Partially Conserved Motifs Support an Alternating Access Model for the Human Mitochondrial NAD+ Transporter

Abstract

SLC25A51 resides in the inner mitochondrial membrane and it is required for cellular respiration and the mitochondrial production of ATP. As a member of the mitochondrial carrier family (MCF), it controls NAD+ concentrations in the mitochondrial matrix by directly importing the oxidized dinucleotide. Nevertheless, the mechanism by which SLC25A51 transports NAD+ across the inner mitochondrial membrane is not known, and its primary sequence lacks key residues that have been otherwise attributed to shared mechanisms of MCF transporters. To investigate this discrepancy, we used Molecular Dynamics simulations to characterize both the outward‐open conformation of SLC25A51 where NAD+ is expected to bind and the inward‐open conformation. We tested predictions from the simulations using a genetically‐encoded fluorescent biosensor that can directly monitor changes in free NAD+ concentrations in the mitochondrial matrix.We found that SLC25A51 lacks a three‐fold symmetry of its pore that is a characteristic of many MCF transporters. Instead, the core of SLC25A51 adopts an oblong and flattened shape. We predict this may be critical for the recognition and discrimination of the oxidized dinucleotide from other structurally related molecules, including its reduced form. A similar asymmetry was observed at the salt‐bridge network on the matrix side, produced by the asymmetric formation of a single salt‐bridge between E139 and K236 that linked helices 3 and 5. This ionic interaction was consistently observed throughout the simulations and mutation of either residue ablated SLC25A51 activity. We identified three cardiolipin binding sites on the membrane‐facing exterior of SLC25A51 that permitted cardiolipin to bridge interactions between the helical chains. We showed that these sites impact SLC25A51 function when mutated. Our simulations further predicted an energetically‐comparable single salt‐bridge on the cytoplasmic side of the transporter that could stabilize a complementary matrix‐open state. Mutation of the predicted cytoplasmic salt bridge between K198 and E291, as well as introduction of an ectopic salt bridge to imbalance the potential energetics of transport, diminished SLC25A51 activity. Together, the data indicate that although SLC25A51 lacks may conserved residues compared to other MCF family members, it functions through an alternating access mechanism and its activity depends on cardiolipin.