Exploring the contributions of scaffold domain residues on ligand interactions in the Na+ /succinate transporter, VcINDY

Members of the divalent anion sodium symporter (DASS) family, known as the SLC13 transporter family in humans, are secondary active transporters responsible for translocating substrates against their concentration gradient using an electrochemical gradient. DASS transporters are essential for regulating metabolic homeostasis and provides a pathway for Krebs cycle intermediates to move into and out of living cells. Disruptions of these transporters affects energy balance and fat storage, resulting in metabolic diseases like obesity and type 2 diabetes. Using VcINDY from Vibrio cholerae, a homologue of NaCT, we have explored the effects of scaffold domain residues on ligand interactions. In this study, we employed a thermostability-based screen using differential scanning fluorimetry (DSF) assays to monitor the stability of single alanine substitutions in the presence of varying concentrations of C4 and C5-dicarboxylates. While most of the residues had minimal effects on ligand interactions according to our DSF assay data, this work revealed 2 residues that appear to play a role in the thermostability and transport activity of VcINDY. We have shown these 2 conserved residues specifically affect only some ligands and that imitating these residues reduces the maximum velocity of transport, likely impacting transport efficiency. This work has revealed new insight into the mechanism of DASS transporters which are targets for the treatment of metabolic diseases.