Histidine-containing Phosphotransfer Research Articles

Crystal Structure of a Putative Histidine-containing Phosphotransfer Protein from Oryza sativa

Os09g0567400 codes for a hypothetical protein from Oryza sativa that is annotated as the “Histidine-containing phosphotransfer (Hpt) protein”. Hpt domain is a protein module with a histidine residue mediating phosphotransfer reaction in the histidine-aspartate phosphorelay system. We report here the crystal structure and analysis of Os09g0567400.

Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Protective roles and Pap1-dependent regulation of the Schizosaccharomyces pombe spy1 gene under nitrosative and nutritional stresses

This work aimed to assess novel protective roles and regulation of Spy1, a histidine-containing phosphotransfer (HPt) protein, in the fission yeast Schizosaccharomyces pombe. The structural gene encoding Spy1 was cloned into the shuttle vector pRS316 to generate the recombinant plasmid pYFSpy1. The spy1(+) mRNA level was notably increased in S. pombe cells harboring the plasmid pYFSpy1. The S. pombe cells harboring pYFSpy1 exhibited higher survival than the vector control cells on the minimal media plates with nitric oxide (NO)-generating sodium nitroprusside (SNP) or without nitrogen source. In the liquid minimal media, they also showed higher viability under nitrosative stress or nitrogen-starved condition. The intracellular reactive oxygen species (ROS) level appeared to be lower in the fission yeast cells harboring pYFSpy1 than in the control yeast cells. Overexpression of the spy1(+) gene showed scavenging effect on NO generated from SNP. Synthesis of β-galactosidase from the spy1(+)-lacZ fusion gene was significantly enhanced by SNP and nitrogen starvation in the Pap1-positive but not in the Pap1-negative cells. The spy1(+) mRNA level in S. pombe was also elevated by SNP and nitrogen starvation in the Pap1-positive but not in the Pap1-negative cells. In summary, Spy1 plays protective roles against nitrosative and nutritional stress in the fission yeast and is transcriptionally up-regulated by nitrosative and nutritional stresses in a Pap1-dependent manner.

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

Author SummaryThe ability to respond to environmental stimuli is a universal feature of living cells. Evolution has created a vast array of signalling mechanisms that enable cells to react in many ways to extracellular changes. In bacteria, two-component signalling mechanisms, comprising a sensor protein kinase paired with its a cognate response regulator, are used widely to sense and respond to environmental changes. Some species of bacteria have over 150 different two-component pairs in a single cell, so the specificity between these pairs has to be tightly controlled to prevent “crossed wires” between signalling pathways. In this study, we have identified the determinants of this specificity in a two-component complex that controls the movement of Rhodobacter sphaeroides along a chemical gradient. By solving and analysing the crystal structure of this complex, we were able to pinpoint the amino acid residues that are crucially involved in formation of the complex. Knowledge of these crucial residues allowed us to convert noncognate response regulators into cognate response regulators simply by changing two amino acids. This reengineering of two-component signalling pathways paves the way for producing custom-designed circuits for applications in synthetic biology.

Structural Insight into Partner Specificity and Phosphoryl Transfer in Two-Component Signal Transduction

The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.

Identification of a new promoter for the response regulatorrcsBexpression inSalmonella entericaserovar Typhimurium

The RcsCDB (Rcs) phosphorelay system regulates capsule synthesis, flagella production and other cellular activities in several enteric bacteria. This system consists of three proteins: the sensor RcsC, the cognate response regulator RcsB and the histidine-containing phosphotransfer protein RcsD (YojN), which is hypothesized to act as an intermediary in the phosphotransfer from RcsC to RcsB. The rcsC gene is convergently transcribed toward rcsB, which follows rcsD in what appears to be a two-gene operon. Here, it is reported that the overproduction of the rcsB gene represses rcsD transcription, but has a weak effect on its own expression. We demonstrated that the differential rcsD and rcsB expression is due to the activity of two promoters to transcribe the rcsB gene: (1) P(rcsDB) located upstream of rcsD and (2) P(rcsB) located within the rcsD coding region. In addition, here it was demonstrated that in Salmonella typhimurium, P(rcsB) is important to activate the rcsB expression during the stationary growth phase.

Effects of Osmolytes on the SLN1-YPD1-SSK1 Phosphorelay System from Saccharomyces cerevisiae

The multistep His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells to adapt to osmotic, oxidative, and other environmental stresses. The pathway consists of a hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1, and two response regulator proteins, SSK1 and SKN7. Under nonosmotic stress conditions, the SLN1 sensor kinase is active, and phosphoryl groups are shuttled through YPD1 to SSK1, therefore maintaining the response regulator protein in a constitutively phosphorylated state. The cellular response to hyperosmotic stress involves rapid efflux of water and changes in intracellular ion and osmolyte concentration. In this study, we examined the individual and combined effects of NaCl and glycerol on phosphotransfer rates within the SLN1-YPD1-SSK1 phosphorelay. The results show that the combined effects of glycerol and NaCl on the phosphotransfer reaction rates are different from the individual effects of glycerol and NaCl. The combinatory effect is likely more representative of the in vivo changes that occur during hyperosmotic stress. In addition, the effect of osmolyte concentration on the half-life of the phosphorylated SSK1 receiver domain in the presence/absence of YPD1 was evaluated. Our findings demonstrate that increasing osmolyte concentrations negatively affect the YPD1 x SSK1-P interaction, thereby facilitating dephosphorylation of SSK1 and activating the HOG1 MAP kinase cascade. In contrast, at the highest osmolyte concentrations, reflective of the osmoadaptation phase of the signaling pathway, the kinetics of the phosphorelay favor production of SSK1-P and inhibition of the HOG1 pathway.

Characterization of NikA Histidine Kinase and Two Response Regulators with Special Reference to Osmotic Adaptation and Asexual Development inAspergillus nidulans

Aspergillus nidulans has many histidine-to-aspartate (His-Asp) phosphorelay components, including 15 histidine kinases (HKs), four response regulators (RRs), and a histidine-containing phosphotransfer intermediate (HPt). Of these, NikA (HK) is highly conserved in many filamentous fungi. It has been found that NikA is responsible for the responses of filamentous fungi to fungicides such as iprodione and fludioxonil. Two RRs, SskA and SrrA, are also involved in the fungicide response, providing a typical example of the His-Asp phosphorelay system, in which NikA functions as a sensor upstream of SskA and SrrA in response to fungicides. To gain further insight into the physiological roles of the NikA-SskA/SrrA phosphorelay system, we constructed a pair of DeltanikADeltasskA and DeltanikADeltasrrA double mutants. Here we provide evidence regarding the crucial involvement of the NikA-SskA/SrrA phosphorelay system in both osmotic adaptation and asexual development, including conidia formation. Based on these results, a general insight into the A. nidulans His-Asp phosphorelay network is also discussed.

The Sensor Kinase TodS Operates by a Multiple Step Phosphorelay Mechanism Involving Two Autokinase Domains

Expression of the Pseudomonas putida tod operon, which encodes enzymes for toluene metabolism, takes place from the P(todX) promoter and is mediated by the TodS/TodT two component system. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor- and an autokinase domain and separated by a receiver domain. Based on site-directed mutagenesis of phosphoaccepting His-190, Asp-500, and His-760 and in vitro transphosphorylation experiments with recombinant TodS fragments, we show that TodS uses a multiple step phosphorelay mechanism to activate TodT. Toluene binding stimulates exclusively phosphorylation of His-190, which is followed by phosphotransfer to Asp-500 and subsequently to His-760 prior to phosphorylation of TodT Asp-57. Mutation of His-190, Asp-500, and H760A prevented up-regulation of toluene-mediated stimulation of TodT transphosphorylation in vitro and reduced in vivo expression of P(todX) to the basal level. Calorimetric studies support that TodT binds to the C-terminal kinase module with a K(D) of approximately 200 nm and 1:1 stoichiometry. This is the first report of a multiple step phosphorelay mechanism of a sensor kinase that involves two autokinase domains.

Involvement of putative response regulator genes of the rice blast fungus Magnaporthe oryzae in osmotic stress response, fungicide action, and pathogenicity

Rice blast fungus (Magnaporthe oryzae) has ten histidine kinases (HKs), one histidine-containing phosphotransfer protein (HPt), and three response regulators (RRs) as putative components of the two-component signal transduction system (TCS). Here, we constructed knockout mutants of two putative RR genes (MoSSK1, MoSKN7) and a RR homolog gene (MoRIM15) to analyze the roles of TCS in environmental adaptation and pathogenicity. The DeltaMossk1 strain had increased sensitivity to high osmolarity and decreased sensitivity to fludioxonil. The DeltaMoskn7 strain had slightly decreased sensitivity to fludioxonil. The involvement of MoSkn7 in the osmoresponse was obvious only on the DeltaMossk1 background. These results show that MoSsk1 and MoSkn7 are major and minor contributors, respectively, in the high osmolarity response and fludioxonil action. The DeltaMossk1 strain was more osmosensitive than the predicted upstream HK gene disruptant Deltahik1, which shows sugar-specific high osmolarity sensitivity. The DeltaMossk1 and DeltaMoskn7 strains showed enhanced hyphal melanization, suggesting that RRs regulate hyphal melanization. MoSsk1 and MoRim15 are required for full virulence, because the DeltaMossk1 and DeltaMorim15 strains exhibited reduced virulence. These results suggest that the putative RRs of the rice blast fungus are involved in the osmotic stress response, fludioxonil action, and pathogenicity.

The Arabidopsis thaliana response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

BackgroundThe 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.ResultsWe present evidence that ARR22 is a preferentially cytoplasmic protein, exclusively expressed in the chalaza of developing seeds. ARR22 specifically interacts with AHP2, AHP3 and AHP5 in yeast and living plant cells. Two new loss-of-function alleles, arr22-2 and arr22-3, were isolated and characterized. With respect to their morphology and metabolite status, no significant difference in the developing seeds of the arr22 mutants was observed compared to wild type. The genetic complementation of the arr22 mutants with a genomic ARR22 fragment resulted in plants (arr22/gARR22) with a pleiotropic phenotype of different penetrance. This phenotype was not observed when the phosphorylatable Asp74 of ARR22 was changed to either a dominant-active Glu or a dominant-inactive Asn. The phenotype of the arr22/gARR22 plants was comparable to that of multiple ahk, ahp and B-type arr mutants.ConclusionOur results favor the model that ARR22 acts as a phospho-histidine phosphatase on specific AHPs in the cytoplasm of Arabidopsis chalaza cells. The lack of any aberrant morphological and metabolite phenotype in the seeds of the arr22 mutants indicates that ARR22 is probably primarily responsible for the fine tuning of specific branches of chalaza-based TCS signalling. Even when slightly mis-expressed, ARR22 interferes with hormone homeostasis in non-chalaza tissues. Our data indicate that the chromatin status might play a crucial role in maintaining the chalaza-restricted expression of ARR22.

Phosphorylation of A-type ARR to function as negative regulator of cytokinin signal transduction

The plant hormone cytokinins regulate diverse aspects of plant growth and development. In Arabidopsis, a multi-step TCS system similar to bacterial and yeast TCS is used for cytokinin signaling. In a TCS system, a His sensor kinase perceives the signal by autophosphorylating on a His residue in response to an output signal, and the phosphate group is transferred to a conserved Asp residue in the receiver domain of the response regulator. The response regulator then modulates downstream signaling. Cytokinin multi-step TCS system utilizes an additional component, histidine-containing phosphotransfer domain protein (HPT) to transfer the phosphate group from a sensor kinase to a response regulator in the nucleus. The typical response regulators are classified into either type A or B. The type-B ARRs are transcription activators that act as positive regulators of cytokinin signaling, whereas most of the type-A ARRs are negative regulators of cytokinin signaling. Histidyl-aspartidyl phosphorelays are presumed to be essential for this cytokinin signal transduction in plants. Our studies have shown that ARR7, an A-type response regulator, negatively regulates cytokinin signaling in various aspects by acting as a transcriptional repressor and that the phosphorylation of ARR7 is required for these ARR7-regulated cytokinin-responses. Here I propose potential mechanisms by which the phosphorylation of ARRs is involved in regulating cytokinin-mediated gene expression, mainly based on biochemical and structural studies of bacterial response regulators. Protein-protein interaction and DNA-binding studies using the phosphorylated and the un-phosphorylated forms of the ARR proteins with their structural determination will provide molecular understanding of cytokinin-responsive gene regulation by ARRs.

Glycolytic Enzyme GAPDH Promotes Peroxide Stress Signaling through Multistep Phosphorelay to a MAPK Cascade

Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.

Arabidopsis Histidine-containing Phosphotransfer Factor 4 (AHP4) Negatively Regulates Secondary Wall Thickening of the Anther Endothecium during Flowering

Arabidopsis Histidine-containing Phosphotransfer Factor 4 (AHP4) Negatively Regulates Secondary Wall Thickening of the Anther Endothecium during Flowering

Characterization of the Histidine-containing Phosphotransfer Protein B-mediated Multistep Phosphorelay System in Pseudomonas aeruginosa PAO1

Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346-PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.

Two-component signaling elements and histidyl-aspartyl phosphorelays.

Two-component systems are an evolutionarily ancient means for signal transduction. These systems are comprised of a number of distinct elements, namely histidine kinases, response regulators, and in the case of multi-step phosphorelays, histidine-containing phosphotransfer proteins (HPts). Arabidopsis makes use of a two-component signaling system to mediate the response to the plant hormone cytokinin. Two-component signaling elements have also been implicated in plant responses to ethylene, abiotic stresses, and red light, and in regulating various aspects of plant growth and development. Here we present an overview of the two-component signaling elements found in Arabidopsis, including functional and phylogenetic information on both bona-fide and divergent elements.

Response Regulators SrrA and SskA Are Central Components of a Phosphorelay System Involved in Stress Signal Transduction and Asexual Sporulation in Aspergillus nidulans

Among eukaryotes, only slime molds, fungi, and plants contain signal transduction phosphorelay systems. In filamentous fungi, multiple sensor kinases appear to use a single histidine-containing phosphotransfer (HPt) protein to relay signals to two response regulators (RR). In Aspergillus nidulans, the RR SskA mediates activation of the mitogen-activated protein kinase SakA in response to osmotic and oxidative stress, whereas the functions of the RR SrrA were unknown. We used a genetic approach to characterize the srrA gene as a new member of the skn7/prr1 family and to analyze the roles of SrrA in the phosphorelay system composed of the RR SskA, the HPt protein YpdA, and the sensor kinase NikA. While mutants lacking the HPt protein YpdA are unviable, mutants lacking SskA (DeltasskA), SrrA (DeltasrrA), or both RR (DeltasrrA DeltasskA) are viable and differentially affected in osmotic and oxidative stress responses. Both RR are involved in osmostress resistance, but DeltasskA mutants are more sensitive to this stress, and only SrrA is required for H(2)O(2) resistance and H(2)O(2)-mediated induction of catalase CatB. In contrast, both RR are individually required for fungicide sensitivity and calcofluor resistance and for normal sporulation and conidiospore viability. The DeltasrrA and DeltasskA sporulation defects appear to be related to decreased mRNA levels of the key sporulation gene brlA. In contrast, conidiospore viability defects do not correlate with the activity of the spore-specific catalase CatA. Our results support a model in which NikA acts upstream of SrrA and SskA to transmit fungicide signals and to regulate asexual sporulation and conidiospore viability. In contrast, NikA appears dispensable for osmotic and oxidative stress signaling. These results highlight important differences in stress signal transmission among fungi and define a phosphorelay system involved in oxidative and osmotic stress, cell wall maintenance, fungicide sensitivity, asexual reproduction, and spore viability.

Signaling via Histidine-Containing Phosphotransfer Proteins in Arabidopsis

The Arabidopsis genome encodes a number of proteins with similarity to two-component phosphorelay signaling elements, including hybrid receptor histidine kinases, two classes of response regulator proteins (type-A and type-B ARRs) and a family of six histidine-containing phosphotransfer proteins (AHPs), five of which contain a conserved His residue that is required for phosphorelay signaling. The current model for cytokinin signaling includes a multistep phosphorelay: three histidine kinases and at least five type-B ARRs have been shown to act as positive regulators of cytokinin signaling, while a number of type-A ARRs, and AHP6, act as negative regulators of the pathway. In our recent Plant Cell paper, we provided genetic evidence that at least four AHPs can act as positive regulators of cytokinin signaling, affecting responses to cytokinin in the root and the shoot. In this addendum, we discuss the role of AHPs in cytokinin signaling and speculate on their potential interactions with other signaling pathways in Arabidopsis.

Functional characterization of the phosphorelay protein Mpr1p fromSchizosaccharomyces pombe

Histidine-containing phosphotransfer (HPt) proteins play an essential role in multistep histidine-aspartate phosphorelay signal transduction systems in prokaryotes and eukaryotes. The putative HPt protein in Schizosaccharomyces pombe, Mpr1p (also known as Spy1p), is a 295 amino acid protein that appears to be composed of more than one functional domain. The amino acid sequence of the N-terminal region of Mpr1p lacks homology to other known proteins, whereas the C-terminal domain is predicted to have structural similarity to the Ypd1p HPt protein from Saccharomyces cerevisiae. This study provides both in vitro and in vivo evidence that the C-terminal domain of Mpr1p indeed functions as an HPt protein in shuttling phosphoryl groups from one response regulator domain to another. Furthermore, we find that various deletions of the N-terminal region diminish both the phosphotransfer activity of Mpr1p and its affinity for response regulator domains, suggesting a possible role for the N-terminal domain in HPt-response regulator domain interactions.

The SskA and SrrA Response Regulators Are Implicated in Oxidative Stress Responses of Hyphae and Asexual Spores in the Phosphorelay Signaling Network ofAspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated DeltasskA and DeltasrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from DeltasskA were hypersensitive not only to treatment with H(2)O(2), but also to treatment at aberrantly low (4 degrees C) and high (50 degrees C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from DeltasskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.