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.     

Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation

The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.     

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.     

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.     

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.     

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.     

Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW

Characterizing protein-protein interactions in a biologically relevant context is important for understanding the mechanisms of signal transduction. Most signal transduction systems are membrane associated and consist of large multiprotein complexes that undergo rapid reorganization—circumstances that present challenges to traditional structure determination methods. To study protein-protein interactions in a biologically relevant complex milieu, we employed a protein footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. ICAT reagents are valuable tools for proteomics. Here, we show their utility in an alternative application—they are ideal for protein footprinting in complex backgrounds because the affinity tag moiety allows for enrichment of alkylated species prior to analysis. We employed a water-soluble ICAT reagent to monitor cysteine accessibility and thereby to identify residues involved in two different protein-protein interactions in the Escherichia coli chemotaxis signaling system. The chemotaxis system is an archetypal transmembrane signaling pathway in which a complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends on the adaptor protein CheW, which mediates a functionally important bridging interaction between transmembrane receptors and histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large multidomain histidine kinase CheA, as well as with the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA-Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite CheW-Tsr surface for the recruitment of the signaling kinase to the chemoreceptor complex.     

Subunit organization in a soluble complex of tar,CheW, and CheA by electron microscopy

The Salmonella and Escherichia coli aspartate receptor, Tar, is representative of a large class of membrane receptors that generate chemotaxis responses by regulating the activity of an associated histidine protein kinase, CheA. Tar is composed of an NH(2)-terminal periplasmic ligand-binding domain linked through a transmembrane sequence to a COOH-terminal coiled-coil signaling domain in the cytoplasm. The isolated cytoplasmic domain of Tar fused to a leucine zipper sequence forms a soluble complex with CheA and the Src homology 3-like kinase activator, CheW. Activity of the CheA kinase in the soluble complex is essentially the same as in fully active complexes with the intact receptor in the membrane. The soluble complex is composed of approximately 28 receptor cytoplasmic domain chains, 6 CheW chains, and 4 CheA chains. It has a molecular weight of 1,400,000 (Liu, I., Levit, M., Lurz, R., Surette, M.G., and Stock, J.B. (1997) EMBO J. 16, 7231-7240). Electron microscopy reveals an elongated barrel-like structure with a largely hollow center. Immunoelectron microscopy has provided a general picture of the subunit and domain organization of the complex. CheA and CheW appear to be in the middle of the complex with the leucine zippers of the receptor construct at the ends. These findings show that the receptor signaling complex forms higher ordered structures with defined geometric architectures. Coupled with atomic models of the subunits, our results provide insights into the functional architecture by which the receptor regulates CheA kinase activity during bacterial chemotaxis.     

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.     

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.     

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.     

Determinants of chemoreceptor cluster formation in Escherichia coli

Chemotactic stimuli in bacteria are sensed by large sensory complexes, or receptor clusters, that consist of tens of thousands of proteins. Receptor clusters appear to play a key role in signal processing, but their structure remains poorly understood. Here we used fluorescent protein fusions to study in vivo formation of the cluster core, which consists of receptors, a kinase CheA and an assisting protein CheW. We show that receptors aggregate through their cytoplasmic domains even in the absence of other chemotaxis proteins. Clustering is further enhanced by the binding of CheW. Surprisingly, we observed that some fragments of CheA bind receptor clusters well in the absence of CheW, although the latter does assist the binding of full-length CheA. The resulting mode of receptor cluster formation is consistent with an experimentally observed flexible stoichiometry of chemosensory complexes and with assumptions of recently proposed computer models of signal processing in chemotaxis.     

Competitive and cooperative interactions in receptor signaling complexes

In bacterial chemotaxis, clustered transmembrane receptors and the adaptor protein CheW regulate the kinase CheA. Receptors outnumber CheA, yet it is poorly understood how interactions among receptors contribute to regulation. To address this problem, receptor clusters were simulated using liposomes decorated with the cytoplasmic domains of receptors, which supported CheA binding and stimulation. Competitive and cooperative interactions were revealed through the use of known receptor signaling mutants, which were used in mixtures with the wild type domain. Competitive effects among the receptor domains sorted cleanly into two categories defined by either stronger or weaker interactions with CheA. Cooperative effects were also evident in CheA binding and activity. In the transition from the stimulating to the inhibiting states, both the cooperativity of the transition and the persistence of stimulation by the wild type domain increased with receptor modification, as in the intact receptor. We conclude that competitive and cooperative receptor interactions both contribute to CheA regulation and that liposome-mediated assembly is effective in addressing these general membrane phenomena.     

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.     

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.     

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm.     

The carboxy-terminal portion of the CheA kinase mediates regulation of autophosphorylation by transducer and CheW.

The CheA kinase is a central protein in the signal transduction network that controls chemotaxis in Escherichia coli. CheA receives information from a transmembrane receptor (e.g., Tar) and CheW proteins and relays it to the CheB and CheY proteins. The biochemical activities of CheA proteins truncated at various distances from the carboxy terminus were examined. The carboxy-terminal portion of CheA regulates autophosphorylation in response to environmental signals transmitted through Tar and CheW. The central portion of CheA is required for autophosphorylation and is also presumably involved in dimer formation. The amino-terminal portion of CheA was previously shown to contain the site of autophosphorylation and to be able to transfer the phosphoryl group to CheB and CheY. These studies further delineate three functional domains of the CheA protein.     

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.     

Covalent modification regulates ligand binding to receptor complexes in the chemosensory system of Escherichia coli

In the Escherichia coli chemosensory pathway, receptor modification mediates adaptation to ligand. Evidence is presented that covalent modification influences ligand binding to receptors in complexes with CheW and the kinase CheA. Kinase inhibition was measured with serine receptor complexes in different modification levels; Ki for serine-mediated inhibition increased 10,000-fold from the lowest to the highest level. Without CheA and CheW, ligand binding is unaffected by covalent modification; thus, the influence of covalent modification is mediated only in the receptor complex, a conclusion supported by an analogy to allosteric enzymes and the observation of cooperative kinase inhibition. Also, the finding that a subsaturating serine concentration accelerates active receptor-kinase complex assembly implies that the assembly/disassembly process may also contribute to kinase regulation.     

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.