Mechanism of sodium-coupling in sodium-coupled transporters

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Membrane protein transporters are integral membrane proteins that enable small molecules such as nutrients, ions, neurotransmitters, and toxins to be transported across cellular membranes. They play a central role in cell physiology and their dysregulation can lead to disease. Conversely, some are potential drug targets and others aid in the delivery of drugs to cells or their action can cause multi-drug resistance.

Transporters can be classified as primary or secondary transporters. Whereas primary transporters are energized by the hydrolysis of ATP, redox reactions or light, secondary transporters harness the energy stored in electrochemical gradients across the membrane to the transport process.

Of particular interest is how the co-transport of ions in secondary transporters causes the substrate to be carried across the membrane. Indeed, this is a major question for all secondary transporters (2). For instance, ASBTNM, a bacterial homologue of the bile acid transporters ASBT and NTCP, co-transports its substrate with two sodium ions (1). Mhp1, another protein being investigated in the lab, transports only one. The project will focus on these transporters and their homologues to investigate the structural consequences of sodium-ion binding.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1782610 Studentship BB/M01116X/1 03/10/2016 31/03/2021 Aurelien Grob
 
Description The major aims of this award are to solve the crystal structures of members of the SLC10 Solute Carrier Transporter Family (notably the bacterial homologues of the SLC10A2 and SLC10A7 members). These membrane protein transporters are involved in a plethora of roles including the transport of bile acids, steroidal hormones, drugs as well as unknown solutes.
SLC10A7 is termed an "Orphan Transporter" (i.e. has no known substrate identified yet!) and as such offers a wide set of challenges both structurally and functionally. Recently, it has been shown in humans that a knockout of this gene leads to severe skeletal abnormalities leading to a stunted growth and curved spine as well as a disruption of the GAG biosynthesis and Amylogenesis Imperfecta. Working with bacterial homologues, successful attempts at expression trials, solubility trials (FSEC) and protein purifications have been completed, however progress with protein Crystallisation and hence structure determination has been hindered. Crystallisation optimisation has been extensively investigated as well as protein stability and buffer optimisation via GFP-TS, CPM and FSEC-TS assays. It was shown that an increase in buffer pH from 7.5 to 9.0 increases overall protein stability (measured by the melting temperature of the protein). However, unfortunately this still has failed to yield any successful protein crystals.

Due to the lack of Crystallisation of the SLC10A7 bacterial homologue, attention has been drawn to the SLC10A2 protein member. This membrane protein is involved in the transport of bile acids in the ileum of the small intestine of humans that get recycled back to the liver via the enterohepatic cycle. These bile acids are vital in the break down of cholesterol and solubilise fatty vitamins by acting as detergents. SLC10A2 is a core pharmaceutical target whereby inhibiting the transporter leads to lowered levels of cholesterol in mice. Significant progress has been made with this project where a 3.3 Angstrom protein crystal dataset has been obtained and current structure solving procedures are still ongoing. Molecular replacement has been successful and we are currently model building in Coot before refinement can be commenced. We aim to be able to collect a higher resolution structure shortly and carry out further functional studies in due course. Furthermore, CPM stability assays have shown great improvement in the melting temperature when coupled with bile acids indication its potential role in transport of these molecules. The addition of 1mM of Deoxycholic acid seems to increase the melting temperature of the protein by roughly 10 degrees!
If time permits, mutations in the protein as well as radio-active transport assays may be carried out.
Exploitation Route Future PhD students in the lab can continue my project and take it to new and unattained levels in the research realm. Mutations in these bacterial homologues can make them resemble as close as possible the human targets and relevant novel research can be carried out!
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description Structural Biology Public Engagement Evening 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Approximately 100 people from the general public attended a Structural Biology Public Engagement evening event organise by the School of Life Science at the University of Warwick. Multiple talks were given by professors and academics before laboratory tours were advertised to the public. I took part in the lab tour explaining the kind of research that is carried out at Warwick University: specifically within the field of Structural Biology and X-ray Crystallography. Using practical procedures, I was able to explain techniques that we use on a regular basis for protein expression and purification in a way that the public can understand. This experience was very fulfilling and very enjoyable and was a really nice environment to share your research with the public in an informal way.
Year(s) Of Engagement Activity 2018