pH-dependent structural dynamics/gating of HpUreI

More than half of the world`s population is infected with Helicobactor pylori, a bacterium that colonizes the gastric mucus or the gastric mucosa. In many patients the infection leads to gastritis or a duodenal ulcer. Since standard therapies have to cope with increasing antibiotic resistance, other strategies to eliminate H. pylori are essential for the future. The urea channel, HpUreI of H. pylori located in the inner of two membranes surrounding the bacterium, represents such a potential drug target. In the acidic environment of the stomach the channel opens and transports gastric urea from the compartment between the two membranes into the cell interior. There, urea is cleaved into ammonia and carbon dioxide by means of an enzyme, the urease. These two substances in turn neutralize the acidic environment thereby creating a viable microenvironment for the pathogen. Thus, the space between the two membranes serves as a buffer zone between the acidic environment of the stomach and the neutral bacterial interior as long as HpUreI channels urea in its open state. To selectively turn off the life insurance of H. pylori with drugs, it is important to understand its opening and closing behavior. In the course of the project, we will establish if HpUreI undergoes major conformational changes during opening and if channel closing involves physical occlusion of channels pore. Moreover, since HpUreI consists of six identical subunits arranged in a hexagonal shape we will investigate if channel opening occurs simultaneously in all six subunits and clarify the involvement of other parts of the protein and its surrounding on this process. To reach these goals we will examine the natural protein and different variants, based on the comparison with homologues proteins of different species, utilizing cell-based assays, high-end protein characterization techniques and molecular dynamics simulations. This will enable us to get a detailed mechanistic view of HpUreI channel opening and closing. Furthermore, we will be the first to visualize the dynamics and cooperativity of HpUreI at video rate and sub-nanometer resolution in a native-like environment using high-speed-atomic force microscopy. The project is conducted in a collaborative effort between the project leader Assoc. Prof. Dr. Andreas Horner (Johannes Kepler University Linz), co-project leader Dr. Johannes Preiner (University of Applied Sciences Upper Austria) and Dr. Daniele Narzi (University of LAquila, Italy).