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John S. Parkinson - One of the best experts on this subject based on the ideXlab platform.
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disruption of chemoreceptor signalling arrays by high levels of CheW the receptor kinase coupling Protein
Molecular Microbiology, 2010Co-Authors: Marcos J. Cardozo, John S. Parkinson, Diego A. Massazza, Claudia A. StuddertAbstract:During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW Protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.
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Disruption of chemoreceptor signalling arrays by high levels of CheW, the receptor–kinase coupling Protein
Molecular microbiology, 2010Co-Authors: Marcos J. Cardozo, John S. Parkinson, Diego A. Massazza, Claudia A. StuddertAbstract:During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW Protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.
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Cysteine-Scanning Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA
Journal of bacteriology, 2006Co-Authors: Jinshi Zhao, John S. ParkinsonAbstract:The C-terminal P5 domain of the histidine kinase CheA is essential for coupling CheA autophosphorylation activity to chemoreceptor control through a binding interaction with the CheW Protein. To locate P5 determinants critical for CheW binding and chemoreceptor control, we surveyed cysteine replacements at 39 residues predicted to be at or near the P5 surface in Escherichia coli CheA. Two-thirds of the Cys replacement Proteins exhibited in vitro defects in CheW binding, either before or after modification with a bulky fluorescein group. The binding-defective sites were widely distributed on the P5 surface and were often interspersed with sites that caused no functional defects, implying that relatively minor structural perturbations, often far from the actual binding site, can influence its conformation or accessibility. The most likely CheW docking area included loop 2 in P5 folding subdomain 1. All but four of the binding-defective P5-Cys Proteins were defective in receptor-mediated activation, suggesting that CheW binding, as measured in vitro, is necessary for assembly of ternary signaling complexes and/or subsequent CheA activation. Other Cys sites specifically affected receptor-mediated activation or deactivation of CheA, demonstrating that CheW binding is not sufficient for assembly and/or operation of receptor signaling complexes. Because P5 is quite similar to CheW, whose structure is known to be dynamic, we suggest that conformational flexibility and dynamic motions govern the signaling activities of the P5 domain. In addition, relative movements of the CheA domains may be involved in CheW binding, in ternary complex assembly, and in subsequent stimulus-induced conformational changes in receptor signaling complexes.
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Mutational Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA
Journal of bacteriology, 2006Co-Authors: Jinshi Zhao, John S. ParkinsonAbstract:During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW Protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and Protein stability tests and in vitro assays with purified mutant Proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA Proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA Proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant Proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant Proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3 interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA. The histidine kinase CheA plays a central signaling role in bacterial chemotaxis pathways (see references 48 and 49 for recent reviews). In Escherichia coli, CheA forms signaling complexes with five membrane-associated chemoreceptors, known as methyl-accepting chemotaxis Proteins (MCPs), that communicate with the flagellar motors by controlling CheA activity. CheA autophosphorylates at a histidine residue, using ATP as the phosphodonor, and subsequently donates those phosphoryl groups to two response regulators, CheY and CheB. Phosphorylated CheY (phospho-CheY) interacts with the switching machinery at the base of the flagellar motors to promote clockwise (CW) rotation, which causes random turning episodes or tumbles during swimming. Phospho-CheB functions in a feedback adaptation circuit that enables the cells to detect temporal changes in attractant and repellent concentrations as they move through spatial chemical gradients. Chemoeffectors, sensed by the chemoreceptors, modulate CheA activity over a wide range. Unliganded receptors activate CheA several hundredfold over its basal, receptor-uncoupled autophosphorylation rate, whereas attractant-bound receptors deactivate CheA to about its basal activity level (8, 9, 26, 33, 45). CheA autophosphorylation control occurs in ternary complexes formed between the cytoplasmic signaling domains of the chemoreceptors, CheA, and the coupling Protein CheW, which is essential for chemotactic behavior in vivo and for receptor-mediated activation of CheA in vitro. The role of CheW in receptor coupling control of CheA is poorly understood, owing to a paucity of structural information about the
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insights into the organization and dynamics of bacterial chemoreceptor clusters through in vivo crosslinking studies
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Claudia A. Studdert, John S. ParkinsonAbstract:The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers that bind the cytoplasmic Proteins CheA and CheW to form ternary signaling complexes clustered at the cell poles. We used a trifunctional crosslinking reagent targeted to cysteine residues in the aspartate (Tar) and serine (Tsr) receptors to obtain in vivo snapshots of trimer composition in the receptor population. To analyze the dynamics of trimer formation, we followed the appearance of mixed trimers when cells expressing Tar were induced for the expression of Tsr and treated with the crosslinker shortly after the onset of induction. In the absence of CheA or CheW, preformed Tar trimers exchanged partners readily with newly made Tsr. Conversely, in the presence of CheA and CheW, receptor trimers seldom exchanged partners, irrespective of the presence or absence of attractants. The C-terminal receptor-coupling domain of the CheA kinase, which contains binding determinants for the CheW Protein, was essential for conferring low exchangeability to the preformed trimers of dimers. CheW also was required for this effect, but, unlike CheA, overexpression of CheW interfered with trimer formation and chemotactic behavior. The CheW effect probably occurs through binding interactions that mask the receptor sites needed for trimer formation. We propose that clustered receptors are organized in mixed trimers of dimers through binding interactions with CheA and CheW, which play distinctly different architectural roles. Moreover, once complete signaling teams have formed, they no longer undergo dynamic exchange of receptor members.
Claudia A. Studdert - One of the best experts on this subject based on the ideXlab platform.
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disruption of chemoreceptor signalling arrays by high levels of CheW the receptor kinase coupling Protein
Molecular Microbiology, 2010Co-Authors: Marcos J. Cardozo, John S. Parkinson, Diego A. Massazza, Claudia A. StuddertAbstract:During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW Protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.
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Disruption of chemoreceptor signalling arrays by high levels of CheW, the receptor–kinase coupling Protein
Molecular microbiology, 2010Co-Authors: Marcos J. Cardozo, John S. Parkinson, Diego A. Massazza, Claudia A. StuddertAbstract:During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW Protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.
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insights into the organization and dynamics of bacterial chemoreceptor clusters through in vivo crosslinking studies
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Claudia A. Studdert, John S. ParkinsonAbstract:The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers that bind the cytoplasmic Proteins CheA and CheW to form ternary signaling complexes clustered at the cell poles. We used a trifunctional crosslinking reagent targeted to cysteine residues in the aspartate (Tar) and serine (Tsr) receptors to obtain in vivo snapshots of trimer composition in the receptor population. To analyze the dynamics of trimer formation, we followed the appearance of mixed trimers when cells expressing Tar were induced for the expression of Tsr and treated with the crosslinker shortly after the onset of induction. In the absence of CheA or CheW, preformed Tar trimers exchanged partners readily with newly made Tsr. Conversely, in the presence of CheA and CheW, receptor trimers seldom exchanged partners, irrespective of the presence or absence of attractants. The C-terminal receptor-coupling domain of the CheA kinase, which contains binding determinants for the CheW Protein, was essential for conferring low exchangeability to the preformed trimers of dimers. CheW also was required for this effect, but, unlike CheA, overexpression of CheW interfered with trimer formation and chemotactic behavior. The CheW effect probably occurs through binding interactions that mask the receptor sites needed for trimer formation. We propose that clustered receptors are organized in mixed trimers of dimers through binding interactions with CheA and CheW, which play distinctly different architectural roles. Moreover, once complete signaling teams have formed, they no longer undergo dynamic exchange of receptor members.
Frederick W. Dahlquist - One of the best experts on this subject based on the ideXlab platform.
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CheW Binding Interactions with CheA and Tar IMPORTANCE FOR CHEMOTAXIS SIGNALING IN ESCHERICHIA COLI
The Journal of biological chemistry, 2002Co-Authors: Marina Boukhvalova, Frederick W. Dahlquist, Richard C. StewartAbstract:The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a Protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter Protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW Proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW Protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.
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Signal transduction in bacteria: CheW forms a reversible complex with the Protein kinase CheA
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Julie A. Gegner, Frederick W. DahlquistAbstract:Abstract An essential step in the signal transduction pathway of Escherichia coli is the control of the Protein kinase activity of CheA by the chemotaxis receptor Proteins. This control requires the participation of the CheW Protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCl/5 mM MgCl2, pH 7.4 at 4 degrees C) was determined to be in the physiologically relevant range of 17 microM. No evidence for cooperativity in the association of CheW with CheA was found.
Jinshi Zhao - One of the best experts on this subject based on the ideXlab platform.
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Cysteine-Scanning Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA
Journal of bacteriology, 2006Co-Authors: Jinshi Zhao, John S. ParkinsonAbstract:The C-terminal P5 domain of the histidine kinase CheA is essential for coupling CheA autophosphorylation activity to chemoreceptor control through a binding interaction with the CheW Protein. To locate P5 determinants critical for CheW binding and chemoreceptor control, we surveyed cysteine replacements at 39 residues predicted to be at or near the P5 surface in Escherichia coli CheA. Two-thirds of the Cys replacement Proteins exhibited in vitro defects in CheW binding, either before or after modification with a bulky fluorescein group. The binding-defective sites were widely distributed on the P5 surface and were often interspersed with sites that caused no functional defects, implying that relatively minor structural perturbations, often far from the actual binding site, can influence its conformation or accessibility. The most likely CheW docking area included loop 2 in P5 folding subdomain 1. All but four of the binding-defective P5-Cys Proteins were defective in receptor-mediated activation, suggesting that CheW binding, as measured in vitro, is necessary for assembly of ternary signaling complexes and/or subsequent CheA activation. Other Cys sites specifically affected receptor-mediated activation or deactivation of CheA, demonstrating that CheW binding is not sufficient for assembly and/or operation of receptor signaling complexes. Because P5 is quite similar to CheW, whose structure is known to be dynamic, we suggest that conformational flexibility and dynamic motions govern the signaling activities of the P5 domain. In addition, relative movements of the CheA domains may be involved in CheW binding, in ternary complex assembly, and in subsequent stimulus-induced conformational changes in receptor signaling complexes.
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Mutational Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA
Journal of bacteriology, 2006Co-Authors: Jinshi Zhao, John S. ParkinsonAbstract:During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW Protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and Protein stability tests and in vitro assays with purified mutant Proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA Proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA Proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant Proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant Proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3 interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA. The histidine kinase CheA plays a central signaling role in bacterial chemotaxis pathways (see references 48 and 49 for recent reviews). In Escherichia coli, CheA forms signaling complexes with five membrane-associated chemoreceptors, known as methyl-accepting chemotaxis Proteins (MCPs), that communicate with the flagellar motors by controlling CheA activity. CheA autophosphorylates at a histidine residue, using ATP as the phosphodonor, and subsequently donates those phosphoryl groups to two response regulators, CheY and CheB. Phosphorylated CheY (phospho-CheY) interacts with the switching machinery at the base of the flagellar motors to promote clockwise (CW) rotation, which causes random turning episodes or tumbles during swimming. Phospho-CheB functions in a feedback adaptation circuit that enables the cells to detect temporal changes in attractant and repellent concentrations as they move through spatial chemical gradients. Chemoeffectors, sensed by the chemoreceptors, modulate CheA activity over a wide range. Unliganded receptors activate CheA several hundredfold over its basal, receptor-uncoupled autophosphorylation rate, whereas attractant-bound receptors deactivate CheA to about its basal activity level (8, 9, 26, 33, 45). CheA autophosphorylation control occurs in ternary complexes formed between the cytoplasmic signaling domains of the chemoreceptors, CheA, and the coupling Protein CheW, which is essential for chemotactic behavior in vivo and for receptor-mediated activation of CheA in vitro. The role of CheW in receptor coupling control of CheA is poorly understood, owing to a paucity of structural information about the
Marcos J. Cardozo - One of the best experts on this subject based on the ideXlab platform.
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disruption of chemoreceptor signalling arrays by high levels of CheW the receptor kinase coupling Protein
Molecular Microbiology, 2010Co-Authors: Marcos J. Cardozo, John S. Parkinson, Diego A. Massazza, Claudia A. StuddertAbstract:During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW Protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.
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Disruption of chemoreceptor signalling arrays by high levels of CheW, the receptor–kinase coupling Protein
Molecular microbiology, 2010Co-Authors: Marcos J. Cardozo, John S. Parkinson, Diego A. Massazza, Claudia A. StuddertAbstract:During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW Protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.