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.     

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).     

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.     

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.     

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.     

Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli.

CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli. CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation. Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells. To find out why, we examined the effects of a purified P1 fragment, CheA[1-149], on CheA-related signaling activities in vitro and devised in vivo assays for those same activities. Although readily phosphorylated by CheA[260-537], the CheA catalytic domain, CheA[1-149], was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center. CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA[1-149], suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect. In agreement with these findings, a 40-fold excess of CheA[1-149] fragments did not impair the CheA autophosphorylation reaction. CheA[1-149] did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules. An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site. The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells. However, an excess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragments have both transphosphorylation and phosphotransfer capability in vivo.     

Determinants of chemotactic signal amplification in Escherichia coli

A well-characterized protein phosphorelay mediates Escherichia coli chemotaxis towards the amino acid attractant aspartate. The protein CheY shuttles between flagellar motors and methyl-accepting chemoreceptor (MCP) complexes containing the linker CheW and the kinase CheA. CheA-CheY phosphotransfer generates phospho-CheY, CheY-P. Aspartate triggers smooth swim responses by inactivation of the CheA bound to the target MCP, Tar; but this mechanism alone cannot explain the observed response sensitivity. Here, we used behavioral analysis of mutants deleted for CheZ, a catalyst of CheY-P dephosphorylation, or the methyltransferase CheR and/or the methylesterase CheB to examine the roles of accelerated CheY-P dephosphorylation and MCP methylation in enhancement of the chemotactic response. The extreme motile bias of the mutants was adjusted towards wild-type values, while preserving much of the aspartate response sensitivity by expressing fragments of the MCP, Tsr, that either activate or inhibit CheA. We then measured responses to small jumps of aspartate, generated by flash photolysis of photo-labile precursors. The stimulus-response relation for Delta cheZ mutants overlapped that for the host strains. Delta cheZ excitation response times increased with stimulus size consistent with formation of an occluded CheA state. Thus, neither CheZ-dependent or independent increases in CheY-P dephosphorylation contribute to the excitation response. In Delta cheB Delta cheR or Delta cheR mutants, the dose for a half-maximal response, [Asp](50), was ca 10 microM; but was elevated to 100 microM in Delta cheB mutants. In addition, the stimulus-response relation for these mutants was linear, consistent with stoichiometric inactivation, in contrast to the non-linear relation for wild-type E. coli. These data suggest that response sensitivity is controlled by differential binding of CheR and/or CheB to distinct MCP signaling conformations.     

Reconstitution of signaling in bacterial chemotaxis.

Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.     

Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro

A chimeric fusion protein consisting of Natronomonas pharaonis sensory rhodopsin II (SRII), fused by a flexible linker to the two transmembrane helices of its cognate transducer protein, HtrII, followed by the HtrII membrane-proximal cytoplasmic fragment joined to the cytoplasmic domains of the Escherichia coli chemotaxis receptor Tsr, was expressed in E. coli. Purified fusion chimera protein reconstituted in liposomes binds to E. coli CheA kinase in the presence of the coupling protein CheW, and activates CheA autophosphorylation activity. CheA kinase activity is stimulated by photoexcitation of the SRII domain of the fusion protein, as shown by the wavelength-dependence of photostimulated phosphotransfer to the E. coli flagellar motor response regulator CheY in the purified in vitro liposomal system. Further confirming the fidelity of the in vitro system, increased and decreased levels of CheA activation in vitro result from overmethylated and undermethylated fusion protein purified from methylesterase and methyltransferase-deficient E. coli, respectively. Photoexcitation of the undermethylated fusion protein resulted in a 3-fold increase in phosphotransfer over that of the dark state. The results directly demonstrate the coupling of SRII photoactivated states to histidine kinase activity, previously predicted on the basis of sequence homologies of the haloarchaeal phototaxis system components to those of E. coli chemotaxis. The fusion chimera provides the first tool for in vitro measurement of photosignaling activity of SRII-HtrII molecular complexes.     

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.     

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.     

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.     

Direction of flagellar rotation in bacterial cell envelopes

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable.     

Structural insight into the rotational switching mechanism of the bacterial flagellar motor

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.     

Structural analysis of bacterial chemotaxis proteins: components of a dynamic signaling system

Most motile bacteria are capable of directing their movement in response to chemical gradients, a behavior known as chemotaxis. The signal transduction system that mediates chemotaxis in enteric bacteria consists of a set of six cytoplasmic proteins that couple stimuli sensed by a family of transmembrane receptors to behavioral responses generated by the flagellar motors. Signal transduction occurs via a phosphotransfer pathway involving a histidine protein kinase, CheA, and a response regulator protein, CheY, that in its phosphorylated state, modulates the direction of flagellar rotation. Two auxiliary proteins, CheW and CheZ, and two receptor modification enzymes, methylesterase CheB and methyltransferase CheR, influence the flux of phosphoryl groups within this central pathway. This paper focuses on structural characteristics of the four signaling proteins (CheA, CheY, CheB, and CheR) for which NMR or x-ray crystal structures have been determined. The proteins are examined with respect to their signaling activities that involve reversible protein modifications and transient assembly of macromolecular complexes. A variety of data suggest conformational flexibility of these proteins, a feature consistent with their multiple roles in a dynamic signaling pathway.     

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.     

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.     

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.     

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.