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.     

Using Structural Information to Change the Phosphotransfer Specificity of a Two-Component Chemotaxis Signalling Complex

Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 Å crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA₃, in complex with its cognate RR, CheY₆. A methionine finger on CheY₆ that nestles in a hydrophobic pocket in CheA₃ was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA₃, CheY₆, and CheB₂. Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA₃-P to CheY₆. Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA₃-P. The structure presented here has allowed us to identify specificity determinants for the CheA–CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction.     

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-component systems and their co-option for eukaryotic signal transduction

Two-component signaling pathways involve histidine kinases, response regulators, and sometimes histidine-containing phosphotransfer proteins. Prevalent in prokaryotes, these signaling elements have also been co-opted to meet the needs of signal transduction in eukaryotes such as fungi and plants. Here we consider the evolution of such regulatory systems, with a particular emphasis on the roles they play in signaling by the plant hormones cytokinin and ethylene, in phytochrome-mediated perception of light, and as integral components of the circadian clock.     

Crystal structure of the histidine-containing phosphotransfer protein ZmHP2 from maize

In higher plants, histidine-aspartate phosphorelays (two-component system) are involved in hormone signaling and stress responses. In these systems, histidine-containing phosphotransfer (HPt) proteins mediate the signal transmission from sensory histidine kinases to response regulators, including integration of several signaling pathways or branching into different pathways. We have determined the crystal structure of a maize HPt protein, ZmHP2, at 2.2 A resolution. ZmHP2 has six alpha-helices with a four-helix bundle at the C-terminus, a feature commonly found in HPt domains. In ZmHP2, almost all of the conserved residues among plant HPt proteins surround this histidine, probably forming the docking interface for the receiver domain of histidine kinase or the response regulator. Arg102 of ZmHP2 is conserved as a basic residue in plant HPt proteins. In bacteria, it is replaced by glutamine or glutamate that form a hydrogen bond to Ndelta atoms of the phospho-accepting histidine. It may play a key role in the complex formation of ZmHP2 with receiver domains.     

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.     

Molecular and biochemical characterization of a novel two-component signal transduction system,amrA- amkA,involved in rifamycin SV production in Amycolatopsis mediterranei U32

Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Although several model systems, including sporulation in Bacillus subtilis and chemotaxis in Escherichia coli, have been extensively studied, the two-component signal transduction systems in industrially important actinomycetes are not well studied. We report the molecular and biochemical characterization of a novel two-component signal system, amrA-amkA,from the rifamycin-SV-producing Amycolatopsis mediterranei U32. The deduced sequences of amkAand amrA contain all the structural features that are highly conserved in the typical bacterial histidine kinases and response regulators, respectively. BLAST analyses showed that AmrA and AmkA displayed high similarities to AfsQ1/AfsQ2 of Streptomyces coelicolor and MtrA/MtrB of Mycobacterium tuberculosis. The amrAand amkA genes were over-expressed and the gene products were purified from E. coli. Biochemical studies showed that AmkA is able to autophosphorylate, supporting its functional assignment as a histidine kinase. That AmrA functions as the cognate response regulator for histidine kinase AmkA was demonstrated by in vitro phosphotransfer from [gamma-(32)P]ATP-labeled AmkA to AmrA. Rifamycin SV production was also decreased by 10-20% in amrAor amkA gene disruption mutants under the tested condition. Although the detailed regulatory mechanism is still unknown, this is the first report regarding the involvement of two-component signal systems in rifamycin biosynthesis in the genus Amycolatopsis.     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His) 蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径。在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统。最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用。     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His)蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径.在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统.最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用.     

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.     

Putative osmosensor – OsHK3b – a histidine kinase protein from rice shows high structural conservation with its ortholog AtHK1 from Arabidopsis

Prokaryotes and eukaryotes respond to various environmental stimuli using the two-component system (TCS). Essentially, it consists of membrane-bound histidine kinase (HK) which senses the stimuli and further transfers the signal to the response regulator, which in turn, regulates expression of various target genes. Recently, sequence-based genome wide analysis has been carried out in Arabidopsis and rice to identify all the putative members of TCS family. One of the members of this family i.e. AtHK1, (a putative osmosensor, hybrid-type sensory histidine kinase) is known to interact with AtHPt1 (phosphotransfer proteins) in Arabidopsis. Based on predicted rice interactome network (PRIN), the ortholog of AtHK1 in rice, OsHK3b, was found to be interacting with OsHPt2. The analysis of amino acid sequence of AtHK1 showed the presence of transmitter domain (TD) and receiver domain (RD), while OsHK3b showed presence of three conserved domains namely CHASE (signaling domain), TD, and RD. In order to elaborate on structural details of functional domains of hybrid-type HK and phosphotransfer proteins in both these genera, we have modeled them using homology modeling approach. The structural motifs present in various functional domains of the orthologous proteins were found to be highly conserved. Binding analysis of the RD domain of these sensory proteins in Arabidopsis and rice revealed the role of various residues such as histidine in HPt protein which are essential for their interaction.     

Kinetic Buffering of Cross Talk between Bacterial Two-Component Sensors

Two-component systems are a class of sensors that enable bacteria to respond to environmental and cell-state signals. The canonical system consists of a membrane-bound sensor histidine kinase that autophosphorylates in response to a signal and transfers the phosphate to an intracellular response regulator. Bacteria typically have dozens of two-component systems. The key questions are whether these systems are linear and, if they are, how cross talk between systems is buffered. In this work, we studied the EnvZ/OmpR and CpxA/CpxR systems from Escherichia coli, which have been shown previously to exhibit slow cross talk in vitro. Using in vitro radiolabeling and a rapid quenched-flow apparatus, we experimentally measured 10 biochemical parameters capturing the cognate and non-cognate phosphotransfer reactions between the systems. These data were used to parameterize a mathematical model that was used to predict how cross talk is affected as different genes are knocked out. It was predicted that significant cross talk between EnvZ and CpxR only occurs for the triple mutant ΔompR ΔcpxA ΔactA-pta. All seven combinations of these knockouts were made to test this prediction and only the triple mutant demonstrated significant cross talk, where the cpxP promoter was induced 280-fold upon the activation of EnvZ. Furthermore, the behavior of the other knockouts agrees with the model predictions. These results support a kinetic model of buffering where both the cognate bifunctional phosphatase activity and the competition between regulator proteins for phosphate prevent cross talk in vivo.     

Identification of a novel two component system in Thermotoga maritima. Complex stoichiometry and crystallization

Two-component signal transduction systems, comprised of histidine kinase and its cognate response regulator, are the predominant mechanism by which microorganisms sense and respond to changes in many different environmental conditions. Different Thermotoga maritima histidine kinases have been used as prototypes; among them, the orphan TM0853 has been presented as a structural model of class I histidine kinases. We used phosphotransfer assays to identify TM0468 as the partner response regulator of TM0853. Since full-length TM0853 can be produced as a soluble protein in Escherichia coli, it was used to analyze the union stoichiometry in an intact two-component system for the first time. We demonstrate that TM0853, or its cytoplasmic catalytic portion, form a 1:1 complex with TM0468 with native PAGE. The complex band is unique, even in the presence of an excess of each individual protein, indicating that the union is cooperative. We corroborated these findings by using ultracentrifugation assays. Therefore, we propose that the general mode of interaction in an orthodox two-component system may be the stoichiometric and cooperative complex between a dimeric histidine kinase and two response regulators. Finally, we have been able to produce protein crystals of the complex between the cytoplasmic portion of TM0853 and TM0468 that diffract to 2.8 A Bragg spacing.     

玉米细胞分裂素反应调节因子基因的克隆与进化分析

在植物的生长发育过程中,细胞分裂素在调节细胞分裂和组织发育中起着非常重要的作用.研究表明细胞分裂素的信号转导是一种双组分信号转导途径,在这个系统中,由3种蛋白组成,分别是组氨酸激酶、磷酸转移蛋白和反应调控因子.利用已经克隆的玉米和水稻细胞分裂素反应调节因子基因,进行BLAST分析从玉米全基因组中获得候选ZmRR类型基因.然后设计全长基因引物,通过Trizol法提取玉米叶片总RNA,利用RT-PCR技术克隆出全长候选基因.序列分析表明所扩增序列含有完整的编码框,共编码156个氨基酸残基.序列比对分析其与ZmRR1-10基因具有较高的同源性,并命名为ZmRR11,系统进化树分析证实其属于A类细胞分裂素调控因子,并对所有ZmRR类型基因进行motif分析,共发现37个保守的motif.该基因的克隆和进化分析对阐明玉米双元信号传导体系具有重要的意义.     

Characterization of genes involved in cytokinin signaling and metabolism from rice

Two-component signaling elements play important roles in plants, including a central role in cytokinin signaling. We characterized two-component elements from the monocot rice (Oryza sativa) using several complementary approaches. Phylogenetic analysis reveals relatively simple orthologous relationships among the histidine kinases in rice and Arabidopsis (Arabidopsis thaliana). In contrast, the histidine-containing phosphotransfer proteins (OsHPs) and response regulators (OsRRs) display a higher degree of lineage-specific expansion. The intracellular localizations of several OsHPs and OsRRs were examined in rice and generally found to correspond to the localizations of their dicot counterparts. The functionality of rice type-B OsRRs was tested in Arabidopsis; one from a clade composed of both monocot and dicot type-B OsRRs complemented an Arabidopsis type-B response regulator mutant, but a type-B OsRR from a monocot-specific subfamily generally did not. The expression of genes encoding two-component elements and proteins involved in cytokinin biosynthesis and degradation was analyzed in rice roots and shoots and in response to phytohormones. Nearly all type-A OsRRs and OsHK4 were up-regulated in response to cytokinin, but other cytokinin signaling elements were not appreciably affected. Furthermore, multiple cytokinin oxidase (OsCKX) genes were up-regulated by cytokinin. Abscisic acid treatment decreased the expression of several genes involved in cytokinin biosynthesis and degradation. Auxin affected the expression of a few genes; brassinosteroid and gibberellin had only modest effects. Our results support a shared role for two-component elements in mediating cytokinin signaling in monocots and dicots and reveal how phytohormones can impact cytokinin function through modulating gene expression.