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Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

ABSTRACT: Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, "dipped" and "undipped," for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units.IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called "dipped" CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

SUBMITTER: Pinas GE

PROVIDER: S-EPMC6832072 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

Piñas Germán E GE Parkinson John S JS

Journal of bacteriology 20191105 23

<i>Escherichia coli</i> chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecu ...[more]

PMID: 31501279

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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:Transfer of a phosphoryl group from autophosphorylated CheA (P-CheA) to CheY is an important step in the bacterial chemotaxis signal transduction pathway. This reaction involves CheY (i) binding to the P2 domain of P-CheA and then (ii) acquiring the phosphoryl group from the P1 domain. Crystal structures indicated numerous side chain interactions at the CheY-P2 binding interface. To investigate the individual contributions of the P2 side chains involved in these contacts, we analyzed the effects of eight alanine substitution mutations on CheA-CheY binding interactions. An F214A substitution in P2 caused ∼1,000-fold reduction in CheA-CheY binding affinity, while Ala substitutions at other P2 positions had small effects (E171A, E178A, and I216A) or no detectable effects (H181A, D202A, D207A, and C213A) on binding affinity. These results are discussed in relation to previous in silico predictions of hot-spot and anchor positions at the CheA-CheY interface. We also investigated the consequences of these mutations for chemotaxis signal transduction in living cells. CheA(F214A) was defective in mediating localization of CheY-YFP to the large clusters of signaling proteins that form at the poles of Escherichia coli cells, while the other CheA variants did not differ from wild-type (wt) CheA (CheA(wt)) in this regard. In our set of mutants, only CheA(F214A) exhibited a markedly diminished ability to support chemotaxis in motility agar assays. Surprisingly, however, in FRET assays that monitored receptor-regulated production of phospho-CheY, CheA(F214A) (and each of the other Ala substitution mutants) performed just as well as CheA(wt). Overall, our findings indicate that F214 serves as an anchor residue at the CheA-CheY interface and makes an important contribution to the binding energy in vitro and in vivo; however, loss of this contribution does not have a large negative effect on the overall ability of the signaling pathway to modulate P-CheY levels in response to chemoattractants.

Project description:Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

Dataset Information

Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

Publications

Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

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