Elucidating molecular level details of eukaryotic nucleobase ascorbate transporter function.

All cells are surrounded by a membrane made up of lipid molecules. This membrane acts as an effective barrier separating the contents of the cell from the external environment. The lipid membrane itself is impermeable to all but a limited number of molecules, however cells need to have a means of efficiently taking up key nutrients and removing waste products. The import and export of a wide range of molecules across the membrane is mediated via a system of specialized proteins called membrane transporters, which are embedded into the lipid layer. These transporter proteins bind a specific substrate or cargo on one side of the membrane, undergo a reconfiguration and then release the substrate on the other side of the membrane. Such a mechanism has the potential to be exploited as a means of getting drugs into cells and this has made transporters the subject of significant interest by pharmaceutical companies. However, our understanding of transporter function for higher-order organisms, the eukaryotes, is currently limited. UapA is an unusually well studied eukaryotic membrane transporter responsible for import of xanthine and uric acid in the fungus Aspergillus nidulans. This fungus is particularly amenable to genetic manipulation and this has allowed extensive analysis of mutants of UapA in its native environment. Such studies have provided insights into UapA function, its location within the cell and which parts of the protein are responsible for trafficking to the correct location. However we still lack a detailed picture of how UapA works. Central to increasing understanding of the operation of UapA is a technique called X-ray crystallography which allows us to obtain very detailed information on the arrangement of the atoms within a protein structure. Obtaining such detailed structures of membrane proteins such as transporters remains very challenging as it is necessary to remove the proteins from their membrane environment into a detergent containing solution so they can be isolated and crystallised. We have made substantial progress towards obtaining a detailed structure of UapA, having isolated and crystallised two different forms of the protein and obtained some high resolution structural data. We have also shown that isolated UapA binds xanthine and forms a complex of two protein molecules, a dimer, in solution. The aim of the research outlined in this proposal is to obtain a high resolution structure of UapA by X-ray crystallography. The structure of UapA obtained will be used to guide further investigations into the roles of key regions of the protein in structure and function. We will combine the structural and mutagenic information obtained as part of this study with currently available data on UapA function in order to build up a uniquely detailed picture of how a eukaryotic membrane transporter works.

Understanding of the mechanism of action of secondary active eukaryotic transporters is limited by the lack of high resolution structures. Here we have a unique opportunity to obtain a high resolution structure of one of the best characterised eukaryotic transporters, the Nucleobase-Ascorbate transporter, UapA, from the filamentous fungus Aspergillus nidulans. UapA is responsible for the uptake of the nucleobases, xanthine and uric acid, via a H+ dependent symport mechanism. The suitability of A. nidulans as a model genetic system has been extensively exploited for the characterization of a large number of UapA mutants and has provided a detailed picture of the roles of key residues in transporter function, localization and cellular expression. However many questions remain about the precise mechanism of action of UapA. The aim of the research described in this proposal is to determine the high resolution structure of UapA. Substantial progress has been made towards this aim. We have obtained well diffracting crystals of two stabilized constructs of UapA in complex with xanthine and collected a complete high quality native dataset to 3.8 Å resolution. Using a range of strategies including detergent screening, lipidic cubic phase crystallization and crystal dehydration we aim to increase the resolution of the current crystals. We will make use of heavy atom derivative crystals in order to obtain phase information and will solve the high resolution structure of UapA by exploiting the substantial expertise of the research team in membrane protein crystallography. The structure of UapA obtained will be used to guide further investigations into the roles of key residues in structure and function of the protein. Together with the substantial body of biochemical and mutagenic data currently available and generated as part of this proposal, the high resolution structure of UapA will provide a uniquely detailed picture of the mechanism of action of a eukaryotic transporter.

The main objective of the proposal is to gain highly detailed insight into the mechanism of action of a eukaryotic transporter protein. Given that this is very much a basic science project the immediate impact of the results will be in scientific advancement in the areas of membrane transporter biology and structural biology. The research undertaken will also have significant impact through the strengthening of collaborative links between the applicants based at Imperial and Diamond as well as with our overseas collaborators in Greece. The results of our research will be disseminated to the wider scientific community through the publication of manuscripts in high impact journals and presentations at national and international meetings. Key findings will also be promoted through press releases to the media and through the Imperial College website. We will also undertake public engagement activities both at Diamond and South Kensington with the aim of making our research findings available and accessible to the general public. The PDRA funded by the proposal will benefit by developing high quality research skills in a world-class research environment as well as having access to the expertise of our collaborators. It is anticipated that they will also gain significant experience of presenting data at conferences and writing manuscripts. As a member of the Imperial College Post-doc Centre they will also gain training in more generic professional development skills including CV preparation and interview training to facilitate their next step in academia, industry or other related careers.
Analogues of the types of molecules which are transported by UapA, nucleobases, are used widely as anti-virals and anti-cancer agents however such compounds have not been exploited as anti-fungals/anti-microbial agents due to a limited understanding of the transport systems involved in uptake. In the longer term, UapA has significant potential as a drug target acting as a gateway to deliver antifungals specifically to pathogenic fungi. Since humans use a different class of purine transporters it may be possible to develop drugs that are recognized by UapA but not by the human transporters. It is too early to identify specific Pharma that might be interested in the findings of the research but the high resolution structure of UapA certainly has the potential to be exploited as a drug target.