Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1

In Arabidopsis, ethylene is perceived by a receptor family consisting of five members, one of these being ETR1. The N-terminal half of ETR1 functions as a signal input domain. The C-terminal region of ETR1, consisting of a His kinase domain and a putative receiver domain, is likely to function in signal output. The role of the proposed signal output region in ethylene signaling was examined in planta. For this purpose, the ability of mutant versions of ETR1 to rescue the constitutive ethylene-response phenotype of the etr1-6;etr2-3;ein4-4 triple loss-of-function mutant line was examined. A truncated version of ETR1 that lacks both the His kinase domain and the receiver domain failed to rescue the triple mutant phenotype. A truncated ETR1 receptor that lacks only the receiver domain restored normal growth to the triple mutant in air, but the transgenic seedlings displayed hypersensitivity to low doses of ethylene. A mutation that eliminated His kinase activity had a modest effect upon the ability of the receptor to repress ethylene responses in air. These results demonstrate that the His kinase domain plays a role in the repression of ethylene responses. The potential roles of the receiver domain and His kinase activity in ethylene signaling are discussed.     

A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA

Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.     

Bacterial histidine kinase as signal sensor and transducer

Adaptation to an environmental stress is essential for cell survival in all organisms, from E. coli to human. To respond to changes in their surroundings, bacteria utilize two-component systems (TCSs), also known as histidyl-aspartyl phosphorelay (HAP) systems that consist of a histidine kinase (HK) sensor and a cognate response regulator (RR). While mammals developed complex signaling systems involving serine/threonine/tyrosine kinases in stress response mechanisms, bacterial TCS/HAP systems represent a simple but elegant prototype of signal transduction machineries. HKs are known as a seductive target for anti-bacterial therapeutic development, because of their significance in pathological virulence in some bacteria such as Salmonella enterica. Recent molecular and structural studies have shed light on the molecular basis of the signaling mechanism of HK sensor kinases. This review will focus on recent advancements in structural investigation of signal sensing and transducing mechanisms by HKs, which is critical to our understanding of bacterial biology and pathology.     

Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC , to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr⁺ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr⁺ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus , and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.     

Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli

AtoSC two-component system participates in many indispensable processes of Escherichia coli. We report here that the AtoSC signal transduction is inhibited by established histidine kinase inhibitors. Closantel, RWJ-49815 and TNP-ATP belonging to different chemical classes of inhibitors, abrogated the in vitro AtoS kinase autophosphorylation. However, when AtoS was embedded in the membrane fractions, higher inhibitor concentrations were required for total inhibition. When AtoS interacted with AtoC forming complex, the intrinsic histidine kinase was protected by the response regulator, requiring increased inhibitors concentrations for partially AtoS autophosphorylation reduction. The inhibitors exerted an additional function on AtoSC, blocking the phosphotransfer from AtoS to AtoC, without however, affecting AtoC~P dephosphorylation. Their in vivo consequences through the AtoSC inhibition were elucidated on atoDAEB operon expression, which was inhibited only in AtoSC-expressing bacteria where AtoSC was induced by acetoacetate or spermidine. The inhibitor effects were extended on the AtoSC regulatory role on cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis. cPHB was decreased upon the blockers only in acetoacetate-induced AtoSC-expressing cells. Increased ATP amounts during bacterial growth reversed the inhibitory TNP-ATP-mediated effect on cPHB. The alteration of pivotal E. coli processes as an outcome of AtoSC inhibition, establish this system as a target of two-component systems inhibitors.     

Cytokinin receptor: just another histidine kinase.

The cytokinin family of plant hormones is involved in diverse aspects of plant growth and development in vivo and in culture. Two groups have recently shown that a two-component histidine kinase functions as a cytokinin receptor specifically required for vascular development.     

Nucleotide binding by the histidine kinase CheA

To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg(2+) or Mn(2+). The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide gamma-phosphate and its associated Mg(2+) ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases.     

The HAMP linker in histidine kinase dimeric receptors is critical for symmetric transmembrane signal transduction

The HAMP linker, a common structural element between a sensor and a transmitter module in various sensor proteins, plays an essential role in signal transduction. Here, by in vivo complementation experiments with Tar-EnvZ hybrid receptor mutants in which the HAMP linker forms a heterodimer with Tar and EnvZ-type subunits, we found that mutations at one linker only affect the function of EnvZ in the same subunit. However, the same mutations affect the EnvZ function of both subunits when only a Tar or EnvZ-type HAMP linker is used. These results suggest that intersubunit interactions in the HAMP linker normally mediate signal transduction through both subunits in a sensor dimer, whereas the signal is asymmetrically transduced through the linker in a heterodimer. This is the first demonstration that two HAMP linkers in a sensor dimer are functionally coupled for normal signal transduction; however, this functional coupling can be reduced when the HAMP linkers lose their symmetric nature.     

The role of protein kinase C activation in signal transmission by interleukin 2

Role of protein kinase C (PKC) in interleukin (IL) 2-induced proliferation was investigated by utilizing two murine IL 2-dependent cell lines, CT6 and CTLL-2 cell lines. CT6 cells showed a marked proliferative response to phorbol 12-myristate 13-acetate (PMA), while CTLL-2 did not. PMA induced PKC translocation from cytosol to membrane only in a PMA-responsive cell line. IL 2 failed to stimulate PKC translocation in both cell lines. H-7, a potent and specific PKC inhibitor, however, inhibited the proliferation of both cell lines induced by IL 2. Taken collectively, IL 2 may induce PKC activation without its translocation.     

PAS-mediated dimerization of soluble guanylyl cyclase revealed by signal transduction histidine kinase domain crystal structure

Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) because it is often associated with the heme-nitric oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 A resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCbeta1 H-NOXA homodimerization, heterodimerization of full-length heterodimeric sGC was mostly unaffected, likely due to the additional dimerization contacts of sGC in the coiled-coil and catalytic domains. Exceptions are mutations sGCalpha1 F285A and sGCbeta1 F217A, which each caused a drastic drop in NO stimulated activity, and mutations sGCalpha1 Q368A and sGCbeta1 Q309A, which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture.     

Identification of a signal transduction switch in the chemokine receptor CXCR1

Chemokine receptors belong to the superfamily of G protein-coupled receptors, which regulate the trafficking and activation of leukocytes, and operate as coreceptors in the entry of HIV-1. To investigate the early steps in the signal transmission from the chemokine-binding site to the G protein-coupling region we engineered metal ion-binding sites at putative extracellular sites in the chemokine receptor CXCR1. We introduced histidines into sites located in the second and third putative extracellular loops of CXCR1, creating single, double, and triple mutant receptors: R199H, R203H, D265H, R199H/R203H, R199H/D265H, R203H/D265H, R203H/H207Q, and R199H/R203H/D265H. Cells expressing the double mutants R199H/D265H and R203H/D265H and the triple mutant R199H/R203H/D265H failed to trigger interleukin 8-dependent calcium responses. Interestingly, calcium responses mediated by the single mutant R203H and the double mutants R199H/R203H and R203H/H207Q were blocked by Zn(II), indicating the creation of a functional metal ion-binding site. On the other hand, cells expressing all single, double, or triple histidine-substituted CXCR1 demonstrated high affinity binding to interleukin 8 in the presence and absence of metal ions. These findings indicate that occupation of the engineered metal-binding site uncouples the chemokine-binding site from the activation mechanism in CXCR1. Most importantly, we identify for the first time elements of an early signal transduction switch of chemokine receptors before the activation of cytoplasmic G proteins.     

Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

Background

A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive.

Findings

Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca²⁺ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca²⁺ in a concentration-dependent manner. Conversely, increasing Ca²⁺ levels inhibits kinase activity up to 500-fold at 100 nM Ca²⁺.

Conclusions

Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

     

Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2

Ethylene plays important roles in plant growth, development, and stress responses. Two ethylene receptors, ETR1 from Arabidopsis and NTHK1 from tobacco (Nicotiana tabacum), have been found to have His kinase (HK) activity and Ser/Thr kinase activity, respectively, although both show similarity to bacterial two-component HK. Here, we report the characterization of another ethylene receptor homolog gene, NTHK2, from tobacco. This gene also encodes a HK-like protein and is induced by dehydration and CaCl(2) but not significantly affected by NaCl and abscisic acid treatments. The biochemical properties of the yeast (Schizosaccharomyces pombe)-expressed NTHK2 domains were further characterized. We found that NTHK2 possessed Ser/Thr kinase activity in the presence of Mn(2+) and had HK activity in the presence of Ca(2+). Several lines of evidence supported this conclusion, including hydrolytic stability, phosphoamino acid analysis, mutation, deletion, and substrate analysis. These properties have implications in elucidation of the complexity of the ethylene signal transduction pathway and understanding of ethylene functions in plants.     

Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phosphotransfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. 15N-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored N(delta1)H tautomer of His-45, thereby rendering the N(epsilon2) imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.     

Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC

Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator ( agr ) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans- autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.     

Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.