Coupling Protein CheW Research Articles

Chemotaxis Coupling Protein CheW2 Is Not Required for the Chemotaxis but Contributes to the Full Pathogenicity of Borreliella burgdorferi.

The bacterial chemotaxis regulatory circuit mainly consists of coupling protein CheW, sensor histidine kinase CheA, and response regulator CheY. Most bacteria, such as Escherichia coli, have a single gene encoding each of these proteins. Interestingly, the Lyme disease pathogen, Borreliella burgdorferi, has multiple chemotaxis proteins, e.g., two CheA, three CheW, and three CheY proteins. The genes encoding these proteins mainly reside in two operons: cheW2-cheA1-cheB2-cheY2 (A-I) and cheA2-cheW3-cheX-cheY3 (A-II). Previous studies demonstrate that all the genes in A-II are essential for the chemotaxis of B. burgdorferi; however, the role of those genes in A-I remains unknown. This study aimed to fill this gap using the CheW2 gene, the first gene in A-I, as a surrogate. We first mapped the transcription start site of A-I upstream of cheW2 and identified a σ70-like promoter (PW2) and two binding sites (BS1 and BS2) of BosR, an unorthodox Fur/Per homolog. We then demonstrated that BosR binds to PW2 via BS1 and BS2 and that deletion of bosR significantly represses the expression of cheW2 and other genes in A-I, implying that BosR is a positive regulator of A-I. Deletion of cheW2 has no impact on the chemotaxis of B. burgdorferi in vitro but abrogates its ability to evade host adaptive immunity, because the mutant can establish systemic infection only in SCID mice and not in immunocompetent BALB/c mice. This report substantiates the previous proposition that A-I is not implicated in chemotaxis; rather, it may function as a signaling transduction pathway to regulate B. burgdorferi virulence gene expression.

Eukaryotic catecholamine hormones influence the chemotactic control of Vibrio campbellii by binding to the coupling protein CheW

SignificanceHost-emitted stress hormones significantly influence the growth and behavior of various bacterial species; however, their cellular targets have so far remained elusive. Here, we used customized probes and quantitative proteomics to identify the target of epinephrine and the α-adrenoceptor agonist phenylephrine in live cells of the aquatic pathogen Vibrio campbellii. Consequently, we have discovered the coupling protein CheW, which is in the center of the chemotaxis signaling network, as a target of both molecules. We not only demonstrate direct ligand binding to CheW but also elucidate how this affects chemotactic control. These findings are pivotal for further research on hormone-specific effects on bacterial behavior.

Determining the role of the coupling protein CheW in signal transduction of chemoreceptor complexes using solid-state NMR

How an organism senses and responds to its environment is one of the fundamental questions in nature. Chemotaxis, the ability for bacterial cells to sense their environment and control their swimming direction, has been widely studied in order to understand cell sensing.Bacterial chemoreceptor complexes are responsible for receiving signals from the environment.However, the mechanism that bacterial chemoreceptor complexes use to transmit the signals is still unknown. The chemoreceptor complexes are made up of the chemoreceptor, the histidine kinase CheA and the coupling protein CheW.

Strategies for identifying dynamic regions in protein complexes: Flexibility changes accompany methylation in chemotaxis receptor signaling states

Bacterial chemoreceptors are organized in arrays composed of helical receptors arranged as trimers of dimers, coupled to a histidine kinase CheA and a coupling protein CheW. Ligand binding to the external domain inhibits the kinase activity, leading to a change in the swimming behavior. Adaptation to an ongoing stimulus involves reversible methylation and demethylation of specific glutamate residues. However, the exact mechanism of signal propagation through the helical receptor to the histidine kinase remains elusive. Dynamics of the receptor cytoplasmic domain is thought to play an important role in the signal transduction, and current models propose inverse dynamic changes in different regions of the receptor. We hypothesize that the adaptational modification (methylation) controls the dynamics by stabilizing a partially ordered domain, which in turn modulates the binding of the kinase, CheA. We investigated the difference in dynamics between the methylated and unmethylated states of the chemoreceptor using solid-state NMR. The unmethylated receptor (CF4E) shows increased flexibility relative to the methylated mimic (CF4Q). Methylation helix 1 (MH1) has been shown to be flexible in the methylated mimic receptor. Our analysis indicates that in addition to MH1, methylation helix 2 also becomes flexible in the unmethylated receptor. In addition, we have demonstrated that both states of the receptor have a rigid region and segments with intermediate timescale dynamics. The strategies used in this study for identifying dynamic regions are applicable to a broad class of proteins and protein complexes with intrinsic disorder and dynamics spanning multiple timescales.

Truncated, Non-networking Versions of the Coupling Protein CheW Retain Chemoreceptor Control of Kinase CheA

Bacterial chemoreceptors control the activity of the associated CheA kinase in response to chemical gradients and, consequently, regulate the swimming behavior of the cell. However, such control is not direct but requires the participation of the essential coupling protein CheW, which is structurally homologous to the carboxy-terminal domain of the kinase. The actual role of this small coupling protein is somehow intriguing. It has been demonstrated that it is absolutely essential for chemoreceptor control of the kinase, in spite of the occurrence of direct contacts between chemoreceptors and CheA. In addition, CheW plays an essential role in the assembly of the large macromolecular arrays that combine chemoreceptors of different specificities, and it is therefore responsible for molecular interactions that provide such arrays with remarkable signaling properties. In this work, we analyze truncated CheW derivatives that are still able to control the kinase but have lost the ability to connect signaling units. We demonstrate that these two activities can work separately and speculate about the significance of the roles of these two different activities in the context of the chemoreceptor cluster.

In Situ Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States.

Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

A dual regulation mechanism of histidine kinase CheA identified by combining network-dynamics modeling and system-level input-output data.

It is challenging to decipher molecular mechanisms in biological systems from system-level input-output data, especially for complex processes that involve interactions among multiple components. We addressed this general problem for the bacterial histidine kinase CheA, the activity of which is regulated in chemotaxis signaling complexes by bacterial chemoreceptors. We developed a general network model to describe the dynamics of the system, treating the receptor complex with coupling protein CheW and the P3P4P5 domains of kinase CheA as a regulated enzyme with two substrates, ATP and P1, the phosphoryl-accepting domain of CheA. Our simple network model allowed us to search hypothesis space systematically. For different and progressively more complex regulation schemes, we fit our models to a large set of input-output data with the aim of identifying the simplest possible regulation mechanisms consistent with the data. Our modeling and analysis revealed novel dual regulation mechanisms in which receptor activity regulated ATP binding plus one other process, either P1 binding or phosphoryl transfer between P1 and ATP. Strikingly, in our models receptor control affected the kinetic rate constants of substrate association and dissociation equally and thus did not alter the respective equilibrium constants. We suggest experiments that could distinguish between the two dual-regulation mechanisms. This systems-biology approach of combining modeling and a large input-output dataset should be applicable for studying other complex biological processes.

Regulatory Role of an Interdomain Linker in the Bacterial Chemotaxis Histidine Kinase CheA.

The histidine kinase CheA plays a central role in signal integration, conversion, and amplification in the bacterial chemotaxis signal transduction pathway. The kinase activity is regulated in chemotaxis signaling complexes formed via the interactions among CheA's regulatory domain (P5), the coupling protein CheW, and transmembrane chemoreceptors. Despite recent advancements in the understanding of the architecture of the signaling complex, the molecular mechanism underlying this regulation remains elusive. An interdomain linker that connects the catalytic (P4) and regulatory domains of CheA may mediate regulatory signals from the P5-CheW-receptor interactions to the catalytic domain. To investigate whether this interdomain linker is capable of both activating and inhibiting CheA, we performed in vivo screens to search for P4-P5 linker mutations that result in different CheA autokinase activities. Several CheA variants were identified with kinase activities ranging from 30% to 670% of the activity of wild-type CheA. All of these CheA variants were defective in receptor-mediated kinase activation, indicating that the natural receptor-mediated signal transmission pathway was simultaneously affected by these mutations. The altered P4-P5 linkers were sufficient for making significant changes in the kinase activity even in the absence of the P5 domain. Therefore, the interdomain linker is an active module that has the ability to impose regulatory effects on the catalytic activity of the P4 domain. These results suggest that chemoreceptors may manipulate the conformation of the P4-P5 linker to achieve CheA regulation in the platform of the signaling complex.IMPORTANCE The molecular mechanism underlying kinase regulation in bacterial chemotaxis signaling complexes formed by the regulatory domain of the histidine kinase CheA, the coupling protein CheW, and chemoreceptors is still unknown. We isolated and characterized mutations in the interdomain linker that connects the catalytic and regulatory domains of CheA and found that the linker mutations resulted in different CheA autokinase activities in the absence and presence of the regulatory domain as well as a defect in receptor-mediated kinase activation. These results demonstrate that the interdomain linker is an active module that has the ability to impose regulatory effects on CheA activity. Chemoreceptors may manipulate the conformation of this interdomain linker to achieve CheA regulation in the platform of the signaling complex.

His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.

Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal “core unit” necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes.

Signaling-Related Mobility Changes in Bacterial Chemotaxis Receptors Revealed by Solid-State NMR.

Bacteria employ remarkable membrane-bound nanoarrays to sense their environment and direct their swimming. Arrays consist of chemotaxis receptor trimers of dimers that are bridged at their membrane-distal tips by rings of two cytoplasmic proteins, a kinase CheA and a coupling protein CheW. It is not clear how ligand binding to the periplasmic domain of the receptor deactivates the CheA kinase bound to the cytoplasmic tip ∼300 Å away, but the mechanism is thought to involve changes in dynamics within the cytoplasmic domain. To test these proposals, we applied solid-state NMR mobility-filtered experiments to functional complexes of the receptor cytoplasmic fragment (U–13C,15N-CF), CheA, and CheW. Assembly of these proteins into native-like, homogeneous arrays is mediated by either vesicle binding or molecular crowding agents, and paramagnetic relaxation enhancement is used to overcome sensitivity challenges in these large complexes. INEPT spectra reveal that a significant fraction of the receptor is dynamic on the nanosecond or shorter time scale, and these dynamics change with signaling state. The mobile regions are identified through a combination of biochemical and NMR approaches (protein truncations and unique chemical shifts). The INEPT spectra are consistent with an asymmetric mobility in the methylation region (N-helix mobility ≫ C-helix mobility) and reveal an increase in the mobility of the N-helix in the kinase-off state. This finding identifies functionally relevant dynamics in the receptor, and suggests that this N-helix segment plays a key role in propagating the signal.

Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation.

Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.

Selective allosteric coupling in core chemotaxis signaling complexes.

Bacterial chemotaxis is mediated by signaling complexes that sense chemical gradients and direct bacteria to favorable environments by controlling a histidine kinase as a function of chemoreceptor ligand occupancy. Core signaling complexes contain two trimers of transmembrane chemoreceptor dimers, each trimer binding a coupling protein CheW and a protomer of the kinase dimer. Core complexes assemble into hexagons, and these form hexagonal arrays. The notable cooperativity and amplification in bacterial chemotaxis is thought to reflect allosteric interactions in cores, hexagons, and arrays, but little is known about this presumed allostery. We investigated allostery in core complexes assembled with two chemoreceptor species, each recognizing a different ligand. Chemoreceptors were inserted in Nanodiscs, which rendered them water soluble and allowed isolation of individual complexes. Neighboring dimers in receptor trimers influenced one another's operational ligand affinity, indicating allosteric coupling. However, this coupling did not include the key function of kinase inhibition. Our data indicated that only one receptor dimer could inhibit kinase as a function of ligand occupancy. This selective allosteric coupling corresponded with previously identified structural asymmetry: only one dimer in a trimer contacts kinase and only one CheW. We suggest one of these dimers couples ligand occupancy to kinase inhibition. Additionally, we found that kinase protomers are allosterically coupled, conveying inhibition across the dimer interface. Because kinase dimers connect core complex hexagons, allosteric communication across dimer interfaces provides a pathway for receptor-generated kinase inhibition in one hexagon to spread to another, providing a crucial step for the extensive amplification characteristic of chemotactic signaling.

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).

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.

Autophosphorylation by the Histidine kinase CheA (LB222)

Chemotaxis is the signal transduction mechanism that allows bacteria to change direction in response to an external stimulus. Detection of an environmental stimulus by the receptors has a cascading effect leading to the flagellar motor changing direction. This results in the tumbling of the bacterium. The receptors form hexagonal lattices at the poles of cells. The histidine kinase CheA and coupling protein CheW are localized to the lattice to form the ternary complex. CheA, the principal enzyme in the chemotaxis signaling pathway, is a multi domain protein comprised of: P1‐histidine phosphotransfer, P2‐regulatory, P3‐dimerization, P4‐kinase, and P5‐regulatory. From previous studies it is known that the transfer of the γ‐phosphate of ATP occurs between the P1 and P4 domain, however how autophosphorylation activity is regulated is not completely understood.To comprehend this transient interaction, variants (ΔP2) of Thermotoga maritima CheA were generated. Small angle X‐ray scattering (SAXS) data has shown that spatial changes occur upon addition of an ATP analog. To determine relative activity of the ΔP2 variants or CheA domains radioisotope assays were used. Point mutations on the full length and ΔP2 CheA showed whether autophosphorylation occurs cis or trans in the dimer. Crosslinking between residues near the active sites in the P4 and P1 domains enabled isolation of the autophosphorylation transfer conformation. The crystal structure of P3P4 domains reveals a more active configuration of the P4 domain. Progress towards understanding how the domains interact to facilitate the transfer of the ATP γ‐phosphate is not clear and will be discussed.

Differences between Signaling States of Chemotaxis Receptors Revealed by Hydrogen Exchange Mass Spectrometry

Bacterial chemotaxis receptors provide the sensory input needed to direct swimming towards favorable environments. Ligand binding to the receptor periplasmic domain modulates activation of a kinase CheA bound at the cytoplasmic tip of the receptor. Receptors operate in complexes with CheA and a coupling protein CheW that together form extended arrays in the membrane. Understanding the differences in conformation and dynamics between the kinase-activating and kinase-deactivating signaling states of the receptor will provide insight into the mechanism of transmembrane signaling. We have used hydrogen exchange mass spectrometry to probe the differences between these signaling states of the receptor cytoplasmic domain assembled on membrane vesicles in functional complexes with CheA and CheW. Local exchange measurements reveal several differences between these states, including differences in the exchange pattern (EX1 vs EX2), the fraction undergoing fast exchange (exchanged in 3 minutes), and the fraction that are protected from exchange at long time (16 hours). Preliminary analysis shows that peptides corresponding to the adaptation region undergo EX1 exchange (long-lived unfolded state with complete exchange during each unfolding event) in the kinase-activating state and EX2 exchange (short-lived unfolded state with partial exchange) in the kinase-deactivating state. This suggests the adaptation region is destabilized in the kinase-activating state. Peptides corresponding to the signaling domain cytoplasmic tip of the receptor show greater protection from exchange in the kinase-activating state, which could be due to changes in interactions with CheA and CheW. This hydrogen exchange mass spectrometry approach is a promising means of detecting changes in structure and dynamics of a functional membrane-bound, multi-protein complex.This research supported by GM 47601, GM085288, and a Fellowship to Seena Koshy from the University of Massachusetts as part of the Chemistry-Biology Interface Training Program (NRSA T32 GM08515).

Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi

In the model organism Escherichia coli, the coupling protein CheW, which bridges the chemoreceptors and histidine kinase CheA, is essential for chemotaxis. Unlike the situation in E. coli, Borrelia burgdorferi, the causative agent of Lyme disease, has three cheW homologues (cheW(1) , cheW(2) and cheW(3) ). Here, a comprehensive approach is utilized to investigate the roles of the three cheWs in chemotaxis of B. burgdorferi. First, genetic studies indicated that both the cheW(1) and cheW(3) genes are essential for chemotaxis, as the mutants had altered swimming behaviours and were non-chemotactic. Second, immunofluorescence and cryo-electron tomography studies suggested that both CheW(1) and CheW(3) are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW(1) and cheW(3) , cheW(2) is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW(1) and CheW(3) interact with CheA(2) whereas CheW(2) binds to CheA(1) . Collectively, our results indicate that CheW(1) and CheW(3) are incorporated into one chemosensory pathway that is essential for B. burgdorferi chemotaxis. Although many bacteria have more than one homologue of CheW, to our knowledge, this report provides the first experimental evidence that two CheW proteins coexist in one chemosensory pathway and that both are essential for chemotaxis.

Self-Association of the Histidine Kinase CheA as Studied by Pulsed Dipolar ESR Spectroscopy

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.

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.

Core unit of chemotaxis signaling complexes

Bacterial chemoreceptors, histidine kinase CheA, and coupling protein CheW form clusters of chemotaxis signaling complexes. In signaling complexes kinase activity is enhanced several hundredfold and placed under receptor control. Activation is necessary to poise enzyme activity such that receptor control has physiologically relevant effects. Thus kinase activation can be considered the underlying core activity of signaling complexes. We defined the minimal physical unit that generates this activity using chemoreceptor Tar from Escherichia coli rendered water soluble by insertion into nanodiscs to (i) measure saturable binding of CheA and CheW to the smallest kinase-activating groups of receptor dimers and (ii) purify and characterize core units of signaling complexes. Purified complexes activated kinase almost as well as signaling complexes formed on arrays of receptors in isolated native membrane. Purified complexes contained two receptor trimers of dimers and two CheW for each CheA dimer, consistent with the approximately 1:1 CheACheW ratio determined by binding measurements. The 2:2:1 stoichiometry implied that CheA dimers, the enzymatically active form, connect two chemoreceptor trimers of dimers by interaction of one CheA protomer and a CheW with each trimer, an organization for which specific molecular interactions have previously been identified. The core unit associates six receptor dimers with a CheA dimer, providing sufficient capacity to account for much of the cooperativity and interdimer influence observed experimentally. We conclude that the 221 organization is the core structural and functional unit of chemotaxis signaling complexes and postulate that hexagonal arrays characteristic of signaling complexes are built from this unit.