Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ

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    Evaluation Summary:

    This paper will be of interest to scientists who think about mechanisms of conformational signaling within transmembrane receptor proteins. It describes a model of signaling by allosteric coupling between individual domains rather than by a concerted conformational change and provides substantial experimental evidence for the model from characterization of over 30 mutational substitutions in the bacterial two-component sensor protein PhoQ. The allosteric coupling model provides a way to understand many diverse observations about signaling by two-component receptors and has the potential to be relevant to conformational signaling by many other transmembrane receptors.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg 2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP domain favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological concentrations of Mg 2+ . Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.

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  1. Author Response:

    Reviewer #1 (Public Review):

    Previous studies have provided crystallographic snapshots of the autokinase domains of several sensor histidine kinases (HK) involved in signal transduction in bacteria. Nevertheless, the lack of a full-length structure of these HK hampered the understanding of the molecular mechanism of signaling. Moreover, how a stimuli perceived by the membrane-bound sensor domain is transmitted to the catalytic cytoplasmic domain of an HK, to modulate its activity is poorly understood. To probe the coupling between the sensor and autokinase domains Mensa et al. used cysteine cross linking and reporter gene assay to probe the signaling state of E. coli PhoQ in a set of several point mutations. Using these data they developed a 3-domain model in which the sensor, HAMP and catalytic domain are in …

  2. Evaluation Summary:

    This paper will be of interest to scientists who think about mechanisms of conformational signaling within transmembrane receptor proteins. It describes a model of signaling by allosteric coupling between individual domains rather than by a concerted conformational change and provides substantial experimental evidence for the model from characterization of over 30 mutational substitutions in the bacterial two-component sensor protein PhoQ. The allosteric coupling model provides a way to understand many diverse observations about signaling by two-component receptors and has the potential to be relevant to conformational signaling by many other transmembrane receptors.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …

  3. Reviewer #1 (Public Review):

    Previous studies have provided crystallographic snapshots of the autokinase domains of several sensor histidine kinases (HK) involved in signal transduction in bacteria. Nevertheless, the lack of a full-length structure of these HK hampered the understanding of the molecular mechanism of signaling. Moreover, how a stimuli perceived by the membrane-bound sensor domain is transmitted to the catalytic cytoplasmic domain of an HK, to modulate its activity is poorly understood. To probe the coupling between the sensor and autokinase domains Mensa et al. used cysteine cross linking and reporter gene assay to probe the signaling state of E. coli PhoQ in a set of several point mutations. Using these data they developed a 3-domain model in which the sensor, HAMP and catalytic domain are in allosteric communication to …

  4. Reviewer #2 (Public Review):

    This manuscript characterized the effects of 35 mutational substitutions in three domains the bacterial transmembrane two-component sensor protein for Mg2+, PhoQ, on the signaling state of the periplasmic sensor domain and the cytoplasmic histidine kinase domain. Signaling state was assayed by a diagnostic cysteine cross-link for the sensor domain and the expression of a coupled beta-galactosidase reporter for the kinase domain. The results of those characterizations were used to develop an allosteric coupling model of conformational signaling from sensor domain to kinase domain, with a key role played by the HAMP domain that connects sensor to kinase. Single-site mutational substitutions were at positions expected to be in the interior of the protein structure in the periplasmic, HAMP and the S-helix …

  5. Reviewer #3 (Public Review):

    This manuscript describes a comprehensive study of kinase activation and allosteric coupling in the sensor histidine kinase (SHKs) PhoQ. Quantitative assays for sensor domain activation and kinase response are used to evaluate a large number of variant proteins that display a range of properties with respect to ligand binding, interdomain coupling and kinase activity. The data is used to construct and fit a conceptually elegant model that provides a thermodynamic explanation for domain interactions, allostery and sensing responses in SHKs. The experiments also demonstrate that sensor kinase domains intrinsically favor their "on" states and that HAMP domains act to deactivate both the sensor and the kinase units. In all it is a very impressive study that sets the bar for enzymatic approaches aimed at …