Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors

Falke, Joseph J.; Kim, Sung‐Hou

doi:10.1016/s0959-440x(00)00115-9

Cited by 48 publications

(46 citation statements)

References 66 publications

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“…In total, the adaptation subdomain contains approximately 100 residues per subunit, of which 21 residues per subunit are acidic (including the four adaptation site glutamates). Figure 2 summarizes the positions of these acidic residues within a structural model of the adaptation subdomain consistent with the chemically determined structure of the native cytoplasmic domain and the crystallographically determined structure of the signaling subdomain (5,9,18). Table 1 lists the acidic residues and indicates their high degree of conservation (20).…”

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confidence: 68%

Adaptation Mechanism of the Aspartate Receptor: Electrostatics of the Adaptation Subdomain Play a Key Role in Modulating Kinase Activity

2005

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The aspartate receptor of the Escherichia coli and Salmonella typhimurium chemotaxis pathway generates a transmembrane signal that regulates the activity of the cytoplasmic kinase CheA. Previous studies have identified a region of the cytoplasmic domain that is critical to receptor adaptation and kinase regulation. This region, termed the adaptation subdomain, contains a high density of acidic residues, including specific glutamate residues that serve as receptor adaptation sites. However, the mechanism of signal propagation through this region remains poorly understood. This study uses site-directed mutagenesis to neutralize each acidic residue within the subdomain to probe the hypothesis that electrostatics in this region play a significant role in the mechanism of kinase activation and modulation. Each point mutant was tested for its ability to regulate chemotaxis in vivo and kinase activity in vitro. Four point mutants (D273N, E281Q, D288N, and E477Q) were found to superactivate the kinase relative to the wild-type receptor, and all four of these kinaseactivating substitutions are located along the same intersubunit interface as the adaptation sites. These activating substitutions retained the wild-type ability of the attractant-occupied receptor to inhibit kinase activity. When combined in a quadruple mutant (D273N/E281Q/D288N/E477Q), the four charge-neutralizing substitutions locked the receptor in a kinase-superactivating state that could not be fully inactivated by the attractant. Similar lock-on character was observed for a charge reversal substitution, D273R. Together, these results implicate the electrostatic interactions at the intersubunit interface as a major player in signal transduction and kinase regulation. The negative charge in this region destabilizes the local structure in a way that enhances conformational dynamics, as detected by disulfide trapping, and this effect is reversed by charge neutralization of the adaptation sites. Finally, two substitutions (E308Q and E463Q) preserved normal kinase activation in vitro but blocked cellular chemotaxis in vivo, suggesting that these sites lie within the docking site of an adaptation enzyme, CheR or CheB. Overall, this study highlights the importance of electrostatics in signal transduction and regulation of kinase activity by the cytoplasmic domain of the aspartate receptor.The aspartate receptor of bacterial chemotaxis is one of the best-characterized members of a large family of cell surface receptors that modulate two-component signaling pathways (reviewed in refs 1-6). Through regulation of the associated histidine kinase CheA, the aspartate receptor controls cellular chemotaxis in response to changing concentrations of the chemoattractant aspartate, allowing the cell to move up an attractant concentration gradient. The receptor is an oligomer composed of three homodimers that assemble to form a trimer of dimers, as illustrated in Figure 1A. The chemotaxis signaling pathway is triggered by aspartate † Support provided by NIH Grant GM ...

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confidence: 68%

Adaptation Mechanism of the Aspartate Receptor: Electrostatics of the Adaptation Subdomain Play a Key Role in Modulating Kinase Activity

2005

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“…The basic mechanism in Bacillus subtilis (reviewed by Aizawa et al, 2001) and in Escherichia coli (reviewed by Bourret & Stock, 2002;Bren & Eisenbach, 2000;Falke & Kim, 2000;Falke & Hazelbauer, 2001;Stock & Levit, 2000;Taylor et al, 1999) involves complexes of transmembrane receptors (called methyl-accepting chemotaxis proteins or MCPs), CheA autokinase and CheW coupling protein, which allows the receptors to activate the kinase (Morrison & Parkinson, 1997). The CheA kinase autophosphorylates at the expense of ATP with production of ADP (Garrity & Ordal, 1997;Hess et al, 1988a, b).…”

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confidence: 99%

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis

2004

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Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli. In order to understand better the role of the novel chemotaxis proteins -CheC, CheD and CheV -mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed. The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type. This result may indicate that in absence of CheC the switch might be in a conformation less resembling the transition state than in presence of CheC. Conversely, the cheB (methylesterase) mutant showed considerably increased switching frequency without affecting CCW bias, compared with wild-type. Removal of all known adaptation systems -the methylation, CheC and CheV systems -resulted in a mutant (cheRBCDV ) that still retained some adaptation following the addition of attractant.

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“…The current model of cytoplasmic domain architecture describes the domain as an extended, antiparallel four-helix bundle (4,39,40). This model has been proposed on the basis of evidence from (i) chemical studies, which mapped out the four-helix bundle of the signaling subdomain in the full-length, membrane-bound receptor using a cysteine and disulfide scanning approach (41), and (ii) crystallographic studies, which confirmed the existence of the bundle and provided a high-resolution view of its structure in a water-soluble fragment of the cytoplasmic domain (39).…”

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Evidence that the Adaptation Region of the Aspartate Receptor Is a Dynamic Four-Helix Bundle: Cysteine and Disulfide Scanning Studies

2005

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The aspartate receptor is one of the ligand-specific, homodimeric chemoreceptors that detects extracellular attractants and triggers the chemotaxis pathway of Escherichia coli and Salmonella typhimurium. This receptor regulates the activity of the histidine kinase CheA, which forms a kinetically stable complex with the receptor cytoplasmic domain. An atomic four-helix bundle model has been constructed for this domain, which is functionally subdivided into the signaling and adaptation subdomains. The proposed four-helix bundle structure of the signaling subdomain, which binds CheA, is fully supported by experimental evidence. Much less evidence is available to test the four-helix bundle model of the adaptation subdomain, which possesses covalent adaptation sites and docking surfaces for adaptation enzymes. The present study focuses on a putative helix near the C terminus of the adaptation subdomain. To probe the structural and functional features of positions G467-A494 in this C-terminal region, a cysteine and disulfide scanning approach has been employed. Measurement of the chemical reactivities of scanned cysteines reveals an α-helical periodicity of exposed and buried residues, confirming α-helical secondary structure and mapping out a buried packing face. The effects of cysteine substitutions on activity in vivo and in vitro highlight the functional importance of the helix, especially its buried face. A scan for disulfide bond formation between symmetric pairs of engineered cysteines reveals promiscuous collisions between subunits, indicating the presence of significant thermal dynamics. A scan for functional disulfides reveals lockon and signal-retaining disulfide bonds formed between symmetric pairs of cysteines at buried positions, indicating that the buried face of the helix lies near the subunit interface of the homodimer in the equilibrium structures of both the apo and aspartate-bound states where it plays a critical role in kinase regulation. These results strongly support the existing four-helix bundle model of the adaptation subdomain structure. A mechanistic model is proposed in which a signal is transmitted through the adaptation subdomain by a change in supercoiling of the four-helix bundle.The mechanism of signal transduction by which cell-surface receptors regulate cytoplasmic kinases is an issue of central importance in signaling biology. Some receptors activate their appropriate kinases by dimerization, but others trigger kinase activation by transmembrane conformational changes. The latter class includes a large superfamily of cell-surface receptors that regulate cytoplasmic histidine kinases in prokaryotic and eukaryotic two-component signaling pathways (1,2). An important subfamily of this histidine kinase-coupled receptor superfamily is responsible for regulating the thermo-, photo-, osmo-, redox-, and chemotaxis pathways of a wide variety of prokaryotic organisms (3-5). These taxis receptors, comprising a group of over 2000 homologues (6), exhibit regions of high conservation in ...

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Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors

Cited by 48 publications

References 66 publications

Adaptation Mechanism of the Aspartate Receptor: Electrostatics of the Adaptation Subdomain Play a Key Role in Modulating Kinase Activity

Adaptation Mechanism of the Aspartate Receptor: Electrostatics of the Adaptation Subdomain Play a Key Role in Modulating Kinase Activity

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis

Evidence that the Adaptation Region of the Aspartate Receptor Is a Dynamic Four-Helix Bundle: Cysteine and Disulfide Scanning Studies

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