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.     

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.     

Conformational Coupling between Receptor and Kinase Binding Sites through a Conserved Salt Bridge in a Signaling Complex Scaffold Protein

Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD), NMR spectroscopy, and circular dichroism (CD), we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.     

Cysteine-scanning analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA

The C-terminal P5 domain of the histidine kinase CheA is essential for coupling CheA autophosphorylation activity to chemoreceptor control through a binding interaction with the CheW protein. To locate P5 determinants critical for CheW binding and chemoreceptor control, we surveyed cysteine replacements at 39 residues predicted to be at or near the P5 surface in Escherichia coli CheA. Two-thirds of the Cys replacement proteins exhibited in vitro defects in CheW binding, either before or after modification with a bulky fluorescein group. The binding-defective sites were widely distributed on the P5 surface and were often interspersed with sites that caused no functional defects, implying that relatively minor structural perturbations, often far from the actual binding site, can influence its conformation or accessibility. The most likely CheW docking area included loop 2 in P5 folding subdomain 1. All but four of the binding-defective P5-Cys proteins were defective in receptor-mediated activation, suggesting that CheW binding, as measured in vitro, is necessary for assembly of ternary signaling complexes and/or subsequent CheA activation. Other Cys sites specifically affected receptor-mediated activation or deactivation of CheA, demonstrating that CheW binding is not sufficient for assembly and/or operation of receptor signaling complexes. Because P5 is quite similar to CheW, whose structure is known to be dynamic, we suggest that conformational flexibility and dynamic motions govern the signaling activities of the P5 domain. In addition, relative movements of the CheA domains may be involved in CheW binding, in ternary complex assembly, and in subsequent stimulus-induced conformational changes in receptor signaling complexes.     

Mutational analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3' interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA.     

Reconstruction of the chemotaxis receptor-kinase assembly

In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.     

The solution structure and interactions of CheW from Thermotoga maritima.

Using protein from the hyperthermophile Thermotoga maritima, we have determined the solution structure of CheW, an essential component in the formation of the bacterial chemotaxis signaling complex. The overall fold is similar to the regulatory domain of the chemotaxis kinase CheA. In addition, interactions of CheW with CheA were monitored by nuclear magnetic resonance (NMR) techniques. The chemical shift perturbation data show the probable contacts that CheW makes with CheA. In combination with previous genetic data, the structure also suggests a possible binding site for the chemotaxis receptor. These results provide a structural basis for a model in which CheW acts as a molecular bridge between CheA and the cytoplasmic tails of the receptor.     

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.     

A fragment liberated from the Escherichia coli CheA kinase that blocks stimulatory, but not inhibitory, chemoreceptor signaling.

CheA, a cytoplasmic histidine autokinase, in conjunction with the CheW coupling protein, forms stable ternary complexes with the cytoplasmic signaling domains of transmembrane chemoreceptors. These signaling complexes induce chemotactic movements by stimulating or inhibiting CheA autophosphorylation activity in response to chemoeffector stimuli. To explore the mechanisms of CheA control by chemoreceptor signaling complexes, we examined the ability of various CheA fragments to interfere with receptor coupling control of CheA. CheA[250-654], a fragment carrying the catalytic domain and an adjacent C-terminal segment previously implicated in stimulatory control of CheA activity, interfered with the production of clockwise flagellar rotation and with chemotactic ability in wild-type cells. Epistasis tests indicated that CheA[250-654] blocked clockwise rotation by disrupting stimulatory coupling of CheA to receptors. In vitro coupling assays confirmed that a stoichiometric excess of CheA[250-654] fragments could exclude CheA from stimulatory receptor complexes, most likely by competing for CheW binding. However, CheA[250-654] fragments, even in vast excess, did not block receptor-mediated inhibition of CheA, suggesting that CheA[250-654] lacks an inhibitory contact site present in native CheA. This inhibitory target is most likely in the N-terminal P1 domain, which contains His-48, the site of autophosphorylation. These findings suggest a simple allosteric model of CheA control by ternary signaling complexes in which the receptor signaling domain conformationally regulates the interaction between the substrate and catalytic domains of CheA.     

Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex

Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a “phosphate sink” possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.     

Different signaling roles of two conserved residues in the cytoplasmic hairpin tip of Tsr, the Escherichia coli serine chemoreceptor

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.     

Solution structure of the bacterial chemotaxis adaptor protein CheW from Escherichia coli

The bacterial chemotaxis adaptor protein CheW physically links the chemoreceptors (MCPs) and the histidine kinase CheA. Extensive investigations using bacterium Escherichia coli have established the central role of CheW in the MCP-modulated activation of CheA. Here we report the solution structure of CheW from E. coli determined by NMR spectroscopy. The results show that E. coli CheW shares an overall fold with previously reported structure of CheW from Thermotoga maritima, whereas local conformational deviations are observed. In particular, the C-terminal alpha-helix is considerably longer in E. coli CheW and appears to shrink the active binding pocket with CheA. Our study provides the structural basis for further investigations in E. coli chemotaxis.     

CheA kinase and chemoreceptor interaction surfaces on CheW

Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar. In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA. We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar. Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar.     

Signalling‐dependent interactions between the kinase‐coupling protein CheW and chemoreceptors in living cells

Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross‐linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal‐sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).     

Cellular stoichiometry of the chemotaxis proteins in Bacillus subtilis

The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B. subtilis. Significantly higher levels of chemoreceptors and much lower levels of CheA kinase were measured in B. subtilis than in Escherichia coli. The resulting cellular ratio of chemoreceptor dimers per CheA dimer in B. subtilis is roughly 23.0 ± 4.5 compared to 3.4 ± 0.8 receptor dimers per CheA dimer observed in E. coli, but the ratios of the coupling protein CheW to the CheA dimer are nearly identical in the two organisms. The ratios of CheB to CheR in B. subtilis are also very similar, although the overall levels of modification enzymes are higher. When the potential binding partners of CheD are deleted, the levels of CheD drop significantly. This finding suggests that B. subtilis selectively degrades excess chemotaxis proteins to maintain optimum ratios. Finally, the two cytoplasmic receptors were observed to localize among the other receptors at the cell poles and appear to participate in the chemoreceptor complex. These results suggest that there are many novel features of B. subtilis chemotaxis compared with the mechanism in E. coli, but they are built on a common core.     

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.     

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.     

Chemotactic Signaling by Single-Chain Chemoreceptors

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.     

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.     

Stimulus response coupling in bacterial chemotaxis: receptor dimers in signalling arrays

In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function.