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Acid-Sensing Histidine Kinase With a Redox Switch.

ABSTRACT: The EvgS/EvgA two-component signal transduction system in Escherichia coli is activated under mildly acidic pH conditions. Upon activation, this system induces the expression of a number of genes that confer acid resistance. The EvgS histidine kinase sensor has a large periplasmic domain that is required for perceiving acidic signals. In addition, we have previously proposed that the cytoplasmic linker region of EvgS is also involved in the activation of this sensor. The cytoplasmic linker region resembles a Per-ARNT-Sim (PAS) domain, which is known to act as a molecular sensor that is responsive to chemical and physical stimuli and regulates the activity of diverse effector domains. Our EvgS/EvgA reporter assays revealed that under EvgS-activating mildly acidic pH conditions, EvgS was activated only during aerobic growth conditions, and not during anaerobic growth. Studies using EvgS mutants revealed that C671A and C683A mutations in the cytoplasmic PAS domain activated EvgS even under anaerobic conditions. Furthermore, among the electron carriers of the electron transport chain, ubiquinone was required for EvgS activation. The present study proposes a model of EvgS activation by oxidation and suggests that the cytoplasmic PAS domain serves as an intermediate redox switch for this sensor.

SUBMITTER: Inada S

PROVIDER: S-EPMC8174306 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this.IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps E. coli to survive exposure to low pH, which it must do before taking up residence in the gastrointestinal tract. We describe a predicted structure for the main sensing part of the protein and identify some key residues within it that are involved in the sensing and signaling processes. We propose a mechanism for how the protein may become activated and present some evidence to support our proposal.

Project description:Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below ∼30°C. Membrane fluidity signals are transmitted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of membrane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased mutagenic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every transmembrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the metastable 2-HCC domain of the DesK signaling state.IMPORTANCE Signal sensing and transduction is an essential biological process for cell adaptation and survival. Histidine kinases (HK) are the sensory proteins of two-component systems that control many bacterial responses to different stimuli, like environmental changes. Here, we focused on the HK DesK from Bacillus subtilis, a paradigmatic example of a transmembrane thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C. DesK provides a tractable system for investigating the mechanism of transmembrane signaling, one of the majors interrogates in biology to date. Our studies demonstrate that transmembrane proline residues modulate the conformational switch of a 2-helix coiled-coil (2-HCC) structural motif that controls input-output in a variety of HK. Our results highlight the relevance of proline residues within sensor domains and could inspire investigations of their role in different signaling proteins.

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Acid-Sensing Histidine Kinase With a Redox Switch.

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