machinery. In this review we summarize the present knowledge about the principles and functions of two-component systems in higher plants and address several critical points with respect to cross talk, signal integration and specificity.Abbreviations AHK Arabidopsis histidine kinase - AHP Arabidopsis histidine-containing phosphotransfer domain protein - APRR Arabidopsis pseudo response regulator - ARR Arabidopsis response regulator - CCT CONSTANS CONSTANS-like TOC1 - CKI Cytokinin insensitive - CRE Cytokinin response - CTR Constitutive triple response - Ehd Early heading date - EIN Ethylene insensitive - ERS Ethylene response sensor - ETR Ethylene resistant - GARP-motif Found in Golden2 of maize, Arabidopsis B-type response regulators and Chlamydomonas Psr1 - HPt Histidine-containing phosphotransfer domain - NLS Nuclear localization signal - phyB Phytochrome B - TCS Two-component signalling - TOC Timing of CAB (chlorophyll a/b-binding protein) expression - WOL Wooden leg     

A remote CheZ orthologue retains phosphatase function

Aspartyl‐phosphate phosphatases underlie the rapid responses of bacterial chemotaxis. One such phosphatase, CheZ, was originally proposed to be restricted to beta and gamma proteobacter, suggesting only a small subset of microbes relied on this protein. A putative CheZ phosphatase was identified genetically in the epsilon proteobacter Helicobacter pylori (Mol Micro 61:187). H. pylori utilizes a chemotaxis system consisting of CheAY, three CheVs, CheW, CheY_HP and the putative CheZ to colonize the host stomach. Here we investigate whether this CheZ has phosphatase activity. We phosphorylated potential targets in vitro using either a phosphodonor or the CheAY kinase and [γ‐³²P]‐ATP, and found that H. pylori CheZ (CheZ_HP) efficiently dephosphorylates CheY_HP and CheAY and has additional weak activity on CheV2. We detected no phosphatase activity towards CheV1 or CheV3. Mutations corresponding to Escherichia coli CheZ active site residues or deletion of the C‐terminal region inactivate CheZ_HP phosphatase activity, suggesting the two CheZs function similarly. Bioinformatics analysis suggests that CheZ phosphatases are found in all proteobacteria classes, as well as classes Aquificae, Deferribacteres, Nitrospira and Sphingobacteria, demonstrating that CheZ phosphatases are broadly distributed within Gram‐negative bacteria.     

Solution structure of a complex of the histidine autokinase CheA with its substrate CheY

In the bacterial chemotaxis two-component signaling system, the histidine-containing phosphotransfer domain (the "P1" domain) of CheA receives a phosphoryl group from the catalytic domain (P4) of CheA and transfers it to the cognate response regulator (RR) CheY, which is docked by the P2 domain of CheA. Phosphorylated CheY then diffuses into the cytoplasm and interacts with the FliM moiety of the flagellar motors, thereby modulating the direction of flagellar rotation. Structures of various histidine phosphotransfer domains (HPt) complexed with their cognate RR domains have been reported. Unlike the Escherichia coli chemotaxis system, however, these systems lack the additional domains dedicated to binding to the response regulators, and the interaction of an HPt domain with an RR domain in the presence of such a domain has not been examined on a structural basis. In this study, we used modern nuclear magnetic resonance techniques to construct a model for the interaction of the E. coli CheA P1 domain (HPt) and CheY (RR) in the presence of the CheY-binding domain, P2. Our results indicate that the presence of P2 may lead to a slightly different relative orientation of the HPt and RR domains versus those seen in such complex structures previously reported.     

Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis

Two-component signal transduction systems, comprised of histidine kinases and their response regulator substrates, are the predominant means by which bacteria sense and respond to extracellular signals. These systems allow cells to adapt to prevailing conditions by modifying cellular physiology, including initiating programs of gene expression, catalyzing reactions, or modifying protein–protein interactions. These signaling pathways have also been demonstrated to play a role in coordinating bacterial cell cycle progression and development. Here we report a system-level investigation of two-component pathways in the model organism Caulobacter crescentus. First, by a comprehensive deletion analysis we show that at least 39 of the 106 two-component genes are required for cell cycle progression, growth, or morphogenesis. These include nine genes essential for growth or viability of the organism. We then use a systematic biochemical approach, called phosphotransfer profiling, to map the connectivity of histidine kinases and response regulators. Combining these genetic and biochemical approaches, we identify a new, highly conserved essential signaling pathway from the histidine kinase CenK to the response regulator CenR, which plays a critical role in controlling cell envelope biogenesis and structure. Depletion of either cenK or cenR leads to an unusual, severe blebbing of cell envelope material, whereas constitutive activation of the pathway compromises cell envelope integrity, resulting in cell lysis and death. We propose that the CenK–CenR pathway may be a suitable target for new antibiotic development, given previous successes in targeting the bacterial cell wall. Finally, the ability of our in vitro phosphotransfer profiling method to identify signaling pathways that operate in vivo takes advantage of an observation that histidine kinases are endowed with a global kinetic preference for their cognate response regulators. We propose that this system-wide selectivity insulates two-component pathways from one another, preventing unwanted cross-talk.     

High Specificity in CheR Methyltransferase Function: CheR2 OF PSEUDOMONAS PUTIDA IS ESSENTIAL FOR CHEMOTAXIS,WHEREAS CheR1 IS INVOLVED IN BIOFILM FORMATION*

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (K_D of 0.14–2.2 μm) than the substrate S-adenosylmethionine (K_D of 22–43 μm), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.     

The <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

Background  

The Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood.     

The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.