Investigation Into the Effects of Lipid Environment on Ligand Interactions of the Na+/Succinate Transporter VcINDY

DASS transporters play a fundamental role in cellular physiology as they are found in all domains of life, including bacteria, archaea, and eukaryotes. Modulation of these transporters can influence fat storage, metabolic homeostasis, and lifespan. Among them, VcINDY serves as a well characterised model, offering structural and functional insights relevant to human SLC13 transporters, which are implicated in metabolic disorders such as obesity and diabetes. Understanding the molecular mechanisms governing VcINDY’s stability, ligand interactions, and environmental adaptability is crucial for developing potential therapeutic modulators of these transporters. A combination of CPM-based thermal stability assays and dynamic light scattering was used to assess the impact of different substrates, lipid environments, and buffer compositions on the stability of VcINDY. Single-point alanine mutants (K117A, V118A, and K122A) were analysed to determine their role in ligand binding and stability. This study revealed that these mutations didn’t not significantly impact binding interactions. Among tested ligands, succinate exhibited the strongest stabilising effect, reinforcing VcINDY’s known preference. Furthermore, buffer composition and ionic strength were found to significantly influence VcINDY stability. High-sodium buffers improved VcINDY’s extraction efficiency into SMALPs and enhanced its thermal stability. Comparative analysis demonstrated that SMALP encapsulation provided superior stability compared to traditional detergent solubilisation, likely due to better preservation of the native lipid bilayer. The increase in Tm observed with SMALPs suggests that this solubilisation method maintains the structural integrity of VcINDY more effectively, making SMALPs a valuable tool for studying membrane transporters in near-native conditions.