Infection of the gastric mucosa by Helicobacter pylori remains a worldwide problem and contributes to peptic ulcer disease and gastric cancer. Without active intervention, at least 20% of the population of developed countries will continue to be infected by this gastric pathogen. Eradication of the organism would contribute to prevention of disease. Current eradication requires triple therapy, a proton-pump inhibitor and two antibiotics given twice a day for 10 to 14 days. Resistance to either clarithromycin or metronidazole is > 20% and rising. No monotherapy is effective. Gastric infection by H. pylori depends on the expression of a channel unique to this pathogen, HpUreI. HpUreI is a proton-gated urea channel necessary for rapid access of urea to intrabacterial urease, essential for maintaining the periplasm at pH 6.1 in the acidic environment of the stomach, as low as pH 2.5, thus allowing colonization of the stomach. Expression of the channel is increased in the stomach. The channel has 195 residues with six transmembrane segments and three periplasmic regions. Mutagenesis studies have shown that the proton gating of HpUreI is regulated by hydrogen bonding between histidines and carboxylic acid residues in these three periplasmic regions. In the first funding cycle we determined the 3-dimensional structure of this channel, and we now propose to exploit that knowledge to increase our understanding of its proton-gating mechanism and to identify binding modes of inhibitors, some of which have been discovered already. Inhibition of this channel would be expected to result in specific and effective monotherapy for eradication of the organism and usher in an era of test and treat, rather than only treating symptomatic patients. This would provide a preventive approach to serious upper gastro-intestinal diseases, particularly stomach cancer, which causes approximately 750,000 deaths annually.
Our specific aims are 1. Generate higher-resolution crystals and analyze the open and closed forms of HpUreI using new N- or C-terminal cleavable 6His- tag constructs of HpUreI. 2. Confirm the urea pathway and selectivity filter suggested by our HpUreI crystal structure by determining the effects of site-directed mutations on transport and on substrate selectivity of HpUreI using Xenopus oocyte and proteoliposome assays for urea transport as well as mutants expressed in H. pylori ureI knock-out strains. 3. Analyze the role of the hexameric arrangement by mutating residues conserved at the protomer interface of the hexamer to disrupt protomer association, followed by activity measurements of the monomeric state using Xenopus oocytes and proteoliposomes. 4. Discover and improve small molecule HpUreI inhibitors based on the structure of channel in the open form; measure their ability to increase the Tm of HpUreI, to inhibit urease activity in intact H. pylori and to affect survival in a novel gastro-mimetic incubation system.
Helicobacter pylori infects about 50% of the world's population and chronic colonization of the stomach is associated with several gastric diseases, including gastritis, peptic and duodenal ulcers, gastric carcinoma, and MALT lymphoma. The first high-resolution structure of the proton-gated urea channel, HpUreI, a validated H. pylori drug target, was determined during the first funding cycle and reveals a novel 3D structure with a unique channel architecture. This structure now provides a template for the development of inhibitors that will allow simple targeted monotherapy, allowing a test-and-treat strategy worldwide to prevent peptic ulcer disease and reduce the incidence of gastric adenocarcinoma.
|Dong, Bamboo; Sánchez-Magraner, Lissete; Luecke, Hartmut (2016) Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights. Biophys J 111:963-72|
|Kryshtafovych, Andriy; Moult, John; Bales, Patrick et al. (2014) Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10. Proteins 82 Suppl 2:26-42|
|McNulty, Reginald; Ulmschneider, Jakob P; Luecke, Hartmut et al. (2013) Mechanisms of molecular transport through the urea channel of Helicobacter pylori. Nat Commun 4:2900|
|Ozorowski, Gabriel; Milton, Saskia; Luecke, Hartmut (2013) Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2. Acta Crystallogr D Biol Crystallogr 69:92-104|
|Strugatsky, David; McNulty, Reginald; Munson, Keith et al. (2013) Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori. Nature 493:255-8|
|Wassman, Christopher D; Baronio, Roberta; Demir, Özlem et al. (2013) Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53. Nat Commun 4:1407|
|Nazmi, Ali Reza; Ozorowski, Gabriel; Pejic, Milena et al. (2012) N-terminal acetylation of annexin A2 is required for S100A10 binding. Biol Chem 393:1141-50|
|Arai, Hiromi; Glabe, Charles; Luecke, Hartmut (2012) Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers. Biochim Biophys Acta 1820:1908-14|
|Ozorowski, Gabriel; Ryan, Christopher M; Whitelegge, Julian P et al. (2012) Withaferin A binds covalently to the N-terminal domain of annexin A2. Biol Chem 393:1151-63|
|Scott, David R; Marcus, Elizabeth A; Wen, Yi et al. (2010) Cytoplasmic histidine kinase (HP0244)-regulated assembly of urease with UreI, a channel for urea and its metabolites, CO2, NH3, and NH4(+), is necessary for acid survival of Helicobacter pylori. J Bacteriol 192:94-103|