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Joseph J. Falke - One of the best experts on this subject based on the ideXlab platform.
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Increasing and Decreasing the Ultrastability of Bacterial Chemotaxis Core Signaling Complexes by Modifying Protein−Protein Contacts
2016Co-Authors: Kene N. Piasta, Joseph J. FalkeAbstract:ABSTRACT: The chemosensory signaling array of bacterial chemotaxis is composed of functional core units containing two receptor trimers of dimers, a homodimeric CheA Kinase, and two CheW adaptor proteins. In vitro reconstitutions generate individual, functional core units and larger functional assemblies, including dimers, hexagons, and hexagonal arrays. Such reconstituted complexes have been shown to have both quasi-stable and ultrastable populations that decay with lifetimes of 1−2 days and ∼3 weeks at 22 °C, respectively, where decay results primarily from proteolysis of the bound kinas
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Defining a Key Receptor–CheA Kinase Contact and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study
2016Co-Authors: Kene N. Piasta, Caleb J. Ulliman, Peter F. Slivka, Brian R. Crane, Joseph J. FalkeAbstract:The three core components of the ubiquitous bacterial chemosensory array the transmembrane chemoreceptor, the histidine Kinase CheA, and the adaptor protein CheW assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate Kinase activity. Both the regulatory domain of the Kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the Kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the Kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor–CheA Kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA Kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the Kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA Kinase binding in the array and a regulatory function central to transmission of the activation signal from receptor to CheA Kinase. On–off switching alters the disulfide formation rates of specific Cys pairs at the interface, but not most Cys pairs, indicating that signaling perturbs localized regions of the interface. The findings suggest a simple model for the rearrangement of the interface triggered by the attractant signal and for longer range transmission of the signal in the chemosensory array
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Increasing and Decreasing the Ultrastability of Bacterial Chemotaxis Core Signaling Complexes by Modifying Protein−Protein Contacts
Biochemistry, 2014Co-Authors: Kene N. Piasta, Joseph J. FalkeAbstract:The chemosensory signaling array of bacterial chemotaxis is composed of functional core units containing two receptor trimers of dimers, a homodimeric CheA Kinase, and two CheW adaptor proteins. In vitro reconstitutions generate individual, functional core units and larger functional assemblies, including dimers, hexagons, and hexagonal arrays. Such reconstituted complexes have been shown to have both quasi-stable and ultrastable populations that decay with lifetimes of 1–2 days and ∼3 weeks at 22 °C, respectively, where decay results primarily from proteolysis of the bound Kinase [Erbse, A. H., and Falke, J. J. (2009) Biochemistry 48, 6975–6987; Slivka, P. F., and Falke, J. J. (2012) Biochemistry 51, 10218–10228]. In this work, we show that the ultrastable population can be destabilized to the quasi-stable level via the introduction of a bulky tryptophan residue at either one of two essential protein–protein interfaces within the core unit: the receptor–Kinase contact or Kinase–adaptor interface 1. Moreo...
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structure function and on off switching of a core unit contact between CheA Kinase and chew adaptor protein in the bacterial chemosensory array a disulfide mapping and mutagenesis study
Biochemistry, 2013Co-Authors: Andrew M Natale, Kene N. Piasta, Jane Duplantis, Joseph J. FalkeAbstract:The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His Kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function, and on-off switching of a key contact between the Kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative Kinase-adaptor ring where subdomain 1 of the Kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches, disulfide mapping and site-directed Trp and Ala mutagenesis, were employed (i) to test the structural model of interface 1 and (ii) to investigate its functional roles in both stable Kinase incorporation and receptor-regulated Kinase on-off switching. Studies were conducted in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its Kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its Kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for incorporation of the stable Kinase into the array, for Kinase activation in the array on state, and likely for attractant-triggered Kinase on-off switching. A model is presented that describes the concerted transmission of a conformational signal among the receptor, the Kinase regulatory domain, and the adaptor protein. In principle, this signal could spread out into the surrounding array via the Kinase-adaptor ring, employing a series of alternating frozen-dynamic transitions that transmit low-energy attractant signals long distances.
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defining a key receptor CheA Kinase contact and elucidating its function in the membrane bound bacterial chemosensory array a disulfide mapping and tam ids study
Biochemistry, 2013Co-Authors: Kene N. Piasta, Caleb J. Ulliman, Peter F. Slivka, Brian R. Crane, Joseph J. FalkeAbstract:The three core components of the ubiquitous bacterial chemosensory array — the transmembrane chemoreceptor, the histidine Kinase CheA, and the adaptor protein CheW — assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate Kinase activity. Both the regulatory domain of the Kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the Kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the Kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor–CheA Kinase interface. Model 2 is based on a newly ...
John S. Parkinson - One of the best experts on this subject based on the ideXlab platform.
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Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations
Communications Biology, 2020Co-Authors: C. Keith Cassidy, John S. Parkinson, Benjamin A. Himes, Gongpu Zhao, Dapeng Sun, Phillip J. Stansfeld, Zaida Luthey-schulten, Peijun ZhangAbstract:C. Keith Cassidy et al. use cryo-electron tomography and subtomogram averaging to examine the structure of the core-signalling units of E. coli chemotaxis arrays at subnanometer resolution. They find multiple distinct conformations of the critical CheA Kinase domain, offering new insights into CheA signalling. To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine Kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.
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Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes
Journal of bacteriology, 2019Co-Authors: Germán E. Piñas, John S. ParkinsonAbstract:ABSTRACT Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine Kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct Kinase-on and Kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, “dipped” and “undipped,” for the ATP-binding CheA.P4 domain that could be related to Kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Forster resonance energy transfer (FRET)-based Kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing Kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA Kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the Kinase-active state in core signaling units. IMPORTANCE Regulation of CheA Kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the Kinase-on or Kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called “dipped” CheA.P4 conformation abolished the Kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the Kinase-active state in chemoreceptor signaling arrays.
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in situ conformational changes of the escherichia coli serine chemoreceptor in different signaling states
Mbio, 2019Co-Authors: Wen Yang, John S. Parkinson, Keith C Cassidy, Peter Ames, Christoph A Diebolder, Klaus Schulten, Zaida Lutheyschulten, Ariane BriegelAbstract:ABSTRACT 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.
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conformational shifts in a chemoreceptor helical hairpin control Kinase signaling in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Anchun Cheng, John S. ParkinsonAbstract:Motile Escherichia coli cells use chemoreceptor signaling arrays to track chemical gradients with exquisite precision. Highly conserved residues in the cytoplasmic hairpin tip of chemoreceptor molecules promote assembly of trimer-based signaling complexes and modulate the activity of their CheA Kinase partners. To explore hairpin tip output states in the serine receptor Tsr, we characterized the signaling consequences of amino acid replacements at the salt-bridge residue pair E385-R388. All mutant receptors assembled trimers and signaling complexes, but most failed to support serine chemotaxis in soft agar assays. Small side-chain replacements at either residue produced OFF- or ON-shifted outputs that responded to serine stimuli in wild-type fashion, suggesting that these receptors, like the wild-type, operate as two-state signaling devices. Larger aliphatic or aromatic side chains caused slow or partial Kinase control responses that proved dependent on the connections between core signaling units that promote array cooperativity. In a mutant lacking one of two key adapter-Kinase contacts (interface 2), those mutant receptors exhibited more wild-type behaviors. Lastly, mutant receptors with charged amino acid replacements assembled signaling complexes that were locked in Kinase-ON (E385K|R) or Kinase-OFF (R388D|E) output. The hairpin tips of mutant receptors with these more aberrant signaling properties probably have nonnative structures or dynamic behaviors. Our results suggest that chemoeffector stimuli and adaptational modifications influence the cooperative connections between core signaling units. This array remodeling process may involve activity-dependent changes in the relative strengths of interface 1 and 2 interactions between the CheW and CheA.P5 components of receptor core signaling complexes.
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Noncritical Signaling Role of a Kinase-Receptor Interaction Surface in the Escherichia coli Chemosensory Core Complex.
Journal of molecular biology, 2018Co-Authors: Germán E. Piñas, Michael D. Desantis, John S. ParkinsonAbstract: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.
Lynmarie K. Thompson - One of the best experts on this subject based on the ideXlab platform.
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Signaling-Related Mobility Changes in Bacterial Chemotaxis Receptors Revealed by Solid-State NMR
The journal of physical chemistry. B, 2017Co-Authors: Maryam Kashefi, Lynmarie K. ThompsonAbstract: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 A 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...
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Signaling-Related Mobility Changes in Bacterial Chemotaxis Receptors Revealed by Solid-State NMR
2017Co-Authors: Maryam Kashefi, Lynmarie K. ThompsonAbstract: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
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Hydrogen Exchange Reveals Differences between Bacterial Chemoreceptor Signaling States
Biophysical Journal, 2015Co-Authors: Seena S. Koshy, Robert M. Weis, Stephen J. Eyles, Lynmarie K. ThompsonAbstract:Bacterial chemotaxis receptors form membrane-bound nanoarrays that sense and adapt to ligands from the environment. These arrays consist of receptors interacting at their cytoplasmic tips with a “baseplate” formed by the CheA Kinase and CheW coupling protein, ≈300 A away from the periplasmic ligand binding sites. Receptor methylation at 4 glutamate residues in the cytoplasmic domain mediates adaptation to ongoing stimuli. To determine what ligand- and methylation-induced changes in the receptor cytoplasmic domain control the Kinase activity, we have developed (1) methods to reconstitute native-like arrays of receptor cytoplasmic fragments, CheA, and CheW, and (2) a hydrogen exchange mass spectrometry (HDX-MS) method applicable to membrane-bound, multi-protein complexes. HDX-MS comparison of complexes with high and low Kinase activity shows that differences localize to two functionally important subdomains of the receptor. Changes in the methylation subdomain that mediates adaptation are complex. The uniform exchange behavior of these peptides in the Kinase-off complexes splits roughly in half in the Kinase-on complexes, one fraction with slower exchange and the other with extremely rapid exchange (complete in 3 min). For the signaling subdomain that binds CheA and CheW, peptides exhibit protection from exchange at long times (16 hours) that is greater in the Kinase-on state. HDX-MS of complexes prepared using different means of shifting the signaling state will reveal which changes correlate with Kinase activity and will quantify stabilization of receptor subdomains and binding interfaces that contribute to receptor control of Kinase activity. Thus HDX-MS provides an important tool in a hybrid approach for understanding structure and mechanism in membrane-bound, multi-protein complexes.This research supported by GM085288, and a University of Massachusetts fellowship to Seena Koshy as part of the Chemistry-Biology Interface Training Program (NRSA T32 GM08515).
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Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes.
Biochemistry, 2013Co-Authors: Seena S. Koshy, Robert M. Weis, Stephen J. Eyles, Lynmarie K. ThompsonAbstract:The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 A) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the Kinase activity of the CheA bound at the membrane-distal tip, nearly 200 A away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA Kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) measure...
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Ligand Affinity and Kinase Activity are Independent of Bacterial Chemotaxis Receptor Concentration: Insight into Signaling Mechanisms
Biochemistry, 2012Co-Authors: Fe C. Sferdean, Robert M. Weis, Lynmarie K. ThompsonAbstract:Two-component signaling pathways, a signal transduction motif that is widespread in prokaryotes and found in some eukaryotes, involve the autophosphorylation of histidine Kinases and phosphotransfer to aspartyl groups of response regulators (1). Bacterial chemotaxis is a well-studied two-component signaling system that enables bacteria to sense chemical gradients and bias swimming toward larger attractant concentrations (2). The methyl-accepting chemotaxis receptors that provide the sensory input for this system have been the focus of numerous investigations that seek to understand the mechanism of transmembrane signaling. An intriguing property of these receptors is the formation of large receptor clusters, typically at the poles of the cell (3). These clusters are thought to be important for receptor cooperativity (4) and adaptation (5) to mediate the sensitivity, dynamic range, and integration of the chemotaxis signaling network (6–9). Clustering may also play a role in receptor activation, as observed for the EGF receptor and other receptors that are activated by ligand-induced dimerization or oligomerization (10, 11). Chemoreceptor clustering has been reported to vary with signaling state, but the evidence has been inconsistent. These data raise the question of whether a clustering equilibrium plays a role in the primary signal, ligand regulation of chemoreceptor activation of the Kinase CheA. Four chemotaxis receptors in E coli share the overall structure and interaction sites shown in Figure 1 (12). The high abundance receptors Tar and Tsr detect aspartate and serine, respectively, and the low abundance receptors Trg and Tap detect ribose/galactose and dipeptides, respectively. These chemoreceptors are transmembrane alpha-helical homodimers, based on crystal and NMR structures of the periplasmic, HAMP, and cytoplasmic domains (13–15). The membrane-distal tip of the cytoplasmic domain binds two proteins, the histidine Kinase CheA and a scaffolding protein CheW. The central region of the receptor cytoplasmic domain contains 4 glutamate residues that are methylated and demethylated, which enables the receptor to adapt to an ongoing stimulus. Both the CheR methyltransferase and the CheB methylesterase that modify these adaptation sites have been shown to bind to the carboxy terminus of the high abundance receptors (16, 17). Replacing Glu with Gln at the adaptation sites mimics the effects of receptor methylation (18), and the wild type receptor is genetically encoded in an intermediate adaptation state, with two Glu and two Gln (Gln are initially deamidated to Glu by CheB). Figure 1 The E. coli chemotaxis pathway depicted as a two state signaling system. Receptor/CheW/CheA complexes are shown in Kinase-stimulating (green/magenta/green) and Kinase-inhibiting (red/magenta/gray) states. The binding of attractant (filled triangles) inhibits ... Although not shown in Figure 1 for simplicity, chemotaxis receptors are interconnected by CheA and CheW into hexagonal arrays that have been observed across a wide range of prokaryotes (19). Furthermore, although the stoichiometry of the proteins in these arrays is not known, recent estimates suggest they contain more receptor than CheA and CheW: based on measured stoichiometries of six Tsr per CheA and CheW, it has been suggested that a pair of receptor trimer-of-dimers is needed to activate the dimeric CheA Kinase (20). Recent cryoelectron tomography studies have yielded models for the arrangement of receptor, CheA and CheW in the signaling arrays (21, 22). Receptor signaling is usually described in terms of a two-state equilibrium, with attractant occupancy and methylation shifting the equilibrium in opposite directions (Figure 1). In the absence of attractant ligand, the receptor stimulates autophosphorylation of the histidine Kinase CheA, which transfers the phosphate to either CheY or CheB. Phospho-CheY binds to the flagellar motor, causing a change from counterclockwise rotation of the flagellar bundle that propels the cell forward, to clockwise rotation that disrupts the flagellar bundle and causes the cell to tumble. Phosphorylation also activates the CheB methylesterase, which decreases the steady state level of receptor methylation that is determined by the relative activities of CheR and CheB. Binding of an attractant ligand to the receptor turns off Kinase activation and thus decreases levels of phospho-CheY and tumbling frequency, so that the cell makes longer runs in the presence of attractants such as Asp or Ser. Following this rapid change in tumbling frequency, adaptation occurs on a slower timescale: decreased levels of phospho-CheB lead to increased levels of receptor methylation, shifting the equilibrium back towards the Kinase-activating state. The attractant-bound receptor is also more efficiently methylated by CheR, which contributes to the adaptation shift back to the Kinase-activating state. The focus of this study is to determine whether the extent of receptor clustering is different in the two signaling states depicted in Figure 1. We use the term cluster to refer to a receptor associated with other receptors, CheA, and CheW into an oligomeric multi-protein complex, with intermolecular contacts among the proteins within the cluster, as distinct from co-localized receptors that lack such contacts. Evidence from microscopy and in vivo cross-linking studies is mixed: some studies have reported that ligand binding decreases receptor clustering or methylation increases receptor clustering in cells. Libermann et al (23) observed an increase in the polar localization of CheA-containing complexes with methylation (Tar2Q2E ≈ half methylated receptor relative to Tar4E = unmethylated adaptation state), using fluorescence microscopy of E. coli expressing CheA fused to YFP (yellow fluorescent protein). However, because these changes were much smaller than the measured changes in Kinase activity, they concluded that changes in assembly of CheA-containing clusters do not control the Kinase (23). Based on immunoelectron microscopy of E. coli cells expressing a single type of chemotaxis receptor, Lyberger et al (24) reported that high abundance receptors are clustered independent of methylation state, but low abundance receptors are significantly less clustered in the unmethylated state. They suggested that both increases in abundance and methylation may shift the equilibrium toward a clustered state, and that such an equilibrium could also regulate Kinase activation (24). Homma at al reported that attractant ligand does not decrease the polar localization of a Tar-GFP construct in E. coli, but attractant does decrease in vivo interdimer crosslinking of Tar (25). By contrast, Lamanna et al. observed that attractant ligand decreases polar clusters in both E. coli and B. subtilis, when receptors are crosslinked with paraformaldehyde and then visualized with a fluorescent antibody (26). Finally, Studdert & Parkinson reported that in vivo interdimer crosslinking of Tsr and Tar is independent of both ligand binding and methylation state (27). Limitations of these studies include the inability of microscopy to distinguish clustering (oligomerization) of receptors from co-localization, and the inability of crosslinking studies to distinguish whether changes in the extent of crosslinking result from conformational changes or dissociation of receptors. Two in vitro studies that correlated receptor concentration with changes in Kinase activity are more suggestive that a clustering equilibrium may control the Kinase. Lai et al. (28) varied the overexpression level of Tsr or Tar and isolated inner membrane vesicles that contained each receptor as a variable fraction of total membrane protein. The Kinase activity per receptor increased linearly with receptor fraction, up to 50% of total membrane protein in these samples (28). Besschetnova et al (29) examined simpler, more defined samples of histidine-tagged cytoplasmic fragments of Tar4E, which were assembled on the surface of liposomes with Ni-chelating lipids, along with CheA and CheW. These template-assembled receptor-signalling arrays displayed a cooperative increase in Kinase activation as the 2-dimensional receptor concentration on the vesicle increased. Moreover, receptor methylation activity was observed to decrease as the two dimensional concentration (density) of receptors increased, in a manner consistent with the signaling equilibrium of Figure 1 (29). The results of these in vitro studies are consistent with a clustering equilibrium model in which high receptor concentrations favor the Kinase-activating state, which would be a larger oligomeric (more clustered) state than the methylation-activating state. Such a model predicts that ligand binding would favor receptor dissociation into the Kinase-inactivating state (less clustered) and thus ligand affinity (which was not measured in either study) would also vary with receptor concentration. To test whether ligand-induced unclustering is an essential element of the mechanism of Kinase regulation, we measured Kinase activity and serine dose-response curves on purified E. coli Tsr reconstituted into liposomes over a range of two-dimensional concentrations of receptors. Our results indicate that the activity equilibrium does not involve a change in receptor oligomerization state. In combination with the previous template-assembly results (29), this indicates that the cytoplasmic domain of the Kinase-off state has an expanded conformation.
Brian R. Crane - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor.
The Journal of biological chemistry, 2016Co-Authors: Dipanjan Samanta, Peter P. Borbat, Jack H. Freed, Joanne Widom, Brian R. CraneAbstract:Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA Kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the Kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases Kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.
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Defining a Key Receptor–CheA Kinase Contact and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study
2016Co-Authors: Kene N. Piasta, Caleb J. Ulliman, Peter F. Slivka, Brian R. Crane, Joseph J. FalkeAbstract:The three core components of the ubiquitous bacterial chemosensory array the transmembrane chemoreceptor, the histidine Kinase CheA, and the adaptor protein CheW assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate Kinase activity. Both the regulatory domain of the Kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the Kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the Kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor–CheA Kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA Kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the Kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA Kinase binding in the array and a regulatory function central to transmission of the activation signal from receptor to CheA Kinase. On–off switching alters the disulfide formation rates of specific Cys pairs at the interface, but not most Cys pairs, indicating that signaling perturbs localized regions of the interface. The findings suggest a simple model for the rearrangement of the interface triggered by the attractant signal and for longer range transmission of the signal in the chemosensory array
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defining a key receptor CheA Kinase contact and elucidating its function in the membrane bound bacterial chemosensory array a disulfide mapping and tam ids study
Biochemistry, 2013Co-Authors: Kene N. Piasta, Caleb J. Ulliman, Peter F. Slivka, Brian R. Crane, Joseph J. FalkeAbstract:The three core components of the ubiquitous bacterial chemosensory array — the transmembrane chemoreceptor, the histidine Kinase CheA, and the adaptor protein CheW — assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate Kinase activity. Both the regulatory domain of the Kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the Kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the Kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor–CheA Kinase interface. Model 2 is based on a newly ...
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The 3.2 Å Resolution Structure of a Receptor:CheA:CheW Signaling Complex Defines Overlapping Binding Sites and Key Residue Interactions within Bacterial Chemosensory Arrays
Biochemistry, 2013Co-Authors: Aaron D. Fleetwood, Joseph J. Falke, Alexandrine M Bilwes, Camille Bayas, Davi R. Ortega, Igor B. Zhulin, Brian R. CraneAbstract:Bacterial chemosensory arrays are composed of extended networks of chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), the histidine Kinase CheA, and the adaptor protein CheW. Models of these arrays have been developed from cryoelectron microscopy, crystal structures of binary and ternary complexes, NMR spectroscopy, mutational, data and biochemical studies. A new 3.2 A resolution crystal structure of a Thermotoga maritima MCP protein interaction region in complex with the CheA Kinase-regulatory module (P4–P5) and adaptor protein CheW provides sufficient detail to define residue contacts at the interfaces formed among the three proteins. As in a previous 4.5 A resolution structure, CheA-P5 and CheW interact through conserved hydrophobic surfaces at the ends of their β-barrels to form pseudo 6-fold symmetric rings in which the two proteins alternate around the circumference. The interface between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous studies, whereas the r...
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Self-association of the histidine Kinase CheA as studied by pulsed dipolar ESR spectroscopy.
Biophysical journal, 2012Co-Authors: Jaya Bhatnagar, Jack H. Freed, Ria Sircar, Peter P. Borbat, Brian R. CraneAbstract: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.
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molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of escherichia coli minicells
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Bo Hu, Michael D. Manson, Dustin R Morado, Sneha Jani, William MargolinAbstract:The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA Kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.
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Not too loose, not too tight--just right. Biphasic control of the Tsr HAMP domain.
Molecular microbiology, 2011Co-Authors: Michael D. MansonAbstract:Summary 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.
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The region preceding the C-terminal NWETF pentapeptide modulates baseline activity and aspartate inhibition of Escherichia coli Tar.
Biochemistry, 2008Co-Authors: Run-zhi Lai, Arjan F. Bormans, Roger Russell Draheim, Gus A. Wright, Michael D. MansonAbstract:The Tar chemoreceptor−CheA−CheW ternary complex of Escherichia coli is a transmembrane allosteric enzyme in which binding of ligands to the periplasmic domain modulates the activity of CheA Kinase. Kinase activity is also affected by reversible methylation of four glutamyl residues in the cytoplasmic domain of the receptor. E. coli Tar contains 553 residues. Residues 549−553 comprise the NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. The crystal structure of the similar Tsr chemoreceptor predicts that residues 263−289 and 490−515 of Tar form the most membrane-proximal portion of the extended CD1−CD2 four-helix bundle of the cytoplasmic domain. The last methylation site, Glu-491, is in the C19 heptad, and the N22-19 and C22-19 heptads are present in all classes of bacterial transmembrane chemoreceptors. Residues 516−548 probably serve as a flexible tether for the NWETF pentapeptide. Here, we present a mutational analysis of residues 505−548. The more of this region that i...
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Mutationally Altered Signal Output in the Nart (NarX-Tar) Hybrid Chemoreceptor
Journal of bacteriology, 2006Co-Authors: Scott M. Ward, Arjan F. Bormans, Michael D. MansonAbstract:Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor Kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA Kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor Kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor Kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.
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Cooperative signaling among bacterial chemoreceptors.
Biochemistry, 2005Co-Authors: Run-zhi Lai, Arjan F. Bormans, Roger Russell Draheim, Josiah Manson, Ngoc T. Nguyen, Michael D. MansonAbstract:Four chemoreceptors in Escherichia coli mediate responses to chemicals in the environment. The receptors self-associate and localize to the cell poles. This aggregation implies that interactions among receptors are important parameters of signal processing during chemotaxis. We examined this phenomenon using a receptor-coupled in vitro assay of CheA Kinase activity. The ability of homogeneous populations of the serine receptor Tsr and the aspartate receptor Tar to stimulate CheA was directly proportional to the ratio of the receptor to total protein in cell membranes up to a fraction of 50%. Membranes containing mixed populations of Tar and Tsr supported an up to 4-fold greater stimulation of CheA than expected on the basis of the contributions of the individual receptors. Peak activity was seen at a Tar:Tsr ratio of 1:4. This synergy was observed only when the two proteins were expressed simultaneously, suggesting that, under our conditions, the fundamental "cooperative receptor unit" is relatively static, even in the absence of CheA and CheW. Finally, we observed that inhibition of receptor-stimulated CheA activity by serine or aspartate required significantly higher concentrations of ligand for membranes containing mixed Tsr and Tar populations than for membranes containing only Tsr (up to 10 2 -fold more serine) or Tar (up to 10 4 -fold more aspartate). Together with recent analyses of the interactions of Tsr and Tar in vivo, our results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis.