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Structural studies of the full-length human Vitamin C transporters: unravelling Vitamin C transport across the membrane

Periodic Reporting for period 1 - VitC (Structural studies of the full-length human Vitamin C transporters: unravelling Vitamin C transport across the membrane)

Reporting period: 2018-04-01 to 2020-03-31

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.
I have established that SVCTs are more stable at lower salt concentrations and when reconstituted to nanodiscs. This stability increase is indicative of a favorable lipid environment relative to detergent purified SVCTs.
With the adaptation to urease I have achieved high resolution of the urease bound to two different inhibitors BME and SHA, providing two structural snapshots previously non-existent for Helicobacter pylori urease that helps in establishing the mechanism of inhibition.
I wrote and submitted the grant for the Research Council of Norway and with the attribution of the Young Talent Grant I will be able to progress in my career as an independent researcher and continue to pursue the objectives of SVCTs functional and structural characterization along with drug development. Furthermore, I have participated in several conferences, workshops, teaching and outreach activities during the time of the fellowship.
I have received multidisciplinary training in transporter biology and functional characterization and have established a protocol for reaching negative staining stage of SVCTs, however further optimization is needed for successful cryo-grid preparation and high-resolution structural determination. The high-resolution structural characterization was done with Helicobacter pylori urease and the results have been published. Both of these subjects have the potential to impact society by improving human health, a goal in line with H2020 objectives. In December 2020 I was awarded the Young Talent Grant (800 000 euros distributed over 4 years) from the Research Council of Norway, that will allow me to jump start my own independent group while continuing to pursue the VitC objectives, leveraging the investment made by the H2020 programme on myself and the VitC project.

Dr. Luecke’s laboratory is now fully setup for single particle cryo-EM sample preparation and data processing, something that was not in place until I joined the laboratory. I have also transmitted the knowledge on molecular and biophysical methods to study protein-protein interactions. This project has therefore had an impact on the scientific community at UiO and in Norway as the first cryo-EM structure that was prepared and processed in the country, bridging knowledge between scientific communities.
Cryo-EM structure of Helicobacter pylori urease at 2.0 Å resolution