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...

The C2 domain is a Ca2+-binding motif of approximately 130 residues in length originally identified in the Ca2+-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin I and phosphoinositide-specific phospholipase C-61, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBrain Behav Immun. Author manuscript; available in PMC 2011 January 1. preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

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 ...

The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.

Intracellular calcium plays an essential role in the transduction of most hormonal, neuronal, visual, and muscle stimuli. (Recent reviews include Putney, 1993; Berridge, 1993a,b; Tsunoda, 1993; Gnegy, 1993; Bachset al.1992; Hanson & Schulman, 1992; Villereal & Byron, 1992; Premack & Gardner, 1992; Meanset al.1991).

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 D-galactose chemosensory receptor of Escherichia coli is a .32 kDa globular protein possessing two distinct structural domains, each organized in an alpha/beta folding motif. Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region. In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to place a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions. Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X. A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule. These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and 19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics. Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor. The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structure, while no disulfide formation is observed for the control pair unless the molecule is unfolded. The minimum translational amplitudes of the observed backbone motions range from 4.5 to 15.2 A, and the minimum rotational amplitudes are as large as 35 degrees. For each motion the rate of intramolecular sulfhydryl-sulfhydryl collision has been estimated from the measured rate of disulfide formation: the 4.5 and 15.2 A translations yield approximately 10(4) and approximately 10 collisions s-1 molecule-1, respectively. These collision rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions. The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations. When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories. Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed.