Mutational Analysis of N381, a Key Trimer Contact Residue in Tsr, the Escherichia coli Serine Chemoreceptor

Gosink, Khoosheh K.; Zhao, Yimin; Parkinson, John S.

doi:10.1128/jb.05887-11

Cited by 18 publications

(30 citation statements)

References 48 publications

(77 reference statements)

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“…1) and can be efficiently cross-linked with the trifunctional thiol-reactive reagent TMEA [19]. In S366C/S366C homodimers, the outer receptor subunits of trimers of dimers are not cross-linked by TMEA, thus producing about 50% unlinked one-subunit products and roughly equivalent numbers of twosubunit and three-subunit forms [11, 12, 19, 22]. Many E402* and R404* receptors formed twoand three-subunit TMEA products less efficiently than did the wild-type control, implying defects in trimer formation or stability (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1a). Receptor trimers-of-dimers and CheA/CheW binding interactions are critical for array formation [10] and kinase control [11–13]. Although the architecture of receptor arrays has been elucidated at the molecular level [14–16], the mechanism(s) of CheA control are still unclear, largely due to the many protein-protein interaction surfaces within the signaling arrays, which confound analysis of any single interaction.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers

Lai

Gosink

Parkinson

2017

Journal of Molecular Biology

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Residues E402 and R404 of the E. coli serine chemoreceptor, Tsr, appear to form a salt bridge that spans the interfaces between neighboring dimers in the Tsr trimer of dimers, a key structural component of receptor core signaling complexes. To assess their functional roles we constructed full sets of single amino acid replacement mutants at E402 and R404 and characterized their signaling behaviors with a suite of in vivo assays. Our results indicate that the E402 and R404 residues of Tsr play their most critical signaling roles at their inner locations near the trimer axis where they likely participate in stabilizing trimer-of-dimer packing and the kinase-ON state of core signaling complexes. Mutant receptors with a variety of sidechain replacements still accessed both the ON and OFF signaling states, suggesting that core signaling complexes produce kinase activity over a range of receptor conformations and dynamic motions. Similarly, the kinase-OFF state may not be a discrete conformation, but rather a range of structures outside the range of those suitable for kinase activation. Consistent with this idea, some structural lesions at both E402 and R404 produced signaling behaviors that are not compatible with discrete two-state models of core complex signaling states. Those lesions might stabilize intermediate receptor conformations along the OFF-ON energy landscape. Amino acid replacements produced different constellations of signaling defects at each residue, indicating that they play distinct structure-function roles. R404, but not E402, was critical for high signal cooperativity in the receptor array.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers

Lai

Gosink

Parkinson

2017

Journal of Molecular Biology

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“…Those mutant receptor complexes were distributed uniformly around the cell membrane, with little tendency to cluster at the poles as wild type arrays do. In the absence of CheA and CheW, receptor molecules alone can form polar clusters through trimer-of-dimers interactions or through nonnative receptor-receptor interactions (21). In dispersed core signaling complexes, the mutant CheA and CheW proteins must shield those receptor clustering sites.…”

Section: Discussionmentioning

confidence: 99%

“…S3). Because trimer-of-dimer formation alone allows receptor molecules to form diffuse polar clusters in cells lacking CheA or CheW (20,21), the clustering defects evident with both the CheA::mYPF and YFP-CheR reporters indicate that interface 2 mutant proteins interact with receptor molecules, but that the resultant complexes cannot efficiently organize into larger clusters.…”

Section: Spatial Organization Of Receptor Complexes In Interface 2 Mumentioning

confidence: 99%

The source of high signal cooperativity in bacterial chemosensory arrays

Piñas

Frank

Vaknin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

127

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The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties.chemotaxis | chemoreceptors | kinase activity | stimulus response

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“…Structural changes in the receptor's hairpin tip region (Fig. 1) that produce a wide range of signaling behaviors did not potentiate the CheR effect (53)(54)(55). Perhaps the receptor methylation helices, whose conformation and packing stability are under direct control by the HAMP domain, are central to the CheR effect.…”

Section: A Possible Nonequilibrium Mechanism For Cher Response Enhancmentioning

confidence: 99%

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

Lai¹,

Han²,

Dahlquist³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON-OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands.bacterial chemotaxis | receptor methyltransferase | dynamic-bundle model | signaling conformation | nonequilibrium mechanism M otile bacteria, despite their small size and simple cellular architecture, track chemical gradients in their living environments with extraordinary precision (see refs. 1 and 2 for reviews). They do so with only a handful of different proteins organized in a sensory signaling network that has stimulus integration, amplification, and memory capabilities. The extensively studied chemotaxis machinery of Escherichia coli has provided the molecular paradigm for transmembrane and intracellular signaling mechanisms in microbial chemosensory systems. This report describes a long-unrecognized activity of a central component of the E. coli chemotaxis machinery, suggesting that there are important new molecular lessons to learn from this structurally simple, yet functionally sophisticated, signaling system. E. coli senses attractant and repellent chemicals with transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs) (3). MCPs form networked arrays of signaling complexes, typically at the cell poles, that produce highly sensitive and cooperative responses to small chemoeffector concentration changes. The four MCPs of E. coli (Tsr, Tar, Tap, and Trg) have a common functional architecture comprising a periplasmic sensing domain and a cytoplasmic signaling domain (Fig. 1). An interposed HAMP domain communicates conformational changes between the sensing and signaling domains through an extended four-helix coiled-coil that contains sites for sensory adaptation adjustments of receptor signal output (4). The chemoreceptors modulate the activity of a histidine autokinase (CheA), which is stably coupled to receptors by a scaffolding protein (CheW). CheA donates its phosphoryl groups to CheY, a response regulator that in its phosphory...

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Mutational Analysis of N381, a Key Trimer Contact Residue in Tsr, the Escherichia coli Serine Chemoreceptor

Cited by 18 publications

References 48 publications

Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers

Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers

The source of high signal cooperativity in bacterial chemosensory arrays

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

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