Examining the Ligand Binding, Transport Cycle, and Lipid Interactions of the DASS Family Secondary Active Transporter, VcINDY

The rising prevalence of obesity and type 2 diabetes, fuelled by an increase in human life expectancy, places a major burden upon modern healthcare systems. It has been found that these conditions can be effectively prevented in mice by the knockout of a single transport protein, named mINDY. It is therefore hoped that targeting mINDY in humans will have similar effects, providing a solution to this modern health crisis. mINDY is a member of the Divalent Anion/Na+ Symporter (DASS) protein family, and despite their promise as future drug targets, very little is known about their molecular mechanisms. In order to better understand the molecular principles behind the function of these transporters, we examined the bacterial family member known as VcINDY from Vibrio cholerae. VcINDY is the best characterised member of the DASS family and shares structural and functional features with the pharmacologically-relevant human homologues, therefore making it an excellent model to probe the underlying mechanistic principles of the wider DASS family. We applied a range of biochemical and biophysical techniques to the study of VcINDY's substrate interactions and transport cycle in order to build a more complete picture of how the DASS family achieves controlled substrate transport. This resulted in a range of novel findings. Previously unobserved intermediate transport states were uncovered through the use of single residue accessibility assays, whereby the binding of VcINDY's substrates, sodium and succinate, to the transporter each resulted in distinct conformational changes. Clear principles of both cation and anion selectivity, along with a clear order of binding, were established through the use of high throughput thermostability screening. During the course of this high throughput thermostability screening, several new and unexpected small molecule interactors were identified, which may prove useful in future DASS family inhibitor design. Binding affinities for both sodium and succinate were also established for the first time through the use of thermophoresis based binding assays. The effect of lipids upon VcINDY were also examined through a range of approaches. Aggregation based thermostability measurements demonstrated for the first time not only that VcINDY preferentially interacts with certain lipids, but that the lipid interactions of VcINDY are radically altered in the presence of substrate. This suggests that lipids may play a complex, dynamic, role in the transport cycle of DASS family transporters. In order to further examine the relationship between VcINDY and lipids, methods for the reconstitution of VcINDY into two distinct nanodisc systems were developed; a Membrane Scaffold Protein based nanodisc, and a polymer based native lipid nanodisc, allowing in vitro testing of this membrane protein within a lipid environment. The work presented in this thesis has allowed the creation of a new, far more detailed, mechanistic model for ligand binding and transport by the DASS family, in addition to developing new tools and techniques that will pave the way for further insights in the future.