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.     

Cellular stoichiometry of the components of the chemotaxis signaling complex

The chemotactic sensory system of Escherichia coli comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins. These components form signaling complexes that mediate sensory excitation and adaptation. Previous determinations of cellular content of individual components provided differing and apparently conflicting values. We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two E. coli strains considered wild type for chemotaxis, grown in rich and minimal media. Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30%. Thus, cellular stoichiometries were almost constant as amounts varied substantially. Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex. The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW. These components may interact in extended arrays and, thus, stoichiometries could be nonintegral. In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively. These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical.     

The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.     

CheZ phosphatase localizes to chemoreceptor patches via CheA-short

We have investigated the conditions required for polar localization of the CheZ phosphatase by using a CheZ-green fluorescent protein fusion protein that, when expressed from a single gene in the chromosome, restored chemotaxis to a DeltacheZ strain. Localization was observed in wild-type, DeltacheZ, DeltacheYZ, and DeltacheRB cells but not in cells with cheA, cheW, or all chemoreceptor genes except aer deleted. Cells making only CheA-short (CheA(S)) or CheA lacking the P2 domain also retained normal localization, whereas cells producing only CheA-long or CheA missing the P1 and P2 domains did not. We conclude that CheZ localization requires the truncated C-terminal portion of the P1 domain present in CheA(S). Missense mutations targeting residues 83 through 120 of CheZ also abolished localization. Two of these mutations do not disrupt chemotaxis, indicating that they specifically prevent interaction with CheA(S) while leaving other activities of CheZ intact.     

Polar localization of CheA2 in Rhodobacter sphaeroides requires specific Che homologs

Rhodobacter sphaeroides is a motile bacterium that has multiple chemotaxis genes organized predominantly in three major operons (cheOp(1), cheOp(2), and cheOp(3)). The chemoreceptor proteins are clustered at two distinct locations, the cell poles and in one or more cytoplasmic clusters. One intriguing possibility is that the physically distinct chemoreceptor clusters are each composed of a defined subset of specific chemotaxis proteins, including the chemoreceptors themselves plus specific CheW and CheA proteins. Here we report the subcellular localization of one such protein, CheA(2), under aerobic and photoheterotrophic growth conditions. CheA(2) is predominantly clustered and localized at the cell poles under both growth conditions. Furthermore, its localization is dependent upon one or more genes in cheOp(2) but not those of cheOp(1) or cheOp(3). In E. coli, the polar localization of CheA depends upon CheW. The R. sphaeroides cheOp(2) contains two cheW genes. Interestingly, CheW(2) is required under both aerobic and photoheterotrophic conditions, whereas CheW(3) is not required under aerobic conditions but appears to play a modest role under photoheterotrophic conditions. This suggests that R. sphaeroides contains at least two distinct chemotaxis complexes, possibly composed of proteins dedicated for each subcellular location. Furthermore, the composition of these spatially distinct complexes may change under different growth conditions.     

Identification and localization of a methyl-accepting chemotaxis protein in Rhodobacter sphaeroides

Genes coding for a classical membrane spanning chemoreceptor (mcpG) and a response regulator (cheY4) were identified in a region of Rhodobacter sphaeroides DNA unlinked to either of the two previously identified chemosensory operons. Immunogold electron microscopy had shown that the expression of chemoreceptors in R. sphaeroides varies with growth conditions. Using GFP fused to the newly identified McpG, we examined the targeting of this single methyl-accepting chemotaxis protein (MCP) under different growth conditions. The gene encoding the C-terminal McpG-GFP fusion was introduced by homologous recombination into the chromosome, replacing the wild-type gene. The resultant protein localized to the poles of the cell under aerobic, photoheterotrophic and anaerobic dark conditions, demonstrating that this MCP is expressed under all three growth conditions. More protein was always found at one pole than the other. The polar fluorescence increased during the cell cycle, with protein becoming evident at the second pole around the time of septation. At division, each daughter cell had a label at one pole, but the intensity of fluorescence was higher in the daughter cell containing the original labelled pole. McpG localization was not altered in a che Operon 1 deletion strain, lacking CheW1 and CheA1, but a che Operon 2 deletion strain, lacking CheW2, CheW3 and CheA2, showed significantly reduced polar localization. This observation indicates that polar localization of McpG depends on Che proteins encoded by Operon 2, but not homologues encoded by Operon 1.     

Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA.

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.     

Targeting of the chemotaxis methylesterase/deamidase CheB to the polar receptor-kinase cluster in an Escherichia coli cell

Chemotactic adaptation to persisting stimulation involves reversible methylation of the chemoreceptors that form complexes with the histidine kinase CheA at a cell pole. The methyltransferase CheR targets to the C-terminal NWETF sequence of the chemoreceptor. In contrast, localization of the methylesterase CheB is largely unknown, although regulation of its activity via phosphorylation is central to adaptation. In this study, green fluorescent protein was fused to full-length CheB or its various parts: the N-terminal regulatory domain (N), the C-terminal catalytic domain (C) and the linker (L). The full-length and NL fusions and, to a lesser extent, the LC fusion localized to a pole. Deletion of the P2 domain from CheA abolished polar localization of the full-length and NL fusions, but did not affect that of the LC fusion. Pull-down assays demonstrated that the NL fragment, but not the LC fragment, binds to the P2 fragment of CheA. These results indicate that binding of the NL domain to the P2 domain targets CheB to the polar signalling complex. The LC fusion, like the chemoreceptor, partially localized in the absence of CheA, suggesting that the LC domain may interact with its substrate sites, either as part of the protein or as a proteolytic fragment.     

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.     

Methylation segments are not required for chemotactic signalling by cytoplasmic fragments of Tsr, the methyl-accepting serine chemoreceptor of Escherichia coli

The serine chemoreceptor Tsr and other methyl-accepting chemotaxis proteins (MCPs) control the swimming behaviour of Escherichia coli by generating signals that influence the direction of flagellar rotation. MCPs produce clockwise (CW) signals by stimulating the autophosphorylation activity of CheA, a cytoplasmic histidine kinase, and counter-clockwise signals by inhibiting CheA. CheW couples CheA to chemoreceptor control by promoting formation of MCP/CheW/CheA ternary complexes. To identify MCP structural determinants essential for CheA stimulation, we inserted fragments of the tsr coding region into an inducible expression vector and used a swimming contest called 'pseudotaxis' to select for transformant cells carrying CW-signalling plasmids. The shortest active fragment we found, Tsr (350–470), stimulated CheA in a CheW-dependent manner, as full-length Tsr molecules do. It spans a highly conserved 'core' (370–420) that probably specifies the CheA and CheW contact sites and other determinants needed for stimulatory control of CheA. Tsr (350–470) also carries portions of the left and right arms flanking the core, which probably play roles in regulating MCP signalling state. However, this Tsr fragment lacks all of the methylation sites characteristic of MCP molecules, indicating that methylation segments are not essential for generating receptor output signals.     

Attractant binding induces distinct structural changes to the polar and lateral signaling clusters in Bacillus subtilis chemotaxis

Bacteria employ a modified two-component system for chemotaxis, where the receptors form ternary complexes with CheA histidine kinases and CheW adaptor proteins. These complexes are arranged in semi-ordered arrays clustered predominantly at the cell poles. The prevailing models assume that these arrays are static and reorganize only locally in response to attractant binding. Recent studies have shown, however, that these structures may in fact be much more fluid. We investigated the localization of the chemotaxis signaling arrays in Bacillus subtilis using immunofluorescence and live cell fluorescence microscopy. We found that the receptors were localized in clusters at the poles in most cells. However, when the cells were exposed to attractant, the number exhibiting polar clusters was reduced roughly 2-fold, whereas the number exhibiting lateral clusters distinct from the poles increased significantly. These changes in receptor clustering were reversible as polar localization was reestablished in adapted cells. We also investigated the dynamic localization of CheV, a hybrid protein consisting of an N-terminal CheW-like adaptor domain and a C-terminal response regulator domain that is known to be phosphorylated by CheA, using immunofluorescence. Interestingly, we found that CheV was localized predominantly at lateral clusters in unstimulated cells. However, upon exposure to attractant, CheV was found to be predominantly localized to the cell poles. Moreover, changes in CheV localization are phosphorylation-dependent. Collectively, these results suggest that the chemotaxis signaling arrays in B. subtilis are dynamic structures and that feedback loops involving phosphorylation may regulate the positioning of individual proteins.     

Stabilization of polar localization of a chemoreceptor via its covalent modifications and its communication with a different chemoreceptor

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.     

Proteomic mapping of a suppressor of non-chemotactic cheW mutants reveals that Helicobacter pylori contains a new chemotaxis protein

Bacterial chemotaxis is a colonization factor for the ulcer-causing pathogen Helicobacter pylori. H. pylori contains genes encoding the chemotaxis signalling proteins CheW, CheA and CheY; CheW couples chemoreceptors to the CheA kinase and is essential for chemotaxis. While characterizing a cheW mutant, we isolated a spontaneous, chemotactic variant (Che+). We determined that this phenotype was caused by a genetic change unlinked to the original cheW mutation. To locate the underlying Che+ mutation, we compared total protein profiles of the non-chemotactic mutant (cheW) with those from the cheW Che+ variant by two-dimensional differential in-gel electrophoresis. One protein was found only in the cheW Che+ variant. This protein was identified by MS/MS as HP0170, a hypothetical protein with no known function. DNA sequencing verified that hp0170 was mutated in the cheW Che+ suppressor, and deletion of this open reading frame in the cheW background nearly recapitulated the Che+ suppressor phenotype. Using hidden Markov models, we found that HP0170 is a remote homologue of E. coli CheZ. CheZ interacts with phosphorylated CheY and stimulates its autodephosphorylation. CheZ was not predicted to be present in epsilon-proteobacteria. We found that chemotaxis in the cheW Che+ suppressor depended on both cheY and cheA. We hypothesize that a small amount of phosphorylated CheY is generated via CheA in the cheW mutant, and this amount is sufficient to affect flagellar rotation when HP0170 is removed. Our results suggest that HP0170 is a remote homologue of CheZ, and that CheZ homologues are found in a broader range of bacteria than previously supposed.     

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.     

Clustering requires modified methyl-accepting sites in low-abundance but not high-abundance chemoreceptors of Escherichia coli

Chemotaxis signalling complexes of Escherichia coli, composed of chemoreceptors, CheA and CheW, form clusters located predominantly at cell poles. As the only kind of receptor in a cell, high-abundance receptors are polar and clustered whereas low-abundance chemoreceptors are polar but largely unclustered. We found that clustering was a function of the cytoplasmic, carboxyl-terminal domain and that effective clustering was conferred on low-abundance receptors by addition of the approximately 20-residue sequence from the carboxyl terminus of either high-abundance receptor. These sequences are different but share a carboxyl-terminal pentapeptide that enhances adaptational covalent modification and allows a physiological balance between modified and unmodified methyl-accepting sites, implying that receptor modification might influence clustering. Thus we investigated directly effects of modification state on chemoreceptor clustering. As the sole receptor type in a cell, low-abundance receptors were clustered only if modified, but high-abundance receptors were clustered independent of extent of modification. This difference could mean that the two receptor types are fundamentally different or that they are poised at different positions in the same conformational equilibrium. Notably, no receptor perturbation we tested altered a predominant location at cell poles, emphasizing a distinction between determinants of clustering and polar localization.     

Large increases in attractant concentration disrupt the polar localization of bacterial chemoreceptors

In bacterial chemotaxis, the chemoreceptors [methyl-accepting chemotaxis proteins (MCPs)] transduce chemotactic signals through the two-component histidine kinase CheA. At low but not high attractant concentrations, chemotactic signals must be amplified. The MCPs are organized into a polar lattice, and this organization has been proposed to be critical for signal amplification. Although evidence in support of this model has emerged, an understanding of how signals are amplified and modulated is lacking. We probed the role of MCP localization under conditions wherein signal amplification must be inhibited. We tested whether a large increase in attractant concentration (a change that should alter receptor occupancy from c. 0% to > 95%) would elicit changes in the chemoreceptor localization. We treated Escherichia coli or Bacillus subtilis with a high level of attractant, exposed cells to the cross-linking agent paraformaldehyde and visualized chemoreceptor location with an anti-MCP antibody. A marked increase in the percentage of cells displaying a diffuse staining pattern was obtained. In contrast, no increase in diffuse MCP staining is observed when cells are treated with a repellent or a low concentration of attractant. For B. subtilis mutants that do not undergo chemotaxis, the addition of a high concentration of attractant has no effect on MCP localization. Our data suggest that interactions between chemoreceptors are decreased when signal amplification is unnecessary.     

Clustering of the Chemoreceptor Complex in Escherichia coli Is Independent of the Methyltransferase CheR and the Methylesterase CheB

The Escherichia coli chemoreceptors and their associated cytoplasmic proteins, CheA and CheW, cluster predominantly at the cell poles. The nature of the clustering remains a mystery. Recent studies suggest that CheR binding to and/or methylation of the chemoreceptors may play a role in chemoreceptor complex aggregation. In this study, we examined the intracellular distribution of the chemoreceptors by immunoelectron microscopy in strains lacking either the methyltransferase CheR or the methylesterase CheB. The localization data revealed that, in vivo, aggregation of the chemoreceptor complex was independent of either CheR or CheB.     

Only one of the five CheY homologs in Vibrio cholerae directly switches flagellar rotation

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.     

Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response

In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.     

The receptor-CheW binding interface in bacterial chemotaxis

The basic structural unit of the signaling complex in bacterial chemotaxis consists of the chemotaxis kinase CheA, the coupling protein CheW, and chemoreceptors. These complexes play an important role in regulating the kinase activity of CheA and in turn controlling the rotational bias of the flagellar motor. Although individual three-dimensional structures of CheA, CheW, and chemoreceptors have been determined, the interaction between chemoreceptor and CheW is still unclear. We used nuclear magnetic resonance to characterize the interaction modes of chemoreceptor and CheW from Thermotoga maritima. We find that chemoreceptor binding surface is located near the highly conserved tip region of the N-terminal helix of the receptor, whereas the binding interface of CheW is placed between the β-strand 8 of domain 1 and the β-strands 1 and 3 of domain 2. The receptor-CheW complex shares a similar binding interface to that found in the "trimer-of-dimers" oligomer interface seen in the crystal structure of cytoplasmic domains of chemoreceptors from Escherichia coli. Based on the association constants inferred from fast exchange chemical shifts associated with receptor-CheW titrations, we estimate that CheW binds about four times tighter to its first binding site of the receptor dimer than to its second binding site. This apparent anticooperativity in binding may reflect the close proximity of the two CheW binding surfaces near the receptor tip or further, complicating the events at this highly conserved region of the receptor. This work describes the first direct observation of the interaction between chemoreceptor and CheW.