Insight into the Alternating Access Mechanism of the Sodium Symporter Mhp1 using Path Sampling Simulations

Abstract

Sodium coupled co-transporters of the LeuT superfamily use an alternating access mechanism to move small molecules across the cell membrane. A key step in the transport cycle involves the transporter undergoing a conformational transition from a state poised to bind extracellular substrates to one that is competent to deliver substrate to the cytoplasm. To investigate this step in semi-atomistic detail, we construct a coarse-grained model of the benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states. We obtain a rigorous sampling of the unbiased outward- to inward-facing transition path ensemble (TPE) using the weighted ensemble path-sampling method. Analysis of the TPE reveals two distinct sets of pathways connecting the inward- and outward-facing conformations. The first is consistent with a strict alternating access mechanism, while in the other, decoupling of the inner and outer gates transiently forms a putative permeation pathway through the transporter. Our simulations additionally show that transitions between the outward- and inward-open states result from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. This methodology provides the groundwork for more chemically detailed investigations of the transport mechanism of superfamily members using fully atomistic simulations.