Elucidating the pH gating mechanism of Helicobacter pyloris urea channel UreI.

Abstract

Over 50% of the world population suffer from chronic gastric infection with Helicobacter pyloris which is linked to peptic ulcer disease and stomach cancer. Yet, the efficacy of the common therapy including antibiotics is decreasing. An alternative drug target could be H. pyloris pH gatedinner-membrane urea channel UreI, which is pivotal for the survival of the pathogen in the acidic environment of the human stomach. However, despite in vivo studies and high-resolution structures in the open and closed state, HpUreIs gating mechanism is still elusive. Using yeast complementation assays, we tested (i) homologues urea channels, (ii) point mutations of charged residues also on the cytoplasmic side, and (iii) variants carrying changes at the periplasmic side at the N- and C-terminus as well as in periplasmic loop 1 (PL1). The functionality and pH gating behaviour of these constructs are compared to the wild type protein in the physiological relevant pH range from 4.0 to 7.0. Our in vivo studies are guided and accompanied by an extensive sequence- and structure-based analysis of homologues urea channels. The results question the hypothesis of PL1 and PL2 constituting the main pH-sensor of HpUreI but confirm the importance of the N- and C-terminus in the pH gating mechanism. For example, additional amino acids at the C-terminus provoke a urea impermeable HpUreI, emphasizing its importance for the stability of the open state. Furthermore, the urea channels from Helicobacter hepaticus and Streptococcus salivarius, lacking such extensive periplasmic loops, show similar pH dependence as HpUreI. Overall, yeast complementation assays are an cost-effective tool for testing qualitative differences in protein variant activity for a broad range of solutes.