Abstract
ABSTRACTThe 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.
Highlights
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
Among the many different two-component systems (TCS) that detect signals and regulate gene expression in bacteria, one intriguing group is typified by histidine kinases (HKs) that possess one or more periplasmic Venus flytrap (VFT) domains [1,2,3]
Several obvious questions can be asked for any HK which functions as part of a TCS, and answering these questions for any one kinase will contribute to our understanding of TCS in general, as well as improving our understanding of the particular system that it regulates
Summary
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. Among the many different two-component systems (TCS) that detect signals and regulate gene expression in bacteria, one intriguing group is typified by histidine kinases (HKs) that possess one or more periplasmic Venus flytrap (VFT) domains [1,2,3]. VFT domains are found in proteins across all kingdoms In bacteria, they are largely a feature of amino acid binding proteins, periplasmic binding proteins (PBPs) that bind solutes for subsequent import into the cell via transporters. The protein has a molecular mass of approximately 135 kDa, of which nearly 60 kDa is in the periplasm, linked to the cytoplasmic part by a single transmembrane helix It is dimeric, and the structure of the periplasmic domain has been solved [2, 3], revealing two VFT domains in each protomer, wrapped around each other with one in the open and one in the closed conformation. A detailed mechanism relating movement of domains in the periplasm to a subsequent alteration in the dynamics of a coiled-coil region immediately after the PAS domain has recently been proposed, which may be generic to all proteins with this domain structure [14]
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