Project No. 2176
Primary Supervisor
Dr Eamonn Reading – University of Southampton
Co-Supervisor(s)
Dr Zainab Ahdash – UCB Biopharma (Industry partner)
Summary
Membrane proteins are biological macromolecules that are present within dynamic and complex cellular membranes.
| Membrane proteins are biological macromolecules that are present within dynamic and complex cellular membranes. They have diverse complex cellular functions and represent more than half of drug targets; however, the molecular mechanisms governing their modes of action remain poorly understood. Most membrane proteins are highly dynamic and undergo structural and conformational rearrangements to perform their function. Whilst Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) and Cryo-Electron Microscopy (cryo‐EM) each provide unique information, both techniques complement and validate each other providing static pictures as well as dynamics and flexibility information of large and complex systems1. This proposal focuses on developing workflows to understand the structure and conformational dynamics of membrane proteins using HDX-MS and cryo-EM.
This project will initially focus on developing the knowledge and expertise of the PhD student in both HDX-MS and cryo-EM by studying the efflux pump (MdtF). Learnings and workflow developments from studying the structure and conformational dynamics of this bacterial membrane protein using HDX-MS and cryo-EM will be utilised to study possible UCB targets. The first HDX-MS and native MS measurements of membrane proteins (including AcrB) in SMALPs2,3 have been published as well as a recent publication demonstrating the impact of a clinically relevant efflux pump mutation in AcrB using HDX-MS4. Bacterial resistance to antibiotics is a key global societal challenge and has been linked to the function of multidrug efflux transporters, which expel a broad range of structurally unrelated toxic substances out of bacteria, resulting in reduced inhibitory effects of antibiotics5. MdtEF (also known as YhiUV)-TolC is a member of the resistance nodulation cell division (RND) family of transporters. MdtEF consists of the trimeric inner membrane protein MdtF and the membrane fusion protein MdtE which connects MdtF to TolC to form a tripartite complex. The MdtF pump has been termed the anaerobic efflux pump due to increased expression and efflux in anaerobic conditions and has been found to enhance drug tolerance in anaerobically grown E. coli (an environmental signature of the mammalian gut)6-8. MdtEF efflux pump remains poorly understood and currently no high-resolution structure-function information exists. Our proposal is to determine the structure of MdtF using cryo-EM and explore its structural dynamics using HDX-MS. Complementary biophysical approaches such as circular dichroism (CD) spectroscopy will be used to gain insights into structural stability. To further understand the multidrug resistance role of MdtF we will investigate the impact of the single point mutant, V610F, which was responsible for acquired antimicrobial resistance8. There is increasing evidence that direct interactions of membrane proteins with their surrounding lipids play critical roles in regulating both the conformational dynamics and function of proteins. To capture a state which is most reflective of its cellular condition, we propose to explore MdtF within lipid nanodiscs, which contain a membrane protein within a lipid bilayer composition2,9. By bringing together conformational readouts from HDX-MS and MD predictions, we can uncover key substrate-protein and lipid–protein interactions important in regulating key functional conformations. References: [1] Engen, J. R. & Komives, E. A. Trends Biochem. Sci. 45, 906-918 (2020). [2] Reading, E. et al. Angew. Chem. Int. Ed. Engl. 56, 15654-15657 (2017). [3] Hellwig, N. et al. Chem. Commun 54, 13702-13705 (2018). [4] Reading, E., Ahdash, Z., et al. Nat. Commun. 11, 1-11 (2020). [5] Blair, J. M., Richmond, G. E. & Piddock, L. J. Future Microbiol. 9, 1165-1177 (2014). [6] Zhang, Y. et al. J. Biol. Chem. 286, 26576-26584 (2011). [7] Novoa, D. & Conroy-Ben, O. bioRxiv, 570408 (2019). [8] Bohnert, J. A., Schuster, S., Fähnrich, E., Trittler, R. & Kern, W. V. J. Antimicrob. Chemother. 59, 1216-1222 (2007). [9] Reading, E. Trends Biochem. Sci. 44, 989-990 (2019). |