Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein

In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA. Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)). Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest. To circumvent these difficulties, we generated a derivative of CheY carrying a covalently attached fluorescent label that serves as a sensitive reporter of phosphorylation and binding events and that absorbs and emits light at wavelengths well removed from potential interference by other proteins. This labeled version of CheY has the (dimethylamino)naphthalene fluorophore from Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] attached to the thiol group of a cysteine introduced at position 17 of CheY by site-directed mutagenesis. Under phosphorylating conditions (or in the presence of beryllofluoride), the fluorescence emission of Badan-labeled CheY(M17C) exhibited an approximately 10 nm blue shift and an approximately 30% increase in signal intensity at 490 nm. The fluorescence of Badan-labeled CheY(M17C) also served as a sensitive reporter of CheY-CheA binding interactions, exhibiting an approximately 50% increase in emission intensity in the presence of saturating levels of CheA. Compared to wild-type CheY, Badan-labeled CheY exhibited reduced ability to autodephosphorylate and could not interact productively with the phosphatase CheZ. However, with respect to autophosphorylation and interactions with CheA, Badan-CheY performed identically to wild-type CheY, allowing us to explore CheA-CheY phosphotransfer kinetics and binding kinetics without interference from the fluorescence/absorbance properties of CheA and ATP. These results provide insights into CheY interactions with CheA, CheZ, and other components of the chemotaxis signaling pathway.     

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.     

Response regulation in bacterial chemotaxis

The signal transduction system that mediates bacterial chemotaxis allows cells to moduate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue-at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY. © 1993 Wiley-Liss, Inc.     

The carboxy-terminal portion of the CheA kinase mediates regulation of autophosphorylation by transducer and CheW.

The CheA kinase is a central protein in the signal transduction network that controls chemotaxis in Escherichia coli. CheA receives information from a transmembrane receptor (e.g., Tar) and CheW proteins and relays it to the CheB and CheY proteins. The biochemical activities of CheA proteins truncated at various distances from the carboxy terminus were examined. The carboxy-terminal portion of CheA regulates autophosphorylation in response to environmental signals transmitted through Tar and CheW. The central portion of CheA is required for autophosphorylation and is also presumably involved in dimer formation. The amino-terminal portion of CheA was previously shown to contain the site of autophosphorylation and to be able to transfer the phosphoryl group to CheB and CheY. These studies further delineate three functional domains of the CheA protein.     

Investigation of the role of electrostatic charge in activation of the Escherichia coli response regulator CheY

In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg(2+) ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg(2+)-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.     

Kinetics of CheY phosphorylation by small molecule phosphodonors.

The chemotaxis response regulator CheY can acquire phosphoryl groups either from its associated autophosphorylating protein kinase, CheA, or from small phosphodonor molecules such as acetyl phosphate. We report a stopped-flow kinetic analysis of CheY phosphorylation by acetyl phosphate. The results show that CheY has a very low affinity for this phosphodonor (K(s)&z.Gt;0.1 M), consistent with the conclusion that, whereas CheY provides catalytic functions for the phosphotransfer reaction, the CheA kinase may act simply to increase the effective phosphodonor concentration at the CheY active site.     

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems.     

Chemical-shift-perturbation mapping of the phosphotransfer and catalytic domain interaction in the histidine autokinase CheA from Thermotoga maritima

Regulating the activity of the histidine autokinase CheA is a central step in bacterial chemotaxis. The CheA autophosphorylation reaction minimally involves two CheA domains, denoted P1 and P4. The kinase domain (P4) binds adenosine triphosphate (ATP) and orients the gamma phosphate for phosphotransfer to a reactive histidine on the phosphoacceptor domain (P1). Three-dimensional triple-resonance experiments allowed sequential assignments of backbone nuclei from P1 and P4 domains as well as the P4 assignments within a larger construct, P3P4, which includes the dimerization domain P3. We have used nuclear magnetic resonance chemical-shift-perturbation mapping to define the interaction of P1 and P3P4 from the hyperthermophile Thermotoga maritima. The observed chemical-shift changes in P1 upon binding suggest that the P1 domain is bound by interactions on the side opposite the histidine that is phosphorylated. The observed shifts in P3P4 upon P1 binding suggest that P1 is bound at a site distinct from the catalytic site on P4. These results argue that the P1 domain is not bound in a mode that leads to productive phosphate transfer from ATP at the catalytic site and imply the presence of multiple binding modes. The binding mode observed may be regulatory or it may reflect the binding mode needed for effective transfer of the histidyl phosphate of P1 to the substrate proteins CheY and CheB. In either case, this work describes the first direct observation of the interaction between P1 and P4 in CheA.     

Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp57, Asp13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp57, and in the three-dimensional structure of CheY the Asp57 carboxylate side chain is in close proximity to the beta-carboxylate of Asp13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at Asp57 is essential for the conformational switch that leads to activation of CheY.     

Interaction of CheY with the C-terminal peptide of CheZ

Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P~CheY). The steady-state level of P~CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P~CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZ_C) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol. 359:624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the CheZ_C peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZ_C interaction. In addition, we present kinetic studies of the CheZ_C-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.     

Chemotactic signaling by an Escherichia coli CheA mutant that lacks the binding domain for phosphoacceptor partners

CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.     

Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation

Chemotaxis, the environment-specific swimming behavior of a bacterial cell is controlled by flagellar rotation. The steady-state level of the phosphorylated or activated form of the response regulator CheY dictates the direction of flagellar rotation. CheY phosphorylation is regulated by a fine equilibrium of three phosphotransfer activities: phosphorylation by the kinase CheA, its auto-dephosphorylation and dephosphorylation by its phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two spatially distinct protein-protein contacts: tethering of the two proteins to each other and formation of an active site for dephosphorylation. The former involves interaction of phosphorylated CheY with the small highly conserved C-terminal helix of CheZ (CheZ(C)), an indispensable structural component of the functional CheZ protein. To understand how the CheZ(C) helix, representing less than 10% of the full-length protein, ascertains molecular specificity of binding to CheY, we have determined crystal structures of CheY in complex with a synthetic peptide corresponding to 15 C-terminal residues of CheZ (CheZ(200-214)) at resolutions ranging from 2.0 A to 2.3A. These structures provide a detailed view of the CheZ(C) peptide interaction both in the presence and absence of the phosphoryl analog, BeF3-. Our studies reveal that two different modes of binding the CheZ(200-214) peptide are dictated by the conformational state of CheY in the complex. Our structures suggest that the CheZ(C) helix binds to a "meta-active" conformation of inactive CheY and it does so in an orientation that is distinct from the one in which it binds activated CheY. Our dual binding mode hypothesis provides implications for reverse information flow in CheY and extends previous observations on inherent resilience in CheY-like signaling domains.     

Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI

The activities of the eight mutant proteins of Escherichia coli RNase HI, in which the four carboxylic amino acids (Asp(10), Glu(48), Asp(70), and Asp(134)) involved in catalysis are changed to Asn (Gln) or Ala, were examined in the presence of Mn(2+). Of these proteins, the E48A, E48Q, D134A, and D134N proteins exhibited the activity, indicating that Glu(48) and Asp(134) are dispensable for Mn(2+)-dependent activity. The maximal activities of the E48A and D134A proteins were comparable to that of the wild-type protein. However, unlike the wild-type protein, these mutant proteins exhibited the maximal activities in the presence of >100 microM MnCl(2), and their activities were not inhibited at higher Mn(2+) concentrations (up to 10 mM). The wild-type protein contains two Mn(2+) binding sites and is activated upon binding of one Mn(2+) ion at site 1 at low ( approximately 1 microM) Mn(2+) concentrations. This activity is attenuated upon binding of a second Mn(2+) ion at site 2 at high (>10 microM) Mn(2+) concentrations. The cleavage specificities of the mutant proteins, which were examined using oligomeric substrates at high Mn(2+) concentrations, were identical to that of the wild-type protein at low Mn(2+) concentrations but were different from that of the wild-type protein at high Mn(2+) concentrations. These results suggest that one Mn(2+) ion binds to the E48A, E48Q, D134A, and D134N proteins at site 1 or a nearby site with weaker affinities. The binding analyses of the Mn(2+) ion to these proteins in the absence of the substrate support this hypothesis. When Mn(2+) ion is used as a metal cofactor, the Mn(2+) ion itself, instead of Glu(48) and Asp(134), probably holds water molecules required for activity.     

Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2'-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.     

Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli.

CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli. CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation. Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells. To find out why, we examined the effects of a purified P1 fragment, CheA[1-149], on CheA-related signaling activities in vitro and devised in vivo assays for those same activities. Although readily phosphorylated by CheA[260-537], the CheA catalytic domain, CheA[1-149], was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center. CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA[1-149], suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect. In agreement with these findings, a 40-fold excess of CheA[1-149] fragments did not impair the CheA autophosphorylation reaction. CheA[1-149] did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules. An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site. The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells. However, an excess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragments have both transphosphorylation and phosphotransfer capability in vivo.     

Sinorhizobium meliloti CheA complexed with CheS exhibits enhanced binding to CheY1, resulting in accelerated CheY1 dephosphorylation

Retrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain is a widespread mechanism for efficient dephosphorylation of the activated response regulator. First discovered in Sinorhizobium meliloti, the main response regulator CheY2-P shuttles its phosphoryl group back to CheA, while a second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA-P. We have identified a new component in this phospho-relay system, a small 97-amino-acid protein named CheS. CheS has no counterpart in enteric bacteria but revealed distinct similarities to proteins of unknown function in other members of the α subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In vitro binding, as well as coexpression and copurification studies, gave evidence of CheA/CheS complex formation. Using limited proteolysis coupled with mass spectrometric analyses, we defined CheA(163-256) to be the CheS binding domain, which overlaps with the N-terminal part of the CheY2 binding domain (CheA(174-316)). Phosphotransfer experiments using isolated CheA-P showed that dephosphorylation of CheY1-P but not CheY2-P is increased in the presence of CheS. As determined by surface plasmon resonance spectroscopy, CheY1 binds ～100-fold more strongly to CheA/CheS than to CheA. We propose that CheS facilitates signal termination by enhancing the interaction of CheY1 and CheA, thereby promoting CheY1-P dephosphorylation, which results in a more efficient drainage of the phosphate sink.     

Mutational Analysis of the P1 Phosphorylation Domain in Escherichia coli CheA,the Signaling Kinase for Chemotaxis

The histidine autokinase CheA functions as the central processing unit in the Escherichia coli chemotaxis signaling machinery. CheA receives autophosphorylation control inputs from chemoreceptors and in turn regulates the flux of signaling phosphates to the CheY and CheB response regulator proteins. Phospho-CheY changes the direction of flagellar rotation; phospho-CheB covalently modifies receptor molecules during sensory adaptation. The CheA phosphorylation site, His-48, lies in the N-terminal P1 domain, which must engage the CheA ATP-binding domain, P4, to initiate an autophosphorylation reaction cycle. The docking determinants for the P1-P4 interaction have not been experimentally identified. We devised mutant screens to isolate P1 domains with impaired autophosphorylation or phosphotransfer activities. One set of P1 mutants identified amino acid replacements at surface-exposed residues distal to His-48. These lesions reduced the rate of P1 transphosphorylation by P4. However, once phosphorylated, the mutant P1 domains transferred phosphate to CheY at the wild-type rate. Thus, these P1 mutants appear to define interaction determinants for P1-P4 docking during the CheA autophosphorylation reaction.     

Mechanism of phosphatase activity in the chemotaxis response regulator CheY

Response regulator proteins are phosphorylated on a conserved aspartate to activate responses to environmental signals. An intrinsic autophosphatase activity limits the duration of the phosphorylated state. We have previously hypothesized that dephosphorylation might proceed through an intramolecular attack, leading to succinimide formation, and such an intramolecular dephosphorylation event is seen for CheY and OmpR during mass spectrometric analysis [Napper, S., Wolanin, P. M., Webre, D. J., Kindrachuk, J., Waygood, B., and Stock, J. B. (2003) FEBS Lett 538, 77-80]. Succinimide formation is usually associated with the spontaneous deamidation of Asn residues. We show here that an Asp57 to Asn mutant of the CheY chemotaxis response regulator undergoes an unusually rapid deamidation back to the wild-type Asp57, supporting the hypothesis that the active site of CheY is poised for succinimide formation. In contrast, we also show that the major route of phosphoaspartate hydrolysis in CheY occurs through water attack on the phosphorus both during autophosphatase activity and during CheZ-mediated dephosphorylation. Thus, CheY dephosphorylation does not usually proceed via a succinimide or any other intramolecular attack.     

Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser-56

The site of phosphorylation of the chemotaxis response regulator CheY is aspartate 57. When Asp-57 is replaced with an asparagine, the resultant protein can be phosphorylated at an alternative site. We report here that phosphorylation of this mutant protein, CheY D57N, at the alternative site affords the protein activity in vivo in the absence of CheZ. Using a direct phosphopeptide mapping approach, we identified the alternate phosphorylation site as serine 56. Introduction of a Ser-->Ala substitution at this position in wild-type CheY had no effect on function. However, replacement of Ser-56 with Ala in CheY D57N abrogated the activity seen in vivo for the CheY D57N single mutant protein, and no phosphorylation of the CheY S56A/D57N double mutant protein was observed in vitro. Construction and analysis of double mutants CheY D57N/T87A and CheY D57N/K109R, which were both inactive, suggested that phosphorylation at Ser-56 or Asp-57 may activate the protein by similar mechanisms. In contrast to CheY D57N, mutant CheY D57E displayed no activity in vivo, despite its ability to be phosphorylated in vitro. Acid-base stability analysis indicated that CheY D57E phosphorylates on an acidic residue, presumably Glu-57. These data suggest that a key determinant of the ability of a phosphoryl group to activate CheY is proximity to the hydrophobic core of the protein, with consequent opportunity to reposition key residues, irrespective of the chemical nature of the linkage attaching the phosphoryl group to CheY.     

Phosphorylation in halobacterial signal transduction.

Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli.