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Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

ABSTRACT: The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an ? helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes.

SUBMITTER: Koshy SS

PROVIDER: S-EPMC4270382 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

Koshy Seena S SS Li Xuni X Eyles Stephen J SJ Weis Robert M RM Thompson Lynmarie K LK

Biochemistry 20141203 49

The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplas ...[more]

PMID: 25420045

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Project description: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.

Dataset Information

Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

Publications

Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

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