Dynamics of Mitochondrial NAD+ Import Reveal Preference for Oxidized Ligand and Substrate Led Transport

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that have been attributed 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 study reconstituted SLC25A51 homology models in lipid bilayers. We observed spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51s central pore and found that mutation of these sites impaired 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 activity assays we identified binding sites in SLC25A51 for NAD+ and showed that its binding was guided by an electrostatic interaction between NAD+ 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. SignificanceNAD+ is an intermediary metabolite whose multiple functions are entwined with respiration, catabolism, and stress responses in cells. Previous sensor measurements had indicated that its continuous biosynthesis was required to sustain mitochondrial matrix levels in respiring cells, and SLC25A51 was identified as the required importer of NAD+ across the inner mitochondrial membrane. However, SLC25A51 has little homology to other nucleotide carriers at its substrate binding site. By combining modeling approaches and experimental assays, this work provides mechanistic insight into how human SLC25A51 recognizes its ligand, how the transporter can be regulated by its lipid environment, and an observation of ligand-induced gate opening. This represents the first description of the ligand binding site for an NAD+ mitochondrial carrier.