Sodium coupled cotransporters of the five-helix inverted repeat (5HIR) superfamily use an alternating access mechanism to transport a myriad of small molecules across the cell membrane. One of the primary steps in this mechanism is the conformational transition from a state poised to bind extracellular substrates to a state that is competent to deliver substrate to the cytoplasm. Here, we construct a coarse-grained model of the 5HIR benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states to investigate the mechanism of this conformational change. Using the weighted ensemble path-sampling method, we rigorously sample the outward- to inward-facing transition path ensemble. The transition path ensemble reveals a heterogeneous set of pathways connecting the two states and identifies two modes of transport: one consistent with a strict alternating access mechanism and another where decoupling of the inner and outer gates causes the transient formation of a continuous permeation pathway through the transporter. We also show that the conformational switch between the outward- and inward-open states results from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. Finally, our methodology provides the groundwork for more chemically detailed investigations of the alternating mechanism.

able to (I) find evidence that the key residue-encompassing core region in the AcrB trans-membrane domain is monomer-specifically connected to bulk water by up to 3 periplasmic and 1 cytoplasmic water channels, suggesting three alternative routes of proton transfer; (II) provide evidence that contrary to the available crystal structures the outer membrane efflux duct TolC, while freely accessible from the extracellular medium, is locked only on periplasmic side in a sodium-dependent manner; (III) show that the spontaneous binding of the isolated AcrB docking domain to TolC does not induce an opening of the efflux duct within 1ms simulation time, suggesting that either a longer response time or an additional key is required to unlock the channel; (IV) provide evidence that the currently proposed model of the assembled AcrAB-TolC complex is most likely not correct proposing an alternative model that is consistent with all experimental data currently available including the crystal structure of the membrane fusion protein / inner membrane translocase complex of the homologue copper transporter CusBA.