CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis

The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild-type B. subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.     

CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis

Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD during B. subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.     

A PAS Domain Binds Asparagine in the Chemotaxis Receptor McpB in Bacillus subtilis

During chemotaxis toward asparagine by Bacillus subtilis, the ligand is thought to bind to the chemoreceptor McpB on the exterior of the cell and induce a conformational change. This change affects the degree of phosphorylation of the CheA kinase bound to the cytoplasmic region of the receptor. Until recently, the sensing domains of the B. subtilis receptors were thought to be structurally similar to the well studied Escherichia coli four-helical bundle. However, sequence analysis has shown the sensing domains of receptors from these two organisms to be vastly different. Homology modeling of the sensing domain of the B. subtilis asparagine receptor McpB revealed two tandem PAS domains. McpB mutants having alanine substitutions in key arginine and tyrosine residues of the upper PAS domain but not in any residues of the lower PAS domain exhibited a chemotactic defect in both swarm plates and capillary assays. Thus, binding does not appear to occur across any dimeric surface but within a monomer. A modified capillary assay designed to determine the concentration of attractant where chemotaxis is most sensitive showed that when Arg-111, Tyr-121, or Tyr-133 is mutated to an alanine, much more asparagine is required to obtain an active chemoreceptor. Isothermal titration calorimetry experiments on the purified sensing domain showed a K_D to asparagine of 14 μm, with the three mutations leading to less efficient binding. Taken together, these results reveal not only a novel chemoreceptor sensing domain architecture but also, possibly, a different mechanism for chemoreceptor activation.     

Selective methylation changes on the Bacillus subtilis chemotaxis receptor McpB promote adaptation

The Bacillus subtilis McpB is a class III chemotaxis receptor, from which methanol is released in response to all stimuli. McpB has four putative methylation sites based upon the Escherichia coli consensus sequence. To explore the nature of methanol release from a class III receptor, all combinations of putative methylation sites Gln(371), Gln(595), Glu(630), and Glu(637) were substituted with aspartate, a conservative substitution that effectively eliminates methylation. McpB((Q371D,E630D,E637D)) in a Delta(mcpA mcpB tlpA tlpB)101::cat mcpC4::erm background failed to release methanol in response to either the addition or removal of the McpB-mediated attractant asparagine. In the same background, McpB((E630D,E637D)) produced methanol only upon asparagine addition, whereas McpB((Q371D,E630D)) produced methanol only upon asparagine removal. Thus methanol release from McpB was selective. Mutants unable to methylate site 637 but able to methylate site 630 had high prestimulus biases and were incapable of adapting to asparagine addition. Mutants unable to methylate site 630 but able to methylate site 637 had low prestimulus biases and were impaired in adaptation to asparagine removal. We propose that selective methylation of these two sites represents a method of adaptation novel from E. coli and present a model in which a charged residue rests between them. The placement of this charge would allow for opposing electrostatic effects (and hence opposing receptor conformational changes). We propose that CheC, a protein not found in enteric systems, has a role in regulating this selective methylation.     

Cooperative signaling among bacterial chemoreceptors

Four chemoreceptors in Escherichia coli mediate responses to chemicals in the environment. The receptors self-associate and localize to the cell poles. This aggregation implies that interactions among receptors are important parameters of signal processing during chemotaxis. We examined this phenomenon using a receptor-coupled in vitro assay of CheA kinase activity. The ability of homogeneous populations of the serine receptor Tsr and the aspartate receptor Tar to stimulate CheA was directly proportional to the ratio of the receptor to total protein in cell membranes up to a fraction of 50%. Membranes containing mixed populations of Tar and Tsr supported an up to 4-fold greater stimulation of CheA than expected on the basis of the contributions of the individual receptors. Peak activity was seen at a Tar:Tsr ratio of 1:4. This synergy was observed only when the two proteins were expressed simultaneously, suggesting that, under our conditions, the fundamental "cooperative receptor unit" is relatively static, even in the absence of CheA and CheW. Finally, we observed that inhibition of receptor-stimulated CheA activity by serine or aspartate required significantly higher concentrations of ligand for membranes containing mixed Tsr and Tar populations than for membranes containing only Tsr (up to 10(2)-fold more serine) or Tar (up to 10(4)-fold more aspartate). Together with recent analyses of the interactions of Tsr and Tar in vivo, our results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis.     

Analysis of chimeric chemoreceptors in Bacillus subtilis reveals a role for CheD in the function of the McpC HAMP domain

Motile prokaryotes use a sensory circuit for control of the motility apparatus in which ligand-responsive chemoreceptors regulate phosphoryl flux through a modified two-component signal transduction system. The chemoreceptors exhibit a modular architecture, comprising an N-terminal sensory module, a C-terminal output module, and a HAMP domain that connects the N- and C-terminal modules and transmits sensory information between them via an unknown mechanism. The sensory circuits mediated by two chemoreceptors of Bacillus subtilis have been studied in detail. McpB is known to regulate chemotaxis towards the attractant asparagine in a CheD-independent manner, whereas McpC requires CheD to regulate chemotaxis towards the attractant proline. Although CheD is a phylogenetically widespread chemotaxis protein, there exists only a limited understanding of its function. We have constructed chimeras between McpB and McpC to probe the role of CheD in facilitating sensory transduction by McpC. We found that McpC can be converted to a CheD-independent receptor by the replacement of one-half of its HAMP domain with the corresponding sequence from McpB, suggesting that McpC HAMP domain function is complex and may require intermolecular interactions with the CheD protein. When considered in combination with the previous observation that CheD catalyzes covalent modification of the C-terminal modules of B. subtilis receptors, these results suggest that CheD may interact with chemoreceptors at multiple, functionally distinct sites.     

Template-directed assembly of receptor signaling complexes

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF. The extent of CheA activation was found to be independent of the level of covalent modification on the CF. Instead, the stability of the complex increased significantly as the level of covalent modification increased. Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR. Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.     

Elucidation of the multiple roles of CheD in Bacillus subtilis chemotaxis

Chemotaxis by Bacillus subtilis requires the CheD protein for proper function. In a cheD mutant when McpB was the sole chemoreceptor in B. subtilis, chemotaxis to asparagine was quite good. When McpC was the sole chemoreceptor in a cheD mutant, chemotaxis to proline was very poor. The reason for the difference between the chemoreceptors is because CheD deamidates Q609 in McpC and does not deamidate McpB. When mcpC‐Q609E is expressed as the sole chemoreceptor in a cheD background, chemotaxis is almost fully restored. Concomitantly, in vitro McpC activates the CheA kinase poorly, whereas McpC‐Q609E activates it much more. Moreover, CheD, which activates chemoreceptors, binds better to McpC‐Q609E compared with unmodified McpC. Using hydroxyl radical susceptibility in the presence or absence of CheD, the most likely sites of CheD binding were the modification sites where CheD, CheB and CheR carry out their catalytic activities. Thus, CheD appears to have two separate roles in B. subtilis chemotaxis – to bind to chemoreceptors to activate them as part of the CheC/CheD/CheYp adaptation system and to deamidate selected residues to activate the chemoreceptors and enable them to mediate amino acid chemotaxis.     

CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxis in Bacillus subtilis

For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB. In this study, we show that rapid net demethylation of B. subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB. We also show that net demethylation of McpB occurs upon both addition and removal of asparagine. After each demethylation event, McpB is remethylated to nearly prestimulus levels. Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested. Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.     

Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway.

We examined the binding interactions of the methylation-dependent chemotaxis receptors Tsr and Tar with the chemotaxis-specific protein kinase CheA and the coupling factor CheW. Receptor directly bound CheW, but receptor-CheA binding was dependent upon the presence of CheW. These observations in combination with our previous identification of a CheW-CheA complex suggest that CheW physically links the kinase to the receptor. The ternary complex of receptor, CheW, and CheA is both kinetically and thermodynamically stable at physiological concentrations. Stability is not significantly altered by changes associated with attractant or repellent binding to the receptor. Such binding greatly modulates the kinase activity of CheA. Our results demonstrate that modulation of the kinase activity does not require association-dissociation of the ternary complex. This suggests that the receptor signal is transduced through conformational changes in the ternary complex rather than through changes in the association of the kinase CheA with receptor and/or CheW.     

Noncritical Signaling Role of a Kinase–Receptor Interaction Surface in the Escherichia coli Chemosensory Core Complex

In Escherichia coli chemosensory arrays, transmembrane receptors, a histidine autokinase CheA, and a scaffolding protein CheW interact to form an extended hexagonal lattice of signaling complexes. One interaction, previously assigned a crucial signaling role, occurs between chemoreceptors and the CheW-binding P5 domain of CheA. Structural studies showed a receptor helix fitting into a hydrophobic cleft at the boundary between P5 subdomains. Our work aimed to elucidate the in vivo roles of the receptor–P5 interface, employing as a model the interaction between E. coli CheA and Tsr, the serine chemoreceptor. Crosslinking assays confirmed P5 and Tsr contacts in vivo and their strict dependence on CheW. Moreover, the P5 domain only mediated CheA recruitment to polar receptor clusters if CheW was also present. Amino acid replacements at CheA.P5 cleft residues reduced CheA kinase activity, lowered serine response cooperativity, and partially impaired chemotaxis. Pseudoreversion studies identified suppressors of P5 cleft defects at other P5 groove residues or at surface-exposed residues in P5 subdomain 1, which interacts with CheW in signaling complexes. Our results indicate that a high-affinity P5–receptor binding interaction is not essential for core complex function. Rather, P5 groove residues are probably required for proper cleft structure and/or dynamic behavior, which likely impact conformational communication between P5 subdomains and the strong binding interaction with CheW that is necessary for kinase activation. We propose a model for signal transmission in chemotaxis signaling complexes in which the CheW–receptor interface plays the key role in conveying signaling-related conformational changes from receptors to the CheA kinase.     

Receptor methylation controls the magnitude of stimulus-response coupling in bacterial chemotaxis

Motile prokaryotes employ a chemoreceptor-kinase array to sense changes in the media and properly adjust their swimming behavior. This array is composed of a family of Type I membrane receptors, a histidine protein kinase (CheA), and an Src homology 3-like protein (CheW). Binding of an attractant to the chemoreceptors inhibits CheA, which results in decreased phosphorylation of the chemotaxis response regulator (CheY). Sensitivity of the system to stimuli is modulated by a protein methyltransferase (CheR) and a protein methylesterase (CheB) that catalyze the methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of the receptors. One of the most fundamental unanswered questions concerning the bacterial chemotaxis mechanism is the quantitative relationship between ligand binding to receptors and CheA inhibition. We show that the receptor glutamyl modifications cause adaptation by changing the gain (magnitude amplification) between attractant binding and kinase inhibition without substantially affecting ligand binding affinity. The mechanism adjusts receptor sensitivity to background stimulus intensity over several orders of magnitude of attractant concentrations. The cooperative effects of ligand binding appear to be minimal with Hill coefficients for kinase inhibition less than 2, independent of the state of glutamyl modification.     

An allosteric model for transmembrane signaling in bacterial chemotaxis

Bacteria are able to sense chemical gradients over a wide range of concentrations. However, calculations based on the known number of receptors do not predict such a range unless receptors interact with one another in a cooperative manner. A number of recent experiments support the notion that this remarkable sensitivity in chemotaxis is mediated by localized interactions or crosstalk between neighboring receptors. A number of simple, elegant models have proposed mechanisms for signal integration within receptor clusters. What is a lacking is a model, based on known molecular mechanisms and our accumulated knowledge of chemotaxis, that integrates data from multiple, heterogeneous sources. To address this question, we propose an allosteric mechanism for transmembrane signaling in bacterial chemotaxis based on the "trimer of dimers" model, where three receptor dimers form a stable complex with CheW and CheA. The mechanism is used to integrate a diverse set of experimental data in a consistent framework. The main predictions are: (1) trimers of receptor dimers form the building blocks for the signaling complexes; (2) receptor methylation increases the stability of the active state and retards the inhibition arising from ligand-bound receptors within the signaling complex; (3) trimer of dimer receptor complexes aggregate into clusters through their mutual interactions with CheA and CheW; (4) cooperativity arises from neighboring interaction within these clusters; and (5) cluster size is determined by the concentration of receptors, CheA, and CheW. The model is able to explain a number of seemingly contradictory experiments in a consistent manner and, in the process, explain how bacteria are able to sense chemical gradients over a wide range of concentrations by demonstrating how signals are integrated within the signaling complex.     

Protein phosphorylation in the bacterial chemotaxis system

Bacterial chemotaxis involves the detection of changes in concentration of specific chemicals in the environment of the cell as a function of time. This process is mediated by a series of cell surface receptors that interact with and activate intracellular protein phosphorylation. Five cytoplasmic proteins essential for chemotaxis have been shown to be involved in a coupled system of protein phosphorylation. Ligand binding to cell surface receptors affects the rate of autophosphorylation of the CheA protein. In the absence of an attractant bound to receptor and in the presence of the CheW protein, the rate of CheA autophosphorylation is markedly increased. Phosphorylated CheA can transfer phosphate to the CheY or CheB proteins; phosphorylation of these "effector" proteins may increase their activity. The CheY protein is thought to regulate flagellar rotation and thus control swimming behavior. The CheB protein modifies the cell surface receptor and thus regulates receptor function. Finally, another chemotaxis protein, CheZ, acts to specifically dephosphorylate CheY-phosphate. This system shows marked similarity to the 2-component sensor-regulator systems found to control specific gene expression in a variety of bacteria.     

Two different Pseudomonas aeruginosa chemosensory signal transduction complexes localize to cell poles and form and remould in stationary phase

Pseudomonas aeruginosa has sets of sensory genes designated che and che2. The che genes are required for flagella-mediated chemotaxis. The che2 genes are expressed in the stationary phase of growth and are probably also involved in flagella-mediated behavioural responses. P. aeruginosa also has 26 chemoreceptor genes, six of which are preferentially expressed in stationary phase. Subcellular localization experiments indicated that Che proteins form signal transduction complexes at cell poles throughout growth. Cyan fluorescent protein (CFP)-tagged McpA, a stationary phase-expressed chemoreceptor, appeared and colocalized with yellow fluorescent protein (YFP)-tagged CheA when cells entered stationary phase. This indicates that P. aeruginosa chemotaxis protein complexes are subject to remoulding by chemoreceptor proteins that are expressed when cells stop growing. CheA-CFP and CheY2-YFP tagged proteins that were coexpressed in the same cell had separate subcellular locations, indicating that Che2 proteins do not enter into direct physical interactions with Che proteins. Che2 protein complex formation required McpB, another stationary phase induced chemoreceptor that is predicted to be soluble. This implies that Che2 complexes have a function that depends on just one chemoreceptor. Our results suggest that motile P. aeruginosa cells have signal transduction systems that are adapted to allow non-growing cells to sense and respond to their environment differently from actively growing cells.     

Receptor conformational changes enhance methylesterase activity during chemotaxis by Bacillus subtilis

Addition and removal of the attractant asparagine causes methanol formation as a consequence of methylation and demethylation of conserved glutamate residues in the Bacillus subtilis chemotaxis receptor McpB C-terminal domain. We found that methanol was released on both addition and removal of asparagine even when the response regulator domain of CheB was removed (to produce CheB(141-357)). Thus, in undergoing the transition from unbound receptor to ligand-bound adapted receptor, the receptor must pass through a state of heightened susceptibility to demethylation by CheB that is independent of phosphorylation. The same result occurred when the aspartate phosphorylation site of CheB, Asp54, had been mutated to an asparagine residue, provided the enzyme was sufficiently induced. However, no methanol release was observed for an active site point mutant, cheB(S173C), in response to addition or removal of asparagine even when induced. Finally, methanol release was observed only for attractant addition in a mutant background lacking the coupling proteins, CheW and CheV, provided CheB(141-357) was present. Thus, on attractant addition, methanol must arise from a transient conformation of the receptor C-terminal domain that is an intrinsic property of the receptor; on attractant removal, however, methanol must arise from a different transient conformation, one dependent on the presence of coupling proteins.     

Conserved glycine residues in the cytoplasmic domain of the aspartate receptor play essential roles in kinase coupling and on-off switching

The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on-off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on-off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on-off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on-off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends required for native receptor structure, and in some cases for switching between the on and off signaling states. An initial working model proposes that the novel bundle hinge enables the four-helix bundle to bend, perhaps during the assembly of the receptor trimer of dimers or during on-off switching. More generally, the findings predict that certain human disease states, including specific cancers, could be triggered by lock-on mutations at essential glycine positions that control the on-off switching of receptors and signaling proteins.     

Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis

The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system of Escherichia coli. However, B. subtilis contains several chemotaxis genes not found in the E. coli genome, such as cheC and cheD, indicating that the B. subtilis chemotactic system is more complex. In B. subtilis, CheD is required for chemotaxis; the cheD mutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli. Homologs of B. subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms. However, the molecular function of CheD has remained unknown. In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. In addition, we present evidence that CheD deamidates other B. subtilis chemoreceptors including McpB and McpC. Previously, deamidation of B. subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E. coli receptors. Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required for B. subtilis chemoreceptors to effectively transduce signals to the CheA kinase.     

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.     

CheA-Receptor Interaction Sites in Bacterial Chemotaxis

In bacterial chemotaxis, transmembrane chemoreceptors, the CheA histidine kinase, and the CheW coupling protein assemble into signaling complexes that allow bacteria to modulate their swimming behavior in response to environmental stimuli. Among the protein-protein interactions in the ternary complex, CheA-CheW and CheW-receptor interactions were studied previously, whereas CheA-receptor interaction has been less investigated. Here, we characterize the CheA-receptor interaction in Thermotoga maritima by NMR spectroscopy and validate the identified receptor binding site of CheA in Escherichia coli chemotaxis. We find that CheA interacts with a chemoreceptor in a manner similar to that of CheW, and the receptor binding site of CheA's regulatory domain is homologous to that of CheW. Collectively, the receptor binding sites in the CheA-CheW complex suggest that conformational changes in CheA are required for assembly of the CheA-CheW-receptor ternary complex and CheA activation.