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

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Abstract

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg²⁺ 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 Mg²⁺. 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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All data generated or analysed during this study are included in the manuscript and supporting file; Source data files have been provided for Figure 8. All source code for modeling work is provided as source code files 1-5.

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Bruk Mensa

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States

For correspondence
bruk.mensa@ucsf.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8777-5946
Nicholas F Polizzi

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Kathleen S Molnar

Codexis Inc., Redwood City, United States

Competing interests
The authors declare that no competing interests exist.
Andrew M Natale

Biophysics PhD program, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Thomas Lemmin

Euler Institute, Università della Svizzera Italiana, Lugano, Switzerland

Competing interests
The authors declare that no competing interests exist.
William F DeGrado

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States

For correspondence
Bill.DeGrado@ucsf.edu

Competing interests
The authors declare that no competing interests exist.

Funding

National Institutes of Health (K99-GM138753)

Bruk Mensa
Nicholas F Polizzi
Andrew M Natale
Thomas Lemmin
William F DeGrado

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.