Molecular Evolution of the C-terminal Cytoplasmic Domain of a Superfamily of Bacterial Receptors Involved in Taxis

Moual, Hervé Le; Koshland, Daniel E.

doi:10.1006/jmbi.1996.0483

Cited by 157 publications

(236 citation statements)

References 64 publications

(90 reference statements)

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“…Analysis of the sequence alignment for the 13 class I bacterial receptors reveals a high degree of conservation for the 17 background acidic residues and the four adaptation site residues (Table 1) (20). At 16 of the 17 positions probed in this study, the degree of conservation of an acidic residue is ≥50%, and at 14 of the 17 positions, the degree of conservation exceeds 75%.…”

Section: Discussionmentioning

confidence: 73%

“…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). Previous studies have shown that neutralization of each of the four adaptation sites by methyl esterification, or by site-directed mutagenesis from Glu to Gln, significantly increases receptor-regulated kinase activity (12,13,21,22).…”

mentioning

confidence: 68%

“…More generally, other, more distantly related classes of prokaryotic receptors also possess adaptation subdomains that exhibit high densities of acidic residues and utilize glutamate residues as adaptation sites (20). Presumably, the adaptation signals of these receptors will share the electrostatic mechanism proposed for class I receptors, although there could be exceptions.…”

Section: Discussionmentioning

confidence: 99%

“…Presumably, the adaptation signals of these receptors will share the electrostatic mechanism proposed for class I receptors, although there could be exceptions. For example, the haloarchaeal taxis receptors exhibit a spatial distribution of adaptation sites within their cytoplasmic domains that differs significantly from the typical class I pattern (20), and these receptors function in a cytoplasmic ionic enviroment that contains high levels of salt (4 M KCl and NaCl) where the resulting high ionic strength would weaken long-range electrostatic interactions (42). These haloarchaeal receptors may use a nonelectrostatic mechanism to trigger adaptation signals, or they may be specialized to enable electrostatic signals to be generated in a high-ionic strength environment.…”

Section: Discussionmentioning

confidence: 99%

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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: 73%

mentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

2005

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“…This region is the most highly conserved among chemotaxis receptor structures (30) and is believed to contain the conserved docking surface for the histidine kinase (CheA) and the coupling protein (CheW) essential for the formation of the receptor-kinase signaling complex based on previous genetic and PICM studies (37,21,22,33). Moreover, this same region provides the bulk of receptor-receptor contacts that stabilize the trimer-of-dimers (8,10).…”

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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pH Sensing in Bacterial Chemotaxis

Levit

Stock

2007

Novartis Foundation Symposium 221 ‐ Bacterial Responses to pH

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Molecular Evolution of the C-terminal Cytoplasmic Domain of a Superfamily of Bacterial Receptors Involved in Taxis

Cited by 157 publications

References 64 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

pH Sensing in Bacterial Chemotaxis

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