Simulations of the Alternating Access Mechanism of the Sodium Symporter Mhp1

Abstract

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.

Simulating the Function of the MjNhaP1 Transporter

“…In this respect, MjNhaP1 offered a great opportunity since it has structures reported for both IF and OF conformations. The transporter was said to alternate its accessibility by means of a “rocking bundle” 13 (see refs 36 and 37 and Supporting Figure S2), where at any time only one side of the membrane can access the transporter’s binding side (also see the works in refs 38 and 39). Recently this view has been challenged and the “elevator model” has been suggested instead 23 (see Supporting Figure S2).…”

Path-sampling strategies for simulating rare events in biomolecular systems

“…In studies involving enzymes, milestoning has generated ms conformational transitions between the open and closed states of the HIV reverse transcriptase [*4,55], yielding rate constants that are consistent with experiment (Figure 3a). In studies involving membrane transport proteins, the WE approach has generated pathways for outward-to-inward-facing transitions in the sodium symporter Mhp1 using coarse-grained simulations [56] and the DIMS approach has generated transitions between the cytoplasmic open conformation and perisplamic open conformation of the lactose permease transporter using atomistic simulations in implicit solvent [57]. For the related problem of ion permeation, the WE approach has enabled the calculation of current-voltage relationships for a simple model ion channel [77].…”

Computational characterization of structural dynamics underlying function in active membrane transporters

“…For instance, implicit membrane simulations (49) and coarse-grained models such as mixed elastic network models (mENM) (50), two-state anisotropic network models (51) and GŌ-like models (52) have been used to probe the OF-IF transition of transporters, often accompanied with the use of an enhanced sampling method such as dynamic importance sampling (DIMS) (53, 54), weighted ensemble (WE) path-sampling (52), and self-guided Langevin dynamics (SGLD) (49). The predicted transition pathways obtained from simplified models are sometimes converted into full-atomic representations and analyzed using all-atom unbiased MD simulations launched from the intermediates (50, 51, 53).…”

“…Using WE path-sampling and a coarse-grained representation, two transport modes for Mhp1 have been suggested: one consistent with a strict alternating access model, and another decoupling the inner and outer gates (52). The IF–OF transition of LacY has been probed by SGLD and implicit/explicit membrane simulations, in which protonation of a glutamate residue and sugar binding are found to trigger the transition toward the OF state, and sugar undergoes an orientational change during the transition (49).…”