How the surrounding environment affects cellular functions is to date poorly understood, therefore it is fundamental to explore membrane-embedded protein transporters and channels and to develop models that enable targeting specific, individual molecular mechanisms. The function of Vitamin C transporters is key for normal metabolism of all mammalian cells, and plays critical roles in cellular balance and in a variety of enzymatic pathways. Deregulation of Vitamin C transport has been associated with several human diseases. Using a multidisciplinary approach, including the powerful structural biology method of single particle cryo-electron microscopy (Cryo-EM), we target the mechanisms of human Vitamin C transporters by determining structural differences between different transporters as well as structural details of their interactions. Despite its importance, the atomic-level mechanism of how Vitamin C crosses the membrane remains unknown.
Due to the COVID-19 pandemic, the objectives of the project were adjusted and updated in a way that would allow me to be able to carry out cryo-EM high resolution structure determination. The strategy was so that instead of having as a subject Vitamin C transporters we adapted the question to another system and performed the tasks and deliverables stated in the VitC project with Helicobacter pylori urease.
With this strategy and given the circumstances I was able to collect and process high-resolution cryo-EM data that resulted in a high-impact publication of which I am first and corresponding author.
The description of the adaptation of the project to the Helicobacter pylori urease follows:
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. 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 >25% and rising and no monotherapy is effective. Gastric infection by H. pylori depends on the expression of a bacterial urea channel (HpUreI) and a cytoplasmic urease (HpUreAB) unique to this pathogen. The aim was to develop an innovative monotherapy to eradicate chronic infection by Helicobacter pylori, a widespread human pathogen. We solved the 2.0 Å resolution structure of the 1.1 MDa urease in complex with a novel inhibitor by cryo-electron microscopy and compare it to a B-mercaptoethanol-inhibited structure at 2.5 Å resolution.
Structure-based drug discovery relies on high resolution maps and on the resulting accurate 3D models. To date, the use of cryo-EM is not yet routine for this purpose as there are less than 10 structures with a resolution of 2 Å or better, and those structures are not medically relevant as they are of reference proteins such as apoferritin and B-galactosidase.
Importantly, we have indeed established structural studies using single particle cryo-EM in Norway. This is in line with the H2020 societal challenges and human health objectives.
