Mutational Analysis of SLC25A51 Residues Important for NAD+ Binding and Transport

Mitochondrial NAD(H) is necessary for cellular respiration and generation of ATP. Apart from its role in energetics, it is also required for the activity of signaling enzymes like Sirtuins that regulate many biological processes and diseases like cancer. These enzymes depend on the spatial and temporal concentration of free NAD[H]. Recently our lab discovered that SLC25A51 is the mitochondrial NAD+ transporter and is necessary for maintaining NAD+ in the mitochondria. Despite the progress, the mechanism of NAD+ transport by SLC25A51 across the mitochondrial membrane is not known. To characterize the transporter we used a combination of MD Simulations, compartmentalized NAD+ measurement using genetically encoded fluorescent NAD+ biosensor and other biochemical methods to determine the structural characteristics of the SLC25A51 important for specific NAD+ transport. Using NAD+ biosensor we tested SLC25A51 mutants relative to the WT and measured in-situ steady-state free NAD+ in mitochondria. We determined that the salt bridge (E139-K236) also predicted to be stable by MD simulations is necessary for the NAD+ import. We also observed in unbiased atomistic simulations that three Cardiolipin molecules spontaneously bind to SLC25A51 indicating a regulatory role of the lipid. We validated the binding poses of NAD+ in SLC25A51 predicted using molecular docking by mutating the residues in the substrate binding site. Many of the conserved binding site residues but not all were required for maintaining steady state NAD+ in mitochondria. The binding site mutations were used to identify the correct binding model from our top candidates of molecular docking. The NAD+ Transporter may represent an important missing mechanistic link for how risk factors such as age, inflammation, and diet influence progression of specific pathologies including cancer, metabolic and neurodegenerative diseases. Our structural characterization of the transporter is an important first step towards understanding its mechanism and substrate specificity of the mitochondrial NAD+ transporter