Cysteine and Disulfide Scanning Reveals a Regulatory α-Helix in the Cytoplasmic Domain of the Aspartate Receptor

Danielson, Mark A.; Bass, Randal B.; Falke, Joseph J.

doi:10.1074/jbc.272.52.32878

Cited by 86 publications

(197 citation statements)

References 89 publications

(144 reference statements)

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“…To date, six lock-on disulfides that constitutively activate the receptor-bound kinase have been identified in the cytoplasmic domain (18,19,30). Of these, five are located at the intersubunit interface within the adaptation subdomain.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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 two symmetric linear faces include the receptor adaptation sites. In the trimer-ofdimers crystal structure, one of the two symmetric linear faces is proposed to be oriented toward solvent where it can provide docking sites for the cytoplasmic proteins CheA, CheW, CheR, and CheB (8,26,33,37). The crystal structure indicates that the other symmetric linear face is oriented toward the center of the trimer where it participates in the receptor-receptor interactions that stabilize trimer-of-dimers formation (8).…”

Section: Discussionmentioning

confidence: 99%

Mapping Out Regions on the Surface of the Aspartate Receptor That Are Essential for Kinase Activation

2003

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The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluoresceinmaleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.The transmembrane chemoreceptors of the Escherichia coli and Salmonella typhimurium chemotaxis pathway are members of a large receptor superfamily that modulates twocomponent signaling systems ubiquitous in prokaryotes and lower eukaryotes (1-4). These chemoreceptors span the bacterial inner membrane where they form a signaling complex with a cytoplasmic histidine kinase (CheA) and a coupling protein (CheW). In the absence of chemoattractant, each chemoreceptor stimulates the histidine kinase, which autophosphorylates itself on a specific His residue. Subsequently, the same phosphoryl group is transferred to an aspartate residue in the active site ...

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“…Receptor purification was performed as previously described (49), with the following modifications. Overnight 2 mL of LB cultures were diluted 1:250 into 500 mL of Vogel Bonner Citrate (VBC) minimal growth medium containing 0.75% glycerol, 200 μg/ mL MgSO 4 ·7H 2 O, 2000 μg/mL citric acid·H 2 O, 10 000 μg/mL K 2 HPO 4 , 3500 μg/mL…”

Section: Expression and Isolation Of Engineered Receptors In Membranesmentioning

confidence: 99%

“…This assay was performed as previously described (20,49,61), with the following modifications. Isolated membranes were diluted to contain 12 μM Tar monomer and either (1) oxidized by the addition of the redox catalyst Cu II (1,10-phenanthroline) 3 (400 μM) in the presence of ambient dissolved oxygen for 20 min at 37 °C or (2) reduced by addition of DTT (100 mM) to ensure elimination of disulfides.…”

Section: In Vitro Receptor-coupled Kinase Assaysmentioning

confidence: 99%

“…Efforts to crystallize larger fragments containing the adaptation subdomain have been unsuccessful, perhaps because of the remarkably dynamic nature of the adaptation subdomain in the isolated cytoplasmic domain (47,48). Cysteine and disulfide scanning studies of the adaptation subdomain have been limited to its N-terminal region and, thus far, have identified only two (CD1 and CD1′) of the four proposed helices in the subdomain (Figure 1) (49). Previous hydrodynamic studies have indicated that the full cytoplasmic domain is highly elongated, and circular dichroism studies revealed a high degree of α helicity (50,51), but these bulk measurements do not possess the resolution needed to test the four-helix bundle model of the adaptation subdomain structure.…”

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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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Cysteine and Disulfide Scanning Reveals a Regulatory α-Helix in the Cytoplasmic Domain of the Aspartate Receptor

Cited by 86 publications

References 89 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

Mapping Out Regions on the Surface of the Aspartate Receptor That Are Essential for Kinase Activation

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

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