Conformational coupling in the chemotaxis response regulator CheY.

CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY. Autodephosphorylation and substrate affinity, however, were not significantly affected. Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling. These findings provide structural and conceptual insights into the mechanism of CheY activation. Our results are consistent with a multistate thermodynamic model of response regulator activation.

Alteration of a nonconserved active site residue in the chemotaxis response regulator CheY affects phosphorylation and interaction with CheZ.

CheY is a response regulator in the well studied two-component system that mediates bacterial chemotaxis. Phosphorylation of CheY at Asp(57) enhances its interaction with the flagellar motor. Asn(59) is located near the phosphorylation site, and possible roles this residue may play in CheY function were explored by mutagenesis. Cells containing CheY59NR or CheY59NH exhibited hyperactive phenotypes (clockwise flagellar rotation), and CheY59NR was characterized biochemically. A continuous enzyme-linked spectroscopic assay that monitors P(i) concentration was the primary method for kinetic analysis of phosphorylation and dephosphorylation. CheY59NR autodephosphorylated at the same rate as wild-type CheY and phosphorylated similarly to wild type with acetyl phosphate and faster (4-14x) with phosphoramidate and monophosphoimidazole. CheY59NR was extremely resistant to CheZ, requiring at least 250 times more CheZ than wild-type CheY to achieve the same dephosphorylation rate enhancement, whereas CheY59NA was CheZ-sensitive. However, several independent approaches demonstrated that CheY59NR bound tightly to CheZ. A submicromolar K(d) for CheZ binding to CheY59NR-P or CheY.BeF(3)(-) was inferred from fluorescence anisotropy measurements of fluoresceinated-CheZ. A complex between CheY59NR-P and CheZ was isolated by analytical gel filtration, and the elution position from the column was indistinguishable from that of the CheZ dimer. Therefore, we were not able to detect large CheY-P.CheZ complexes that have been inferred using other methods. Possible structural explanations for the specific inhibition of CheZ activity as a result of the arginyl substitution at CheY position 59 are discussed.

Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli.

The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.

Correlated switch binding and signaling in bacterial chemotaxis.

In Escherichia coli, swimming behavior is mediated by the phosphorylation state of the response regulator CheY. In its active, phosphorylated form, CheY exhibits enhanced binding to a switch component, FliM, at the flagellar motor, which induces a change from counterclockwise to clockwise flagellar rotation. When Ile(95) of CheY is replaced by a valine, increased clockwise rotation correlates with enhanced binding to FliM. A possible explanation for the hyperactivity of this mutant is that residue 95 affects the conformation of nearby residues that potentially interact with FliM. In order to assess this possibility directly, the crystal structure of CheY95IV was determined. We found that CheY95IV is structurally almost indistinguishable from wild-type CheY. Several other mutants with substitutions at position 95 were characterized to establish the structural requirements for switch binding and clockwise signaling at this position and to investigate a general relationship between the two properties. The various rotational phenotypes of these mutants can be explained solely by the amount of phosphorylated CheY bound to the switch, which was inferred from the phosphorylation properties of the mutant CheY proteins and their binding affinities to FliM. Combined genetic, biochemical, and crystallographic results suggest that residue 95 itself is critical in mediating the surface complementarity between CheY and FliM.

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.

Throwing the switch in bacterial chemotaxis.

In Escherichia coli chemotaxis, the switch from counterclockwise to clockwise rotation of the flagella occurs as a result of binding of the phosphorylated CheY protein to the base of the flagellum. Analysis of CheY variants has provided a picture of the surface of CheY that undergoes conformational shifts, as a result of phosphorylation, to interact directly with the flagellum. Whether phospho-CheY binding and flagellar switching are sequential steps or can occur in a concerted fashion has yet to be determined.

Computer-aided resolution of an experimental paradox in bacterial chemotaxis.

Escherichia coli responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190-5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703-8707, 1989). For example, "building up" the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas "breaking down" the pathway by deleting chemotaxis genes except cheA and cheY resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the che genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks.

Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet.

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr-->Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13-->Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.

Acetylation at Lys-92 enhances signaling by the chemotaxis response regulator protein CheY.

When Escherichia coli cells lacking all chemotaxis proteins except the response regulator CheY are exposed to acetate, clockwise flagellar rotation results, indicating the acetate stimulus has activated signaling by CheY. Acetate can be converted to acetyl-CoA by either of two different metabolic pathways, which proceed through acetyl phosphate or acetyl-AMP intermediates. In turn, CheY can be covalently modified by either intermediate in vitro, leading to phosphorylation or acetylation, respectively. Either pathway is sufficient to support the CheY-mediated response to acetate in vivo. Whereas phosphorylation of Asp-57 is a recognized mechanism for activation of CheY to stimulate clockwise flagellar rotation, acetylation of CheY is less well characterized. We found evidence for multiple CheY acetylation sites by mass spectrometry and directly identified Lys-92 and Lys-109 as acetylation sites by Edman degradation of peptides from [14C]acetate-labeled CheY. Replacement of CheY Lys-92, the preferred acetylation site, with Arg has little effect on chemotaxis but completely prevents the response to acetate via the acetyl-AMP pathway. Thus acetylation of Lys-92 activates clockwise signaling by CheY in vivo. The mechanism by which acetylation activates CheY apparently is not simple charge neutralization, nor does it involve enhanced binding to the FliM flagellar switch protein. Thus acetylation probably affects signal generation by CheY at a step after switch binding.

Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY.

The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57. However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses. Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage. Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees C. Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.

Chemotactic response regulator mutant CheY95IV exhibits enhanced binding to the flagellar switch and phosphorylation-dependent constitutive signalling.

CheY, a response regulator protein in bacterial chemotaxis, mediates swimming behaviour through interaction with the flagellar switch protein, FliM. In its active, phosphorylated state, CheY binds to the motor switch complex and induces a change from counterclockwise (CCW) to clockwise (CW) flagellar rotation. The conformation of a conserved aromatic residue, tyrosine 106, has been proposed to play an important role in this signalling process. Here, we show that an isoleucine to valine substitution in CheY at position 95--in close proximity to residue 106--results in an extremely CW, hyperactive phenotype that is dependent on phosphorylation. Further biochemical characterization of this mutant protein revealed phosphorylation and dephosphorylation rates that were indistinguishable from those of wild-type CheY. CheY95IV, however, exhibited an increased binding affinity to FliM. Taken together, these results show for the first time a correlation between enhanced switch binding and constitutive signalling in bacterial chemotaxis. Considering present structural information, we also propose possible models for the role of residue 95 in the mechanism of CheY signal transduction.

Origins of individual swimming behavior in bacteria.

Cells in a cloned population of coliform bacteria exhibit a wide range of swimming behaviors--a form of non-genetic individuality. We used computer models to examine the proposition that these variations are due to differences in the number of chemotaxis signaling molecules from one cell to the next. Simulations were run in which the concentrations of seven gene products in the chemotaxis pathway were changed either deterministically or stochastically, with the changes derived from independent normal distributions. Computer models with two adaptation mechanisms were compared with experimental results from observations on individuals drawn from genetically identical populations. The range of swimming behavior predicted for cells with a standard deviation of protein copy number per cell of 10% of the mean was found to match closely the experimental range of the wild-type population. We also make predictions for the swimming behaviors of mutant strains lacking the adaptational mechanism that can be tested experimentally.

Catalytic mechanism of phosphorylation and dephosphorylation of CheY: kinetic characterization of imidazole phosphates as phosphodonors and the role of acid catalysis.

Kinetic and equilibrium measurements of phosphotransfer events involving CheY carried out over a range of pH conditions elucidated several features of the phosphotransfer mechanism. Using tryptophan fluorescence intensity measurements as a monitor of phosphorylation, we showed that phosphorylation using small molecule phosphodonors occurred by fast association of CheY with the phosphodonor, followed by rate-limiting phosphotransfer. Two previously uncharacterized phosphodonors, monophosphoimidazole and diphosphoimdazole, were able to phosphorylate CheY at a concentration about 6-fold lower than that of the previously described phosphodonors acetyl phosphate and phosphoramidate. This was shown to be due to tighter binding of the imidazole phosphates to CheY and implied the presence of binding interactions between CheY and the imidazole group. The ability of CheY to autophosphorylate through the pH range of 5-10 differed for various phosphodonors. Acetyl phosphate and diphosphoimidazole were unaffected by pH over this range, whereas phosphoramidate and monophosphoimidazole showed a steep dependence on pH with a loss of phosphorylation ability at about pH 7.4 (midpoint) for monophosphoimidazole and pH 7.8 (midpoint) for phosphoramidate. This behavior correlated with the loss of the positive charge on the nitrogen atom in the nitrogen-phosphorus bond in both monophosphoimidazole and phosphoramidate and implied that CheY was not capable of donating a proton to the leaving group in phosphotransfer with small molecules. The rate of phosphotransfer from [32P]CheA-phosphate to wild type CheY also decreased markedly (> 150 times) between pH 7.5 and 10. Because the mutant CheY proteins K109R and T87A showed the same pH dependence as the wild type, the loss of activity in the alkaline range could not be attributed to deprotonation of either of these active site residues. This observation, combined with the moderate decreases in phosphotransfer rates for these mutants relative to that of wild type CheY, indicated that it is unlikely that either Thr87 or Lys109 plays a direct role in the catalysis of phosphotransfer. Finally, we showed that the rate of autodephosphorylation of CheY was independent of pH over the range of 4.5-11. Together, these studies led to a model with CheY playing a largely entropic role in its own phosphorylation and dephosphorylation.

Uncoupled phosphorylation and activation in bacterial chemotaxis. The 2.3 A structure of an aspartate to lysine mutant at position 13 of CheY.

An aspartate to lysine mutation at position 13 of the chemotaxis regulatory protein CheY causes a constitutive tumbly phenotype when expressed at high copy number in vivo even though the mutant protein is not phosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylation, so knowledge of its structure could explain the activation mechanism of CheY. The x-ray crystallographic structure of the CheY D13K mutant has been solved and refined at 2.3 A resolution to an R-factor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg2+-free structure, but there are localized changes within the active site. The side chain of lysine 13 blocks access to the active site, whereas its epsilon-amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspartate 57 is weakened, and the solvent structure is perturbed. Although the D13K mutant has the inactive conformation in the crystalline form, rearrangements in the active site appear to weaken the overall structure of that region, potentially creating a metastable state of the molecule. If a conformational change is required for signaling by CheY D13K, then it most likely proceeds dynamically, in solution.

Computer analysis of the binding reactions leading to a transmembrane receptor-linked multiprotein complex involved in bacterial chemotaxis.

The chemotactic response of bacteria is mediated by complexes containing two molecules each of a transmembrane receptor and the intracellular signaling proteins CheA and CheW. Mutants in which one or the other of the proteins of this complex are absent, inactive, or expressed at elevated amounts show altered chemotactic behavior and the phenotypes are difficult to interpret for some overexpression mutants. We have examined the possibility that these unexpected phenotypes might arise from the binding steps that lead to active complex formation. A limited genetic algorithm was used to search for sets of binding reactions and associated binding constants expected to give mutant phenotypes in accord with experimental data. Different sets of binding equilibria and different assumptions about the activity of particular receptor complexes were tried. Computer analysis demonstrated that it is possible to obtain sets of binding equilibria consistent with the observed phenotypes and provided a simple explanation for these phenotypes in terms of the distribution of active and inactive complexes formed under various conditions. Optimization methods of this kind offer a unique way to analyze reactions taking place inside living cells based on behavioral data.

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ.

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow counter-clockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephosphorylation rates of these mutants. CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of cheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephosphorylation rates. Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.

Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance.

We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli. A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY. Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate. This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems. The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.

Purification and characterization of Bacillus subtilis CheY.

Amino acid sequence comparison suggests that numerous proteins are common to the signal transduction pathways controlling chemotaxis in Bacillus subtilis and Escherichia coli. However, previous work has indicated several differences between the two systems. We have undertaken a comparative study of the roles of the CheY protein in chemotaxis by B. subtilis and E. coli. Although CheY from the two species share only 36% amino acid sequence identity, purified B. subtilis CheY was phosphorylated in vitro by E. coli CheA, and dephosphorylation of CheY-P was enhanced by E. coli CheZ. Alteration of the putative site of phosphorylation in B. subtilis CheY, Asp54, eliminated chemotaxis in vivo, further confirming that phosphorylation is important for B. subtilis chemotaxis. Loss of CheY function resulted in tumbling behavior in B. subtilis. Introduction of positively charged residues in place of Asp10 of B. subtilis CheY abolished function, whereas the corresponding changes in E. coli CheY apparently result in constitutive activation. The B. subtilis CheY Asp10 mutant proteins also failed to cause tumbling in E. coli, consistent with a different interaction between CheY and the flagellar switch in the two species. Finally, B. subtilis adapted more rapidly to positive stimuli than negative stimuli, whereas the opposite is true of E. coli. We conclude that B. subtilis regulates its response to positive chemotactic stimuli by enhancing phosphorylation of chemotaxis proteins, whereas E. coli reduces phosphorylation in the same circumstance.

Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering.

CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation. 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper. Together these results may give insight into the activation of other prokaryotic response regulators.