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.     

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.     

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.     

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.     

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.     

Mutational analysis of N381, a key trimer contact residue in Tsr, the Escherichia coli serine chemoreceptor

Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery.     

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.     

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.     

Organization of the receptor-kinase signaling array that regulates Escherichia coli chemotaxis

Motor behavior in prokaryotes is regulated by a phosphorelay network involving a histidine protein kinase, CheA, whose activity is controlled by a family of Type I membrane receptors. In a typical Escherichia coli cell, several thousand receptors are organized together with CheA and an Src homology 3-like protein, CheW, into complexes that tend to be localized at the cell poles. We found that these complexes have at least 6 receptors per CheA. CheW is not required for CheA binding to receptors, but is essential for kinase activation. The kinase activity per mole of bound CheA is proportional to the total bound CheW. Similar results were obtained with the E. coli serine receptor, Tsr, and the Salmonella typhimurium aspartate receptor, Tar. In the case of Tsr, under conditions optimal for kinase activation, the ratio of subunits in complexes is approximately 6 Tsr:4 CheW:1 CheA. Our results indicate that information from numerous receptors is integrated to control the activity of a relatively small number of kinase molecules.     

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 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.     

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.     

The dynamics of protein phosphorylation in bacterial chemotaxis

The chemotaxis signal transduction pathway allows bacteria to respond to changes in concentration of specific chemicals (ligands) by modulating their swimming behavior. The pathway includes ligand binding receptors, and the CheA, CheY, CheW, and CheZ proteins. We showed previously that phosphorylation of CheY is activated in reactions containing receptor, CheW, CheA, and CheY. Here we demonstrate that this activation signal results from accelerated autophosphorylation of the CheA kinase. Evidence for a second signal transmitted by a ligand-bound receptor, which corresponds to inhibition of CheA autophosphorylation, is also presented. We postulate that CheA can exist in three forms: a "closed" form in the absence of receptor and CheW; an "open" form that results from activation of CheA by receptor and CheW; and a "sequestered" form in reactions containing ligand-bound receptor and CheW. The system's dynamics depends on the relative distribution of CheA among these three forms at any time.     

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.     

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).     

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.     

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.     

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.     

CheA Kinase of bacterial chemotaxis: chemical mapping of four essential docking sites

The chemotaxis pathway of Escherichia coli and Salmonella typhimurium is the paradigm for the ubiquitous class of 2-component signaling pathways in prokaryotic organisms. Chemosensing begins with the binding of a chemical attractant to a transmembrane receptor on the cell surface. The resulting transmembrane signal regulates a cytoplasmic, multiprotein signaling complex that controls cellular swimming behavior by generating a diffusible phosphoprotein. The minimal functional unit of this signaling complex, termed the core complex, consists of the transmembrane receptor, the coupling protein CheW, and the histidine kinase CheA. Though the structures of individual components are largely known and the core complex can be functionally reconstituted, the architecture of the assembled core complex has remained elusive. To probe this architecture, the present study has utilized an enhanced version of the protein-interactions-by-cysteine-modification method (PICM-beta) to map out docking surfaces on CheA essential for kinase activity and for core complex assembly. The approach employed a library of 70 single, engineered cysteine residues, scattered uniformly over the surfaces of the five CheA domains in a cysteine-free CheA background. These surface Cys residues were further modified by the sulfhydryl-specific alkylating agent, 5-fluorescein-maleimide (5FM). The functional effects of individual Cys and 5FM-Cys surface modifications were measured by kinase assays of CheA activity in both the free and core complex-associated states, and by direct binding assays of CheA associations with CheW and the receptor. The results define (i) two mutual docking surfaces on the CheA substrate and catalytic domains essential for the association of these domains during autophosphorylation, (ii) a docking surface on the CheA regulatory domain essential for CheW binding, and (iii) a large docking surface encompassing regions of the CheA dimerization, catalytic, and regulatory domains proposed to bind the receptor. To test the generality of these findings, a CheA sequence alignment was analyzed, revealing that the newly identified docking surfaces are highly conserved among CheA homologues. These results strongly suggest that the same docking sites are widely utilized in prokaryotic sensory pathways. Finally, the results provide new structural constraints allowing the development of improved models for core complex architecture.     

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.