Mutational analyses of HAMP helices suggest a dynamic bundle model of input–output signalling in chemoreceptors

Dynamics are hypothesized to play an important role in the transmission of signals across membranes by receptors. Bacterial chemoreceptors are long helical proteins that consist of a periplasmic ligand-binding domain; a transmembrane region; a cytoplasmic HAMP (histidine kinase, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain; and a kinase-control module (KCM). The KCM is further composed of adaptation, hinge, and protein interaction regions (PIRs), the latter of which binds the histidine kinase CheA and adaptor CheW. Fusions of the Escherichia coli aspartate receptor KCM to HAMP domains of defined structure (H1-Tar vs. H1-2-Tar) give opposite responses in phosphotransfer and cellular assays, despite similar binding to CheA and CheW. Pulsed dipolar ESR spectroscopy (PDS) of these isolated on and off dimeric effectors reveals that, in the kinase-on state, the HAMP is more conformationally destabilized compared with the PIR, whereas in the kinase-off state, the HAMP is more compact, and the PIR samples a greater breadth of conformations. On and off HAMP states produce different conformational effects at the KCM junction, but these differences decrease through the adaptation region and into the hinge only to return with the inverted relationship in the PIR. Continuous wave-ESR of the spin-labeled proteins confirms that broader PDS distance distributions correlate with increased rates of dynamics. Conformational breadth in the adaptation region changes with charge alterations caused by modification enzymes. Activating modifications broaden the HAMP conformational ensemble but correspondingly, compact the PIR. Thus, chemoreceptors behave as coupled units, in which dynamics in regions proximal and distal to the membrane change coherently but with opposite sign.chemotaxis | transmembrane signaling receptor | allostery | dynamics | adaptation T he ability of localized dynamics to modulate the function of transmembrane receptors is an emerging theme in signal transduction (1-4). These ideas have been largely supported by computational studies (2), although direct measurements also correlate dynamics with activity (1-4). Nonetheless, we are only beginning to address the link between conformational heterogeneity and signal propagation in complex proteins. Bacterial chemotaxis, the process by which cells modulate their motility in response to the chemical environment, provides an important model system to explore receptor dynamics experimentally (5, 6). During chemotaxis, attractant-bound chemoreceptors cause counterclockwise (CCW) flagella rotation and smooth swimming, whereas repellant-bound receptors cause clockwise flagella rotation and cell tumbling. Chemoreceptors, also termed methyl-accepting chemotaxis proteins, form extended arrays in the cytoplasmic membrane to communicate ligand binding (6) to the histidine kinase CheA and the coupling protein CheW. Great progress has been made in understanding how receptors communicate ligand-binding events across the cytoplasmic membrane, but h...

Section: Resultsmentioning

confidence: 98%

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confidence: 99%

Bacterial chemoreceptor dynamics correlate with activity state and are coupled over long distances

Samanta

Borbat

Dzikovski

et al. 2015

“…1A). CqsS lacks a HAMP domain, which is important in signal transduction in many histidine kinases (32)(33)(34)(35)(36)(37)(38). Thus, interactions between CAI-1 and the final transmembrane domain suggest that this transmembrane domain serves not only in ligand binding but as a critical regulatory region for CqsS kinase activity.…”

Section: Discussionmentioning

confidence: 99%

Probing bacterial transmembrane histidine kinase receptor–ligand interactions with natural and synthetic molecules

Wei

Perez³

et al. 2010

136

Bacterial histidine kinases transduce extracellular signals into the cytoplasm. Most stimuli are chemically undefined; therefore, despite intensive study, signal recognition mechanisms remain mysterious. We exploit the fact that quorum-sensing signals are known molecules to identify mutants in the Vibrio cholerae quorum-sensing receptor CqsS that display altered responses to natural and synthetic ligands. Using this chemical-genetics approach, we assign particular amino acids of the CqsS sensor to particular roles in recognition of the native ligand, CAI-1 (S-3 hydroxytridecan-4-one) as well as ligand analogues. Amino acids W104 and S107 dictate receptor preference for the carbon-3 moiety. Residues F162 and C170 specify ligand head size and tail length, respectively. By combining mutations, we can build CqsS receptors responsive to ligand analogues altered at both the head and tail. We suggest that rationally designed ligands can be employed to study, and ultimately to control, histidine kinase activity.agonist | antagonist | quorum-sensing | two-component protein

“…Those structural differences probably involve changes in substrate helix stability and changes in the packing interactions between helices of the methylation bundle (9,(16)(17)(18)(19).…”

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confidence: 99%

“…Adaptation site residues lie on solventexposed helix faces and share a consensus nine-residue motif at which CheR and CheB must bind for catalysis (13)(14)(15). Differences in higher-order structures of the methylation helix bundle presumably govern state-specific access to the substrate sites.Those structural differences probably involve changes in substrate helix stability and changes in the packing interactions between helices of the methylation bundle (9,(16)(17)(18)(19).The critical sensory adaptation roles of the CheR and CheB enzymes have been known for decades (20)(21)(22)(23)(24)(25). Here, we report that CheR has a second function that opposes the signaling consequences of its catalytic activity.…”

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confidence: 99%

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

Lai¹,

Han²,

Dahlquist³

et al. 2017

Self Cite

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...