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FloCEN Florence Center for Electron Nanoscopy


Friday, 20 March 2020 (postponed due to COVID-19 pandemic)

Title: TBA

Prof. Poul Nissen (Aarhus University, Denmark)




Monday, 23 March 2020 - 11:00 (postponed due to COVID-19 pandemic)

Helicobacter pylori Acid Acclimation: The Evil Duo of a pH-Gated Urea Channel and a Cytoplasmic Urease

Prof. Hartmut Leucke (Oslo University, Norway)


Helicobacter pylori’s proton-gated plasma membrane urea channel and cytoplasmic urease are essential for its survival in the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease hydrolyzes urea into NH3 and CO2, neutralizing entering protons and thus buffering the cytoplasm even in gastric juice at a pH below 2.0. We determined the crystal structure of the channel, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp153 contributes specificity. The structure suggests a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea. Follow-up microsecond-scale unrestrained molecular dynamics studies provide a detailed mechanism of urea and water transport by the channel.  More recently, we have determined the structure of the 1.1 MDa urease with a bound inhibitor to a resolution of 2.17 Å using cryo electron microscopy. Inhibitor discovery against both targets is in progress.





Monday, 23 March 2020 - 11:30 (postponed due to COVID-19 pandemic)

Cryo EM Structure of Helicobacter pylori inhibitor-bound urease at 2.3 Å resolution

Dr. Eva Cunha (Oslo University, Norway)


Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics given twice a day for 10 to 14 days. Resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore there is a clear need for more specific drugs to combat the increasing resistance. Gastric colonization by H. pylori requires the expression of a bacterial urea channel and a cytoplasmic urease and we purpose to target this acid acclimation system. The proton-gated urea channel is necessary for delivery of urea to the acidsensitive intra-bacterial urease, essential for maintaining the periplasm at pH 6.1 in the acidic environment of the stomach (as low as pH 1), thus enabling colonization of the stomach. Previously, we determined the crystal structure of the channel to 3.2 Å. Here, we report a 2.3 Å structure of the 1.1 MDa urease with bound novel inhibitor by cryo electron microscopy. Comparison with the apo form of urease shows a large movement of the flap region near the active site, highlighting its importance on the mechanism of activation/inhibition of Helicobacter pylori urease. These atomic resolution structures enable the unraveling of the mechanism of urease inhibition by elucidating different binding modes that will lead to development of more specific inhibitors and structure based rational drug design.

ultimo aggiornamento: 28-Mag-2020
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