Intermolecular complementation of the kinase activity of CheA

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis In Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheA_s) were found to phosphorylate the kinase-defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer. The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphoryltion requires the presence of both carboxy-terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.     

Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.     

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.     

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.     

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.     

Mechanism of CheA protein kinase activation in receptor signaling complexes.

The chemotaxis receptor for aspartate, Tar, generates responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an extracellular sensory domain connected by a transmembrane sequence to a cytoplasmic signaling domain. The cytoplasmic domain fused to a leucine zipper dimerization domain forms soluble active ternary complexes with CheA and an adapter protein, CheW. The kinetics of kinase activity within these complexes compared to CheA alone indicate approximately a 50% decrease in the KM for ATP and a 100-fold increase in the Vmax. A truncated CheA construct that lacks the phosphoaccepting H-domain and the CheY/CheB-binding domain forms an activated ternary complex that is similar to the one formed by the full-length CheA protein. The Vmax of H-domain phosphorylation by this complex is enhanced approximately 60-fold, the KM for ATP decreased to 50%, and the KM for H-domain decreased to 20% of the values obtained with the same CheA construct in the absence of receptor and CheW. The kinetic data support a mechanism of CheA regulation that involves perturbation of an equilibrium between an inactive form where the H-domain is loosely bound and an active form where the H-domain is tightly associated with the CheA active site and properly positioned for phosphotransfer. The data are consistent with an asymmetric mechanism of CheA activation [Levit, M., Liu, I., Surette, M. G., and Stock, J. B. (1996) J. Biol. Chem. 271, 32057-32063] wherein only one phosphoaccepting domain of CheA at a time can interact with an active center within a CheA dimer.     

Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis

Six cytoplasmic che gene products are required for signal transduction in bacterial chemotaxis, but the nature of their biochemical interactions is not known. We show that in vitro the CheA protein becomes autophosphorylated in the presence of ATP. In addition, the phosphate group on CheA can be rapidly transferred to CheB, a protein involved in adaptation to stimuli, or to CheY, a protein involved in the excitation response. The phosphorylation of CheB and CheY is transient; they readily dephosphorylate. We have also found that CheZ, a protein that appears to antagonize CheY function in vivo, accelerates the hydrolysis of the phosphate on CheY. These results suggest that signal transduction in bacterial chemotaxis may involve the flow of phosphate through a cascade of phosphorylated protein intermediates.     

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.     

Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions

We prepared fusions of yellow fluorescent protein [the YFP variant of green fluorescent protein (GFP)] with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild-type and mutant cells of Escherichia coli. All but the CheA fusions were functional. The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl-accepting chemotaxis proteins (MCPs), but only if MCPs were present. Co-localization of CheY and CheZ with MCPs was CheA dependent, and co-localization of CheA with MCPs was CheW dependent, as expected. Co-localization with MCPs was confirmed by immunofluorescence using an anti-MCP primary antibody. The motor protein FliM appeared as discrete spots on the sides of the cell. These were seen in wild-type cells and in a fliN mutant, but not in flhC or fliG mutants. Co-localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody. Surprisingly, we did not observe co-localization of CheY with motors, even under conditions in which cells tumbled.     

Auto‐activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Many Ser/Thr protein kinases are activated by autophosphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the mycobacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative ‘substrate’ KD was disordered, with the ends positioned at the entrance to the partner KD active site. Single amino‐acid substitutions in the G‐helix interface reduced activation‐loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. Phosphorylation of an inactive mutant KD was reduced by G‐helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans‐autophosphorylation complex. These results support a model in which a structurally and functionally asymmetric, ‘front‐to‐front’ association mediates autophosphorylation of PknB and homologous kinases.     

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.     

Mutants defective in bacterial chemotaxis show modified protein phosphorylation

To examine the correlation between CheA phosphorylation and bacterial chemotaxis, cheA mutations leading to defects in chemotaxis were mapped and characterized. Mutant CheA proteins were tested in vitro for phosphorylation and were grouped into four classes: nonphosphorylated, partially phosphorylated, phosphorylated but not dephosphorylated by CheB and CheY, and phosphorylated and dephosphorylated. Nearly all the mutants were found to be defective in an aspect of phosphorylation. Furthermore, the mutant phenotypes were found to cluster in different regions of the cheA gene. We suggest that the CheA protein has three functional domains: one for interaction with CheB and CheY, a second for regulating phosphorylation and controlling the stability of the protein, and a third for receiving input signals regulating CheA activity.     

Regulation of the Escherichia coli antiterminator protein BglG by phosphorylation at multiple sites and evidence for transfer of phosphoryl groups between monomers

Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101.     

Ezrin mutants affecting dimerization and activation

ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane-associated proteins and the actin cytoskeleton. Previous work has shown that ezrin can exist in a dormant monomeric state in which the N-terminal FERM domain is tightly associated with the C-ERMAD (carboxyl-terminal ERM association domain), masking binding sites for at least some ligands, including F-actin and the scaffolding protein EBP50. Activation of ezrin requires relief of the intramolecular association, and this is believed to involve phosphorylation of threonine 567. Studies have therefore employed the T567D phosphomimetic mutant to explore the consequences of ezrin activation in vivo. Ezrin also exists as a stable dimer, in which the orientation of the two subunits is unknown, but might involve the central alpha-helical region predicted to form a coiled-coil. By characterization of ezrin mutants, we show that relief of the intramolecular association in the monomer results in unmasking of ligand binding sites and a significant conformational change, that the T567D mutation has a small effect on the biochemical activation of ezrin, and that the predicted coiled-coil region does not drive dimer formation. These results provide strong support for the conformational activation model of ezrin, elucidate the basis for dimer formation, and reveal that a mutant generally considered to be fully activated is not.     

Mutations that affect control of the methylesterase activity of CheB, a component of the chemotaxis adaptation system in Escherichia coli.

Sensory adaptation by the chemotaxis system of Escherichia coli requires adjustments of the extent of methyl esterification of the chemotaxis receptor proteins. One mechanism utilized by E. coli to make such adjustments is to control the activity of CheB, the enzyme responsible for removing receptor methyl ester groups. Previous work has established the existence of a multicomponent signal transduction pathway that enables the chemotaxis receptor proteins to control the methylesterase activity in response to chemotactic stimuli. We isolated and characterized CheB mutants that do not respond normally to this control mechanism. In intact cells these CheB variants could not be activated in response to negative chemotaxis stimuli. Further characterization indicated that these CheB variants could not be phosphorylated by the chemotaxis protein kinase CheA. Disruption of the mechanism responsible for regulating methylesterase activity was also observed in cells carrying chromosomal deletions of either cheA or cheW as well as in cells expressing mutant versions of CheA that lacked kinase activity. These results provide further support for recent proposals that activation of the methylesterase activity of CheB involves phosphorylation of CheB by CheA. Furthermore, our findings suggest that CheW plays an essential role in enabling the chemotaxis receptor proteins to control the methylesterase activity, possibly by controlling the CheA-CheB phosphotransfer reaction.     

The noncatalytic site-deficient alpha3beta3gamma subcomplex and FoF1-ATP synthase can continuously catalyse ATP hydrolysis when Pi is present.

We investigated ATP hydrolysis by a mutant (DeltaNC) alpha3beta3gamma subcomplex of F0F1-ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites. This mutant subcomplex was activated by inorganic phosphate ions (Pi) and did not show continuous ATP hydrolysis activity in the absence of Pi. Pi also activated the wild-type alpha3beta3gamma subcomplex in a similar manner. Sulphate activated wild-type alpha3beta3gamma but not DeltaNC alpha3beta3gamma, indicating that Pi activation did not involve noncatalytic sites but that sulphate activation did. Pi also activated ATP hydrolysis and coupled proton translocation by the wild-type and DeltaNC F0F1-ATP synthases reconstituted into vesicle membranes.     

The dynamics of protein phosphorylation in bacterial chemotaxis

The chemotaxis signal transduction pathway allows bacteria to respond to changes in concentration of specific chemicals (ligands) by modulating their swimming behavior. The pathway includes ligand binding receptors, and the CheA, CheY, CheW, and CheZ proteins. We showed previously that phosphorylation of CheY is activated in reactions containing receptor, CheW, CheA, and CheY. Here we demonstrate that this activation signal results from accelerated autophosphorylation of the CheA kinase. Evidence for a second signal transmitted by a ligand-bound receptor, which corresponds to inhibition of CheA autophosphorylation, is also presented. We postulate that CheA can exist in three forms: a "closed" form in the absence of receptor and CheW; an "open" form that results from activation of CheA by receptor and CheW; and a "sequestered" form in reactions containing ligand-bound receptor and CheW. The system's dynamics depends on the relative distribution of CheA among these three forms at any time.     

Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3beta identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes

In vitro phosphorylation of recombinant wild-type 2N4R tau and FTDP-17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3beta (GSK3beta) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild-type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild-type, P301L or V337M tau by GSK3beta but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long-range conformational effects on the structure of tau.