Ligand-Dependent Conformational Cycle of the Na+/Hydantoin Transporter Mhp1

Abstract

The LeuT Fold is characterized by a five transmembrane helix, inverted repeat structure that has been observed in an increasing number of physiologically important transporter families. The recurrence of this structural scaffold among the LeuT Fold transporters has stimulated conjectures of a unified mechanism of alternating access despite significant diversity in sequence, function, and ion coupling stoichiometries. Mhp1, a Na+-coupled symporter of the Nucleobase:Cation Symporter 1 (NCS1) family, was the first LeuT Fold member to be structurally characterized by a full complement of canonical states including outward-facing, inward-facing, and occluded conformations, which implied a rocking bundle mechanism. Here we report an investigation of Mhp1 dynamics by electron paramagnetic resonance (EPR) spectroscopy. In this analysis, double electron-electron resonance (DEER) distance distributions between pairs of spin labels monitored conformational changes in response to ligand-binding. These experimental distance distributions were directly compared with the Mhp1 crystal structures by applying MD simulations of spin label rotamers at the double mutant sites and simulating distance histograms. The results of this investigation support the assertion that the crystallographically-captured conformations of Mhp1 are reflected in solution as major conformations and that Mhp1 operates primarily through a rocking bundle-like mechanism. However, unlike LeuT, Na+ binding does not induce transitions between inward- and outward-facing conformations, rather it appears that Na+ powers transport exclusively through modulation of substrate binding affinity. The model of Mhp1 transport proposed here depends critically on the description of conformational equilibria, as the Mhp1 transport cycle progresses through states using low probability transitions. A comparative analysis of the LeuT and Mhp1 mechanisms highlight significant mechanistic divergence that we speculate may in part be explained by the differential mechanisms of coupling to the Na+ gradient.