Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS

Abstract

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 twocomponent 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.

Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

“…Such breaking of symmetry is thought to be essential for activation of HisKA-type HKs (8,9,14,15). Much of this view is rooted in the fact that most HKs are dimeric; however, it is becoming clear that a wider variety of oligomeric assemblies are used in HK signaling-from the monomeric EL346 to the trimeric SasA component of cyanobacterial signaling systems (11) to the monomeric/dimeric acidsensing EvgS in Escherichia coli (46). We assert that this variety of quaternary structure used for HK regulation suggests that the root element-destabilization of the DHp (or DHpL) interactions with the CA domain-is conserved, even if the precise mechanism of triggering that destabilization is different (i.e., symmetry-breaking vs. sensor/DHpL interface breaking).…”

An acid-tolerance response system protecting exponentially growing Escherichia coli

“…Due to the involvement of CRP, the AR1 system is repressed by glucose. The AR2−AR5 systems are all dependent on a specific extracellular amino acid, and consist of an antiporter as well as a decarboxylase enzyme that is usually induced by low pH and extracellular amino acid 3,7 , except that AR2 can be induced at acidic pH in the absence of glutamate 8 . They confer acid resistance by consumption of intracellular protons in amino acid decarboxylation reaction to produce a less acidic internal pH, using glutamate, arginine, lysine and ornithine as their corresponding substrates, respectively 1,5,[9][10][11][12] .…”

The Signaling Molecule Indole Inhibits Induction of the AR2 Acid Resistance System in Escherichia coli

“…A striking feature of the EvgS sensor kinase is that it has a large periplasmic domain of approximately 61 kDa (out of a total molecular mass of 135 kDa). Structural predictions for this domain reveal that it contains two Venus fly-trap domains, a feature that it shares with several other sensor kinases in pathogenic bacteria, including the well-studied BvgS from Bordetella pertussis (Herrou et al, 2010;Dupré et al, 2015a;Sen et al, 2017). Venus fly-trap domains typically show a structural transition from an open to closed conformation when they bind their ligand, a process that in turn can trigger a signal transduction cascade (Mao et al, 1982;Tam and Saier, 1993).…”

“…It is reasonable to propose that the activity of EvgS and related sensor kinases with large periplasmic domains are modulated by the binding of a periplasmic ligand or ligands, but the nature of these is not known. We therefore screened a range of small molecules (cadaverine, gamma-aminobutyric acid, glycine betaine, indole, ornithine, sarcosine, and spermine) which we considered to be potential candidates for modulators of EvgS activity, based both on consideration of the predicted EvgS periplasmic domain structure (Sen et al, 2017) and knowledge of metabolites produced by E. coli. We used a simple promoter probe to assay the effects of these molecules on induction of AR2 promoters at pH 5.5.…”