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Transmembrane region of bacterial chemoreceptor is capable of promoting protein clustering.

ABSTRACT: Many membrane proteins are known to form higher-order oligomers, but the degree to which membrane regions could facilitate protein complex assembly remains largely unclear. Clusters of chemotaxis receptors are among the most prominent structures in the bacterial cell membrane, and they play important functions in processing of chemotactic signals. Although much work has been done to elucidate mechanisms of cluster formation, it almost exclusively focused on cytoplasmic interactions among receptors and other chemotaxis proteins, whereas involvement of membrane-mediated interactions was only hypothesized. Here we used imaging of constructs composed of only a fluorescent protein and the TM helices of Tar to demonstrate that interactions between the lipid bilayer and transmembrane (TM) helices of Escherichia coli chemoreceptors alone are sufficient to mediate clustering. We found that the ability to cluster depends on the sequence or length of the TM helices, implying that certain conformations of these helices facilitate clustering, whereas others do not. Notably, observed sequence specificity was apparently consistent with differences in clustering between native E. coli receptors, with the TM sequence of better-clustering high-abundance receptors being more efficient in promoting membrane-mediated complex formation. These results indicate that being more than just membrane anchors, TM helices could play an important role in the clustering and organization of membrane proteins in bacteria.

SUBMITTER: Pollard AM

PROVIDER: S-EPMC5808774 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Transmembrane region of bacterial chemoreceptor is capable of promoting protein clustering.

Pollard Abiola M AM Sourjik Victor V

The Journal of biological chemistry 20171219 6

Many membrane proteins are known to form higher-order oligomers, but the degree to which membrane regions could facilitate protein complex assembly remains largely unclear. Clusters of chemotaxis receptors are among the most prominent structures in the bacterial cell membrane, and they play important functions in processing of chemotactic signals. Although much work has been done to elucidate mechanisms of cluster formation, it almost exclusively focused on cytoplasmic interactions among recepto ...[more]

PMID: 29259129

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Project description:UnlabelledThe transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements.ImportanceThe Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston displacements of a membrane-spanning helix and an adjoining five-residue control cable segment. We characterized the signaling properties of Tsr variants to elucidate the transmembrane signaling role of the control cable, an element present in many microbial sensory proteins. Both the kinase-on and kinase-off output states of Tsr depended on control cable helicity, but only one residue, I214, was critical for triggering responses to attractant inputs. These findings suggest that signal transmission in Tsr involves modulation of control cable helicity through interaction of the I214 side chain with the cytoplasmic membrane.

Project description:Soil bacteria adapt to diverse and rapidly changing environmental conditions by sensing and responding to environmental cues using a variety of sensory systems. Two-component systems are a widespread type of signal transduction system present in all three domains of life and typically are comprised of a sensor kinase and a response regulator. Many two-component systems function by regulating gene expression in response to environmental stimuli. The bacterial chemotaxis system is a modified two-component system with additional protein components and a response that, rather than regulating gene expression, involves behavioral adaptation and results in net movement toward or away from a chemical stimulus. Soil bacteria generally have 20 to 40 or more chemoreceptors encoded in their genomes. To simplify the identification of chemoeffectors (ligands) sensed by bacterial chemoreceptors, we constructed hybrid sensor proteins by fusing the sensor domains of Pseudomonas putida chemoreceptors to the signaling domains of the Escherichia coli NarX/NarQ nitrate sensors. Responses to potential attractants were monitored by β-galactosidase assays using an E. coli reporter strain in which the nitrate-responsive narG promoter was fused to lacZ Hybrid receptors constructed from PcaY, McfR, and NahY, which are chemoreceptors for aromatic acids, tricarboxylic acid cycle intermediates, and naphthalene, respectively, were sensitive and specific for detecting known attractants, and the β-galactosidase activities measured in E. coli correlated well with results of chemotaxis assays in the native P. putida strain. In addition, a screen of the hybrid receptors successfully identified new ligands for chemoreceptor proteins and resulted in the identification of six receptors that detect propionate.IMPORTANCE Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors.

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Transmembrane region of bacterial chemoreceptor is capable of promoting protein clustering.

Publications

Transmembrane region of bacterial chemoreceptor is capable of promoting protein clustering.

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