A conserved cytoplasmic glutamic acid mediates pH-dependent gating in bacterial urea channel UreI

The bacterial UreI channel family enables rapid urea uptake, essential for urease activity and acid resistance. While pH-dependent gating in Helicobacter pylori UreI (HpUreI) is attributed to periplasmic histidines, the role of cytoplasmic residues remains unexplored. The UreI homolog from Streptococcus salivarius (SsUreI), lacking periplasmic histidines, serves as a simplified model to identify gating determinants. Here, we combine yeast complementation assays, in vitro studies, and MD simulations to show that cytoplasmic glutamic acid E136 mediates pH-dependent gating in SsUreI. Mutagenesis in H. pylori/H. hepaticus homologs confirm E136 as a conserved pH sensor across UreI channels. Protonation of E136 disrupts its salt bridge with R20, increasing cytoplasmic-loop flexibility and urea-permeable filter conformations. These findings challenge the paradigm of exclusive periplasmic pH sensing, supporting a Gram-negative-specific dual-sensor model (E136 + histidines). By elucidating this molecular mechanism, we identify E136 as a therapeutic target to disrupt UreI-mediated acid resistance in pathogenic bacteria.