Elucidating the pH gating mechanism of the inner membrane urea channel UreI from Helicobacter pylori using MAVE

Around 50% of the world population suffer from chronic infection with Helicobacter pylori. The gram-negative bacterium colonizes the stomach mucosa and is a main driver for gastric diseases like gastric ulcer disease and stomach cancer. The standard treatment against H. pylori, a combination of a proton-pump-inhibitor and two antibiotics, struggles with increasing therapy failures. An alternative therapeutic toehold might pose H. pylori’s life insurance, HpUreI, a small proton-gated inner membrane channel. It is open at acidic pH and closes at neutral pH, conducting urea from the periplasm into the cytoplasm. Urea is further hydrolysed by the cytosolic urease into ammonia and carbon dioxide. Both products in turn buffer the cytosol and the periplasm, ensuring the survival of the pathogen in the acidic gastric juice.
However, even after recent cryoEM structures in the open and closed state, the mechanism of channel gating remains elusive due to a protein affinity tag comprised of 6 His residues located within the proton sensing periplasmic loop 1 (PL1), causing a shift in pH gating in oocyte measurements. Furthermore, experiments with protein chimeras combining different homologs of UreI indicate that other parts like the ransmembrane and cytosolic domains are also involved in the inactivation at neutral pH, additionally to the C-terminus, PL1 and PL2. Current approaches to explore the gating mechanism of HpUreI are limited to the WT protein using high-resolution structures or MD simulations. Experimental studies using oocytes and H.pylori as model systems tested the influence of whole domains or of predicted single amino
acid positions in the periplasmic domain of HpUreI. These approaches are very elaborate, time consuming and resource intensive though, as each mutant has to be characterized in a separate experiment.
In contrast, this project aims to explore the influence of several dozens of amino acids on HpUreIs pH gating mechanism in a single experiment. In the first set of experiments it is planned to focus on the impact of the cytosolic domains in relation to the periplasmic domains. If it is possible within the time frame of the project, the analysis will further be expanded to transmembrane regions which are involved in intra- and interprotomer interactions of the hexameric channel. Therefore, a variant library with every possible single point mutation for the amino acids in the respective regions is constructed, and the variants are tested for their functionality in a single assay. This will be carried out with multiplexed assays for variant effects (MAVE). Here the functionality is tested for the whole library in one experiment at amino acid resolution in an in-vivo system. The main steps of MAVE are the 1) construction of a variant library; 2) transformation of the variant plasmid library into cells; 3) functional assay to enrich functional proteins; 4) sequencing the cells after the functional assay to quantify each variants representation and 5) functional scoring of each variant by calibration. In the end the amino acid changes are mapped to their functionality in a heat map visualizing gain and loss of function mutations. The most crucial step in MAVE is the enrichment of gain of function mutations/variants by functional assays, which leads to a higher representation in the final variant pool. Within this project it is planned to overcome the challenge of designing/developing a selective functional assay to be able to rate the functionality of each protein mutation/variant by using yeast complementation assays. Hereby, growth or survival of yeast deletion strains is linked to the permeability of the channel/mutant of interest for different solutes. These assays are suited for the functional enrichment in MAVE and will allow us to investigate the pH gating of HpUreI for different solutes like urea, ammonia and water. After screening with MAVE, candidate variants and combinations of these, will be subjected to quantitative yeast complementation assays to perform more detailed characterization. According to our preliminary data of HpUreIs pH dependent permeability for urea and ammonia, pH gating seems to be more complex as anticipated as it differs for both solutes. Hence, the current project proposal aims
to pursue two main hypotheses: (i) Cytoplasmic regions are crucial for pH gating; (ii)
HpUreIs pH gating mechanism is solute specific. In this regard, MAVE assays selective for urea, ammonia and water will allow us to reveal essential amino acids and crucial amino acid properties for the pH gating of HpUreI. Within the framework of this project proposal, the functionality of HpUreI and transmembrane channels in general will be screened with MAVE for the first time. This type of screening assay will allow to elucidate fundamental properties of single amino acids and amino acid positions within the structural arrangement of the respective protein on protein function which would hardly be revealed with conventional approaches. We are convinced that the potential of MAVE to get a deeper understanding of selectivity, permeability or gating of transmembrane channels at amino acid resolution in only a single experiment should be started to be explored now.