A Non-hydrolyzable ATP Derivative Generates a Stable Complex in a Light-inducible Two-component System

Isothermal calorimetry (ITC) measurements yielded the binding constants during complex formation of light-inducible histidine kinases (HK) and their cognate CheY-type response regulators (RR). HK-RR interactions represent the core function of the bacterial two-component system, which is also present in many bacterial phytochromes. Here, we have studied the recombinant forms of phytochromes CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate RRs RcpA and RcpB. The interaction between the two reaction partners (HK and RR) was studied in the presence and absence of ATP. A complex formation was observable in the presence of ATP, but specific interactions were only found when a non-hydrolyzable ATP derivative was added to the mixture. Also, the incubation of the HK domain alone (expressed as a recombinant protein) with the RR did not yield specific interactions, indicating that the HK domain is only active as a component of the full-length phytochrome. Considering also previous studies on the same proteins (Hübschmann, T., Jorissen, H. J. M. M., Börner, T., Gärtner, W., and de Marsac, N. (2001) Eur. J. Biochem. 268, 3383–3389) we now conclude that the HK domains of these phytochromes are active only when the chromophore domain is in its Pr form. The formerly documented phosphate transfer between the HK domain and the RR takes place via a transiently formed protein-protein complex, which becomes detectable by ITC in the presence of a non-hydrolyzable ATP derivative. This finding is of interest also in relation to the function of some (blue light-sensitive) photoreceptors that carry the HK domain and the RR fused together in one single protein.     

A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue‐light receptors such as aureochromes to absorb shorter‐wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV‐HK) that functions as a new class of eukaryotic blue‐light receptor in the pico‐phytoplanktonic cell Ostreococcus tauri. This LOV‐HK is related to the large family of LOV‐HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV‐HKs, including O. tauri LOV‐HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV‐HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV‐HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV‐HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV‐HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV‐HK alters CCA1 rhythmicity under constant red light, irrespective of LOV‐HK’s photochemical reactivity, suggesting that O. tauri LOV‐HK also participates in regulation of the circadian clock independent of its blue‐light‐sensing property. Molecular characterization of O. tauri LOV‐HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.     

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.     

Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.     

Domain Swapping to Assess the Mechanistic Basis of Arabidopsis Phototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

Phototropins (phot1 and phot2) are plasma membrane–associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.     

PAS/LOV proteins: A proposed new class of plant blue light receptor

The light, oxygen or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor.Key words: PAS, LOV, blue light, protein-protein interaction, photoreceptor     

Factors That Control the Chemistry of the LOV Domain Photocycle

Algae, plants, bacteria and fungi contain Light-Oxygen-Voltage (LOV) domains that function as blue light sensors to control cellular responses to light. All LOV domains contain a bound flavin chromophore that is reduced upon photon absorption and forms a reversible, metastable covalent bond with a nearby cysteine residue. In Avena sativa LOV2 (AsLOV2), the photocycle is accompanied by an allosteric conformational change that activates the attached phototropin kinase in the full-length protein. Both the conformational change and formation of the cysteinyl-flavin adduct are stabilized by the reduction of the N5 atom in the flavin’s isoalloxazine ring. In this study, we perform a mutational analysis to investigate the requirements for LOV2 to photocycle. We mutated all the residues that interact with the chromophore isoalloxazine ring to inert functional groups but none could fully inhibit the photocycle except those to the active-site cysteine. However, electronegative side chains in the vicinity of the chromophore accelerate the N5 deprotonation and the return to the dark state. Mutations to the N414 and Q513 residues identify a potential water gate and H₂O coordination sites. These residues affect the electronic nature of the chromophore and photocycle time by helping catalyze the N5 reduction leading to the completion of the photocycle. In addition, we demonstrate that dehydration leads to drastically slower photocycle times. Finally, to investigate the requirements of an active-site cysteine for photocycling, we moved the nearby cysteine to alternative locations and found that some variants can still photocycle. We propose a new model of the LOV domain photocycle that involves all of these components.     

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains,LOV/LOV Proteins

LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly⁶⁶ in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.     

Phototropin receptor kinase activation by blue light

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that function to mediate a variety of adaptive processes that serve to optimize the photosynthetic efficiency of plants and thereby promote their growth. Light sensing by the phototropins is mediated by a repeated motif located within the N-terminal region of the protein designated the LOV domain. Although phototropins possess two LOV photosensors (LOV1 and LOV2), recent biophysical and structure-function analyses clearly indicate that the LOV2 domain plays a predominant role in regulating phototropin kinase activity owing to specific protein changes that occur in response to LOV2 photoexcitation. In particular, the central β-sheet scaffold plays a role in propagating the photochemical signal generated from within LOV2 to protein changes at the surface that are necessary for kinase activation.Key words: phototropin, LOV domain, FMN, cysteinyl adduct, amphipathic helix, receptor autophosphoryation     

Estimation of the available free energy in a LOV2-J alpha photoswitch

Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins--plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent alpha-helix (J alpha) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain-J alpha-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by approximately 3.8 kcal mol(-1), thus quantifying the energy available through LOV-J alpha for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-J alpha interaction.     

NTP-binding properties of the blue-light receptor YtvA and effects of the E105L mutation

YtvA is a blue-light-sensing protein from Bacillus subtilis related to plant phototropins. It carries a LOV (light, oxygen and voltage) domain, binding FMN (flavin mononucleotide) as chromophore, and a STAS (sulphate transporters and antisigma-factor antagonists) domain with poorly characterized function. We have recently shown that YtvA binds triphosphate nucleotides (NTP) and highlighted a structural similarity between the STAS domain and small GTP-binding proteins. In this work we further investigated the NTP-binding properties of YtvA, employing a fluorescent derivative of GTP (GTP_TR) and mutagenesis experiments. The main results are as follows: (a) competition experiments indicate that the affinity of YtvA for GTP is much higher than that for GDP and GMP. (b) Blue-light-induced structural changes are transmitted from the LOV core to the NTP-binding cavity, establishing a possible intraprotein signal-transduction pathway. (c) A mutation in the central β-scaffold of the LOV core, E105L, impairs the light-driven spectroscopic changes of bound GTP_TR. This result is supported by circular dichroism data, in that YtvA-E105L does not show the light-induced conformational change in the turn fraction that characterizes YtvA, implying that E105 is functionally important. (d) In the structural model of the LOV-STAS complex, based on docking algorithms, the interface includes the Iβ–Hβ loop on the LOV core, as well as parts of the central β-scaffold. E105 is predicted to interact with the LOV-STAS linker region, suggested to play a role in phototropin signaling. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity

Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.     

Light Signal Transduction Pathway from Flavin Chromophore to the Jα Helix of Arabidopsis Phototropin1

In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jα helix) for the function of phototropin; however, it remains unclear how the Jα helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jα) of the Jα helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (−)/1625 (+) cm⁻¹) of LOV2-Jα at physiological temperatures (≥260 K), corresponding to structural perturbation of the α-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the β-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jα helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jα helix in Arabidopsis phot1.     

Novel blue light-sensitive proteins from a metagenomic approach

A microarray‐based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor‐encoding genes. The microarray carried 149 different 54‐mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2–5 μg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 μg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid‐cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per‐Arnt‐Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht‐met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht‐met1 is followed by a second open reading frame encoding a Fe‐chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht‐met1 was subcloned and expressed yielding a fully functional, flavin‐containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin‐protein photoadduct. The dark recovery lifetime was found as τ_REC = 120 s (20°C) and is among the fastest ones determined so far for bacterial LOV domains.     

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light-sensitive phototropins harboring a light-oxygen-voltage (LOV) domain coupled to various effector domains. Photon absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of an LOV and a sulfate transporter/anti-sigma factor antagonist domain, and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle X-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does not change its oligomerization state or its overall shape upon light activation.     

Re-engineering the two-component systems as light-regulated in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase (HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach to re-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from Cyanobacteria Synechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs and constructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarX-NarL system. The constructed library serves as a toolkit for future TCS study using optogenetics approach.     

Photosensitivity of Kinase Activation by Blue Light Involves the Lifetime of a Cysteinyl-Flavin Adduct Intermediate,S390, in the Photoreaction Cycle of the LOV2 Domain in Phototropin,a Plant Blue Light Receptor

Phototropin (phot) is a light-regulated protein kinase that mediates a variety of photoresponses in plants, such as phototropism, chloroplast positioning, and stomata opening. Arabidopsis has two homologues, phot1 and phot2, that share physiological functions depending on light intensity. A phot molecule has two photoreceptive light oxygen voltage-sensing domains, LOV1 and LOV2, and a Ser/Thr kinase domain. The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase. To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett. 585, 3391–3395). In the present study, we extended the assay system to phot2 and compared the photochemistry and kinase activation by BL between phot1 and phot2 to gain insight into the molecular basis for the different photosensitivities of phot1 and phot2. Photosensitivity of kinase activation by BL and the lifetime of S390 of phot1 were 10 times higher and longer, respectively, than those of phot2. This correlation was confirmed by an amino acid substitution experiment with phot1 to shorten the lifetime of S390. The present results demonstrated that the photosensitivity of kinase activation in phot involves the lifetime of S390 in LOV2, suggesting that the lifetime is one of the key factors for the different photosensitivities observed for phot1 and phot2.     

Optogenetic Navigation of Routes Leading to Protein Amyloidogenesis in Bacteria

Modulation of liquid–liquid and liquid–hydrogel phase transitions is central to avoid the cytotoxic aggregation of proteins in eukaryotic cells, but knowledge on its relevance in bacteria is limited. Here the power of optogenetics to engineer proteins as light-responsive switches has been used to control the balance between solubility and aggregation for LOV2–WH1, a chimera between the plant blue light-responsive domain LOV2 and the bacterial prion-like protein RepA-WH1. These proteins were first linked by fusing, as a continuous α-helix, the C-terminal photo-transducer Jα helix in LOV2 with the N-terminal domain-closure α1 helix in RepA-WH1, and then improved for light-responsiveness by including mutations in the Jα moiety. In the darkness and in a crowded solution in vitro, LOV2–WH1 nucleates the irreversible assembly of amyloid fibers into a hydrogel. However, under blue light illumination, LOV2–WH1 assembles as soluble oligomers. When expressed in Escherichia coli, LOV2–WH1 forms in the darkness large intracellular amyloid inclusions compatible with bacterial proliferation. Strikingly, under blue light, LOV2–WH1 aggregates decrease in size, while they become detrimental for bacterial growth. LOV2–WH1 optogenetics governs the assembly of mutually exclusive inert amyloid fibers or cytotoxic oligomers, thus enabling the navigation of the conformational landscape of protein amyloidogenesis to generate potential photo-activated anti-bacterial devices (optobiotics).     

Genetic and Biochemical Dissection of a HisKA Domain Identifies Residues Required Exclusively for Kinase and Phosphatase Activities

Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK–RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the α1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria.