Structural and functional analysis of the Escherichia coli acid-sensing histidine kinase EvgS

The EvgS/EvgA two component system of E. coli is activated in response to low pH and alkali metals, and regulates many genes including 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 single 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 build a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide site directed 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 mutants are present. These findings are consistent with a model where 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 purified cytoplasmic portion of EvgS that supports this.