Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Chervitz, Stephen A.; Lin, Christina M.; Falke, Joseph J.

doi:10.1021/bi00030a010

Cited by 98 publications

(209 citation statements)

References 55 publications

(95 reference statements)

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“…Model of aspartate receptor structure and function. (A) Full-length model based on crystallographic and chemical data (18,29,(37)(38)(39). Recent data support the model, except for the indicated helical conformation of the linker region (40,41).…”

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 strain and plasmid used to generate CheY (RBB455/pRBB40) were kindly provided by Robert Bourret, University of North Carolina. The methods and materials used to express and isolate purified CheA, CheW, and CheY have been previously described (24). [γ-32 P]-ATP was purchased from New England Nuclear.…”

Section: Methodsmentioning

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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“…All E. coli strains were provided by Dr. John S. Parkinson (57), expressing the plasmid pSCF6 previously described (58). CheA (HB101/pMO4) and CheW (HB101/pME5) were made from strains and plasmids provided by Jeff Stock (Princeton).…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

“…The standard in vivo swarm-plate assay was used to quantitate the ability of each receptor to restore aspartate sensing and chemotaxis to cells swimming in a self-generated aspartate gradient (60). This assay can detect receptor perturbations that block receptor regulation of kinase activity or inhibit normal receptor adaptation (58). Inhibitory substitutions were operationally defined as those that reduced the aspartate-specific swarm rate by a factor of 5-fold or more relative to the rate observed for overexpression of the wild-type receptor.…”

Section: Effects Of Cysteine Substitutions On Receptor Function In Vivomentioning

confidence: 99%

“…, yielding a covalent cross-link between a pair of symmetric, engineered cysteines in different subunits (58). Disulfide formation was detected by the appearance of a covalent dimer band in SDS-PAGE and was found to reach between 80 and 95% completion, enabling analysis of the effect of each disulfide on kinase regulation in the in vitro assay.…”

Section: Effects Of Cysteine Substitutions and Disulfide Bonds On Recmentioning

confidence: 99%

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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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Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Cited by 98 publications

References 55 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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