Chemoreceptors are crucial components in the bacterial sensory systems that mediate chemotaxis. Chemotactic responses exhibit exquisite sensitivity, extensive dynamic range and precise adaptation. The mechanisms that mediate these high-performance functions involve not only actions of individual proteins but also interactions among clusters of components, localized in extensive patches of thousands of molecules. Recently, these patches have been imaged in native cells, important features of chemoreceptor structure and on-off switching have been identified, and new insights have been gained into the structural basis and functional consequences of higher order interactions among sensory components. These new data suggest multiple levels of molecular interactions, each of which contribute specific functional features and together create a sophisticated signaling device. High-performance signaling in bacterial chemotaxisThe high-performance chemotaxis signaling system of Escherichia coli (Box 1) involves a limited number of components but notable sophistication [1][2][3][4]. This system has become a paradigm for molecular characterization of biological signaling mechanisms. Transmembrane chemoreceptors, known as methyl-accepting chemotaxis proteins (MCPs), direct cell locomotion by regulating the histidine kinase CheA; CheA phosphorylates a response regulator, which in turn controls the rotational direction of the flagellar motor. Chemotactic sensitivity and the range of signal detection are regulated by adaptational modification of chemoreceptors via reversible glutamyl methylation. The resulting interplay between motor control and sensory adaptation produces directed motile behavior. E. coli chemoreceptors are the most extensively studied representatives of the MCP super-family, central components of homologous systems that mediate tactic responses [5,6] across the phylogenetic diversity of bacteria and archaea [7].In E. coli chemotaxis proteins cluster in membrane-associated patches [8]. Interaction within patches is thought to contribute to notable features of the signaling system: high sensitivity, wide dynamic range, extensive cooperativity and precise adaptation. Delineating the molecular mechanisms underlying these features will require knowledge of structures of the components, complexes and higher order arrays. In addition it will require definition of organization at the multiple levels of interaction among signaling components and an understanding of the changes in components and their interactions that mediate signaling at each level. In the past few years, notable progress has been made toward acquiring the NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript information and insights necessary for understanding these molecular mechanisms. This review describes that progress. Here we follow the lead of most investigators in the area, and do not distinguish between studies of first cousins E. coli and Salmonella enterica serovar Typhimurium. Thus, referenc...

Bacterial chemoreceptors mediate chemotaxis by recognizing specific chemicals and regulating a noncovalently associated histidine kinase. Ligand binding to the external domain of the membranespanning receptor generates a transmembrane signal that modulates kinase activity inside the cell. This transmembrane signaling is being investigated by novel strategies, which have revealed a remarkably subtle conformational signal carried by a signaling helix that spans the entire length of the >350-Å-long receptor. Multiple, independent lines of evidence indicate that, in the periplasmic and transmembrane domains, conformational signaling is a piston-type sliding of the signaling helix towards the cytoplasm.Like other motile bacteria, Escherichia coli and Salmonella typhimurium respond to chemical gradients by moving towards higher concentrations of attractants and lower concentrations of repellents (reviewed in Refs [1][2][3][4] ). This behavior, termed chemotaxis, is mediated by a dedicated sensory system comprising transmembrane chemoreceptors, histidine and aspartate kinases, an SH3-like coupling protein, and two enzymes that mediate sensory adaptation by covalently modifying the chemoreceptors (Box 1). Homologs of these sensory components occur in virtually every motile bacterium or archaeon investigated to date, making this type of sensory pathway one of the most prevalent in nature. It is likely that the homologous components possess conserved molecular mechanisms. For example, chemoreceptors are expected to share similar mechanisms of transmembrane signaling.Chemoreceptors are stable homodimers both in the absence and presence of ligands 5 . Each homodimer is an elongated helical bundle thought to be oriented normal to the membrane ( Fig. 1) [6][7][8][9][10][11] . The periplasmic domain consists of eight helices arranged in two symmetric four-helix bundles, one per subunit (helices α1-α4, α1′-α4′). Two helices from each subunit span the bilayer, where they form a transmembrane four-helix bundle (helices TM1, TM2, TM1′, TM2′). The cytoplasmic domain is a distinct four-helix bundle, formed by association of two helical hairpins, one per subunit (helices CD1, CD2, CD1′, CD2′). One helix in each subunit extends the entire length of the structure (helix α4/TM2/linker/CD1), connecting the ligand-binding site at the membrane-distal end of the periplasmic domain with the kinase-interaction region at the opposite end of the receptor. The only major region that has not yet been shown experimentally to be helical is the conserved linker connecting the transmembrane and cytoplasmic domains 12 but, regardless of its structure, the linker is stably folded and thus can communicate signals between receptor domains 13 .The first step in signal transduction is the binding of attractant or attractant-occupied binding protein to the periplasmic domain at one of two interfacial sites between the two symmetric four-helix bundles. Much evidence (summarized below) indicates that attractant binding sends a conformational signal ...

Motile Escherichia coli cells track gradients of attractant and repellent chemicals in their environment with transmembrane chemoreceptor proteins. These receptors operate in cooperative arrays to produce large changes in the activity of a signaling kinase CheA in response to small changes in chemoeffector concentration. Recent research has provided much deeper understanding of the structure and function of core receptor signaling complexes and the architecture of higher-order receptor arrays, which in turn has led to new insights into the molecular signaling mechanisms of chemoreceptor networks. Current evidence supports a new view of receptor signaling in which stimulus information travels within receptor molecules through shifts in the dynamic properties of adjoining structural elements rather than through a few discrete conformational states.

The chemotactic sensory system of Escherichia coli comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins. These components form signaling complexes that mediate sensory excitation and adaptation. Previous determinations of cellular content of individual components provided differing and apparently conflicting values. We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two E. coli strains considered wild type for chemotaxis, grown in rich and minimal media. Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30%. Thus, cellular stoichiometries were almost constant as amounts varied substantially. Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex. The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW. These components may interact in extended arrays and, thus, stoichiometries could be nonintegral. In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively. These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical.Motile bacterial cells respond to chemical gradients by moving toward favorable environments. The phenomenon, termed chemotaxis, is mediated by a set of modular components found across the taxonomic diversity of prokaryotes. The molecular and mechanistic basis of chemotaxis has been most extensively defined in Escherichia coli. In this species, the chemotactic sensory system consists of five related, membrane-embedded chemoreceptors and six different soluble proteins, the Che proteins (see reference 15 for an overview). These components interact, forming signaling complexes that mediate sensory excitation and adaptation. Some components interact to create a stable core complex, and others interact more weakly with this core. How many copies of the components of the signaling complex are contained in a cell? Several studies have reported values for one or a few of these proteins. However, as documented in an informative website (www.anat.cam.ac.uk/comp -cell), published values for a given protein differ, sometimes by as much as 10-fold. The origin and significance of this variation has been unknown and, thus, there has been no basis for choosing from among the different values. In addition, the stoichiometry of signaling complexes is an area of active investigation, and independent investigators have come to...

Bacterial chemoreceptors are transmembrane homodimers that can form trimers, higher order arrays, and extended clusters as part of signaling complexes. Interactions of dimers in oligomers are thought to confer cooperativity and cross-receptor influences as well as a 35-fold gain between ligand binding and altered kinase activity. In addition, higher order interactions among dimers are necessary for the observed patterns of assistance in adaptational modification among different receptors. Elucidating mechanisms underlying these properties will require defining which receptor functions can be performed by dimers and which require specific higher order interactions. However, such an assignment has not been possible. Here, we used Nanodiscs, an emerging technology for manipulating membrane proteins, to prepare small particles of lipid bilayer containing one or only a few chemoreceptor dimers. We found that receptor dimers isolated in individual Nanodiscs were readily modified, bound ligand, and performed transmembrane signaling. However, they were hardly able to activate the chemotaxis histidine kinase. Instead, maximal activation and thus full-range control of kinase occurred preferentially in discs containing approximately three chemoreceptor dimers. The sharp dependence of kinase activation on this number of receptors per dimer implies that the core structural unit of kinase activation and control is a trimer of dimers. Thus, our observations demonstrate that chemoreceptor transmembrane signaling does not require oligomeric organization beyond homodimers and implicate a trimer of dimers as the unit of downstream signaling. membrane protein ͉ nanoparticle ͉ self-assembly ͉ transmembrane receptor ͉ transmembrane signaling M otile bacteria move to favorable chemical environments through chemotaxis. The phenomenon and its mechanisms have been extensively characterized in Escherichia coli and its relatives (1-3). Transmembrane chemoreceptors form signaling complexes with the autophosphorylating histidine kinase CheA and coupling protein CheW. Phospho-CheA mediates phosphorylation of response regulator CheY. Phospho-CheY binds to the flagellar rotary motor, causing rotational reversal, which creates tumbles that alter swimming direction. Formation of signaling complexes activates CheA autophosphorylation and places that activity under the control of chemoreceptors. The sensory system directs cells toward favorable environments by modulating kinase activity, thus controlling the probability of tumbles and resulting directional changes. Transmembrane SignalingTransmembrane signaling by chemoreceptors (3) couples binding of stimulant molecules by its periplasmic domain with changes in its cytoplasmic domain that alter activation of the kinase and change the propensity for covalent modification of that domain, specifically methylation and demethylation at several glutamyl residues (four to six, depending on the specific receptor). Methylation is catalyzed by methyltransferase CheR. Demethylation is catalyzed by methyleste...