Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Na⁺-coupled symporters use the cellular Na⁺ gradient to power transport of physiologically important molecules across the lipid membrane. However, the mechanism by which binding and dissociation of Na⁺ drive transport remains undefined. This work investigated the Na⁺/hydantoin transporter Mhp1, a member of the LeuT-fold class of transporters, to describe the conformations sampled during its transport cycle and elucidate the ligand-induced shifts in its conformational equilibrium. The results of this study suggest that Mhp1 isomerization between inward- and outward-facing conformations are Na⁺-independent and that coupling to the Na⁺ gradient occurs through modulation of substrate affinity by Na⁺ coordination. A previously unidentified model of Mhp1 transport defined by ligand-independent equilibrium fluctuations emerges from this work, offering a new perspective on Na⁺-coupled symport in the LeuT-fold.