Bacterial chemoreceptors of different length classes signal independently

Bacterial chemoreceptors sense environmental stimuli and govern cell movement by transmitting the information to the flagellar motors. The highly conserved cytoplasmic domain of chemoreceptors consists in an alpha‐helical hairpin that forms in the homodimer a coiled‐coil four‐helix bundle. Several classes of chemoreceptors that differ in the length of the coiled‐coil structure were characterized. Many bacterial species code for chemoreceptors that belong to different classes, but how these receptors are organized and function in the same cell remains an open question. E. coli cells normally code for single class chemoreceptors that form extended arrays based on trimers of dimers interconnected by the coupling protein CheW and the kinase CheA. This structure promotes effective coupling between the different receptors in the modulation of the kinase activity. In this work, we engineered functional derivatives of the Tsr chemoreceptor of E. coli that mimic receptors whose cytoplasmic domain is longer by two heptads. We found that these long Tsr receptors did not efficiently mix with the native receptors and appeared to function independently. Our results suggest that the assembly of membrane‐bound receptors of different specificities into mixed clusters is dictated by the length‐class to which the receptors belong, ensuring cooperative function only between receptors of the same class.     

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.     

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.     

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.     

Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor.

Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro. Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.     

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.     

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.     

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.     

Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

Background  

Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr), which contributes to an increase in the steady-state (adapted) methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA) and an adaptor protein (CheW), but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation.     

Chemoreceptors in signalling complexes: shifted conformation and asymmetric coupling

Bacterial chemotaxis is mediated by signalling complexes of chemoreceptors, histidine kinase CheA and coupling protein CheW. Interactions in complexes profoundly affect the kinase. We investigated effects of these interactions on chemoreceptors by comparing receptors alone and in complexes. Assays of initial rates of methylation indicated that signalling complexes shifted receptor conformation towards the methylation-on, higher-ligand-affinity, kinase-off state, tuning receptors for greater sensitivity. In contrast, transmembrane and conformational signalling within chemoreceptors was essentially unaltered, consistent with other evidence identifying receptor dimers as the fundamental units of such signalling. In signalling complexes, coupling of ligand binding to kinase activity is cooperative and the dynamic range of kinase control expanded > 100-fold by receptor adaptational modification. We observed no cooperativity in influence of ligand on receptor conformation, only on kinase activity. However, receptor modification generated increased dynamic range in a stepwise fashion, partly in coupling ligand to receptor conformation and partly in coupling receptor conformation to kinase activity. Thus, receptors and kinase were not equivalently affected by interactions in signalling complexes or by ligand binding and adaptational modification, indicating asymmetrical coupling between them. This has implications for mechanisms of precise adaptation. Coupling might vary, providing a previously unappreciated locus for sensory control.     

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.     

Cooperative signaling among bacterial chemoreceptors

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

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 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.     

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.     

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.     

Not too loose, not too tight--just right. Biphasic control of the Tsr HAMP domain

HAMP domains communicate between input and output signalling modules in a wide variety of bacterial sensor proteins. In the Tsr chemoreceptor, they convert a signal initiated by binding of serine to the periplasmic domain of the protein into regulation of receptor control of the CheA kinase, and ultimately of the direction of flagellar rotation. In this issue, Zhou et al. report an extensive mutational analysis of the Tsr HAMP domain that shows that it can assume a number of different signalling states, which presumably correspond to a variety of different conformations. The two conformational extremes of a tightly packed and a loosely packed HAMP four‐helix bundle support only low levels of CheA activity. Thus, Tsr HAMP does not function as a simple on‐off, two‐state device but rather as a dynamic structure with biphasic control. The normal physiological operating range of Tsr is proposed to be at intermediate degrees of packing of the HAMP four‐helix bundle, but HAMP domains in other proteins could occupy different portions of the conformational spectrum.     

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.     

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.     

Hypoxia‐induced localization of chemotaxis‐related signaling proteins in Vibrio cholerae

Vibrio cholerae has three sets of chemotaxis‐related signaling proteins, of which only System II has been shown to be involved in chemotaxis. Here, we examined localization of green fluorescent protein (GFP)‐fused components of System I. The histidine kinase (CheA1) and the adaptor (CheW0) of System I localized to polar and lateral membrane regions with standing incubation (microaerobic conditions), but their localization was lost after shaking (aerobic conditions). A transmembrane receptor of System I also showed polar and lateral localization with standing incubation. By contrast, GFP‐fused components of System II localized constitutively to the flagellated pole. Nitrogen gas, sodium azide or carbonylcyanide m‐chlorophenylhydrazone induced localization of CheA1‐GFP even with shaking incubation, suggesting that the localization is controlled in response to changes in energy metabolism. Fluorescently labeled tetracysteine‐tagged CheA1 also showed azide‐induced localization, arguing against artifactual effects of GFP fusions. These results suggest that System I components are assembled into the supramolecular signaling complex in response to reduced cellular energy states, raising the possibility that the System I complex plays a role in sensing and signaling under microaerobic environments, such as in the host intestine.