Dynamics of SLC25A51 Reveal Preference for Oxidized NAD+ and Substrate Led Transport

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that have been attributed contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we used Molecular Dynamic simulations to reconstitute used Molecular Dynamic simulations to study reconstituted SLC25A51 homology models into lipid bilayers and generated hypotheses to test. We observed spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51’s central pore and found that mutation of these sites impaired cardiolipin binding and transporter activity. We also observed that stable formation of the required matrix gate was controlled by a single salt bridge. Using simulation data and in-cell activitybiochemical assays we We identified binding sites in SLC25A51 for NAD+ and showed that its binding and specificity selectivity for NAD+ was guided by an electrostatic interaction between NAD+ the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guided the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.