Capturing eukaryotic transporters in action

All cells are surrounded by a membrane made up of fatty 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 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 specialised 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. The ability to transport key nutrients into and out of cells is fundamental to cellular function. These inbuilt transport mechanisms also represent a potential means of direct drug delivery into cells. Substantial progress is being made in understanding the mechanism of action of individual membrane transporters in particular through structural studies, however these approaches fail to capture the full dynamic range of different protein conformations required for transport activity. Here we will use a novel technique, Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) which provides information on which regions of a protein are accessible to the external environment. These regions change as the protein changes conformations and this information can be captured as a change in the level of deuterium labelling. Thus, differences in the deuterium labelling of different forms of the protein can allow researchers to build up a picture of precisely how a protein changes its shape to performs its function. The application of HDX-MS to membrane proteins represents an emerging technology and this is particularly true with regard to membrane proteins from complex organisms. The protein we will use for these studies is a nucleobase transporter from a fungus, UapA, which is an important transporter in its own right but is also an excellent model for a large number of human and other mammalian transporters. Our groups have studied UapA for a number of years and have many tools including a range of different substrates, inhibitors and mutant forms that can be used to obtain information on the different conformations adopted by the protein. The lipid molecules that make up the membrane are known to have important effects on structure and function of many membrane proteins including UapA. We have previously identified a series of lipids important for UapA, however it is not known what effect these lipids have on the conformational state of the protein. We will use HDX-MS to interrogate the conformational mechanism of UapA in artificial lipid environments. Our research will provide a uniquely detailed picture of how UapA carries out its transport function and how this is affected by its surrounding environment. This information is also relevant to other closely related proteins and may facilitate drug discovery efforts targeting UapA and other membrane transporters.

Transporters are essential proteins mediating the movement of molecules across biological membranes and have potential as routes of drug delivery. High resolution structures have provided a large body of information about how these important molecules function. However, this information is often limited to single snapshots of individual proteins. It remains challenging to obtain information on all the different conformational states of a transporter and how these are affected by binding of substrates and co-transported ions using standard structural approaches.

Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) is a sensitive, solution-based method which can provide molecular level information on local protein structure and dynamics. HDX occurs when backbone amides are made accessible to deuterium through structure unfolding and H-bond breakage. Here, we use HDX-MS to explore the conformational dynamics of uric acid xanthine transporter, UapA, from Aspergillus nidulans. UapA is a well-studied eukaryotic transporter which, due to the availability of a wide range of tools (mutants, substrates, inhibitors) and relatedness to proteins from other transporter families, represents an excellent model system for unveiling molecular mechanisms associated with transport. We have established conditions for the successful HDX-MS analysis of UapA constructs, both wild-type and conformationally restricted mutants, following reconstitution into nanodiscs. We will explore the effects of the addition of different substrates, native and non-native, to the protein both in the presence and absence of mutations including mutations that mimic protonation. The use of nanodiscs as a membrane mimetic system will allow us to explore the impact of specific lipid compositions on the structural dynamics and function of UapA. Our approach will deliver a uniquely detailed picture of the conformational changes associated with the transport cycle of UapA and other related eukaryotic systems.