Transient formation of water-conducting states in membrane transporters

Abstract

Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane--a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mechanical coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equilibrium molecular dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.

Keywords: ABC transporters; LeuT-fold transporters; major facilitator superfamily; neurotransmitter transporters.

Fig. 1.

Water-conducting states and water transport in vSGLT. (A) Water-permeable state of vSGLT. (Upper) Overall view of the water-conducting state and the continuous water channel formed within the transporter lumen. The protein is drawn in white cartoon, with some of the helices removed for clarity. Water molecules are drawn in both sticks and surface (red) representations, and the substrate in orange van der Waals. The three gating residues controlling water flow— namely, Y87, F424, and Q428—are shown in sticks and colored by atom type. (Lower) Close-up view of the same conformation, highlighting the narrowest part of the water permeation pathway. Protein is shown in white surface, except for the three gating residues, which are in cyan surface. (B) Water-impermeable conformation of vSGLT. (Upper and Lower) Same as in A, but for a water-impermeable state formed due to side-chain motions of the gating residues (cyan surface) closing the narrowest point along the water permeation pathway. (C) Protein dynamics and water transport. The number of observed water permeation events in either direction (influx or efflux) is plotted against the simulation time in the top two panels. The third panel depicts the time series of the minimum radius of the aqueous pathway. The fourth panel shows the rmsd of gating residues and of the core transmembrane domain (10 TMs). The bottom panel shows the time evolution of the distances between the gating residues, i.e., Q428:N_ε2–F424:C_ε2, F424:C_ε2–Y87:C_ε1, and Q428:N_ε2–Y87:C_ε1. The vertical black dashed lines in C mark the snapshots shown in A and B, respectively, and the horizontal red dashed line is used to indicate the minimum radius needed to accommodate a water molecule.

Fig. 2.

Water-conducting states for Glt_ph (EAAT) of the SLC1 family (Upper) and GlpT from the MFS superfamily (Lower). (Left) Topology of the protein using the crystal structure, highlighting in different colors functionally relevant domains and repeats. Though Glt_ph is simulated as a trimer, here only the monomer in the intermediate state is depicted, with the transport and trimerization domains colored in green and purple, respectively. (Center) Representative water-conducting frame from the simulations. (Right) Time series for the number of water permeation events along the efflux and influx directions, radius of the narrowest part along of the aqueous lumen, and C_α rmsd of the TM region. The vertical black dashed lines represent the snapshots shown in the molecular images, and the horizontal red dashed lines show the minimal radius to accommodate a water molecule.

Fig. 3.

Water-conducting states for Mhp1 of the NCS1 family (Upper) and maltose transporter of the ABC superfamily (Lower). The panels are arranged in the same manner as in Fig. 2. (Left) crystal structure. (Center) Representative water-conducting state. (Right) Time series for key events. For clarity here, only the TM domains and NBDs are depicted in maltose transporter.

Fig. 4.

Formation of water-conducting states during the transport cycle. The core transport cycle of a membrane transporter (dark green region) relies on interconversion of the protein structure between major known functional states—namely, outward-facing open (OF-o), outward-facing occluded (OF-occ), inward-facing open (IF-o), and inward-facing occluded (IF-occ) states, which have been observed in various crystal structures (24, 34, 40, 41, 45). An expanded view of the transport cycle (light green area) would involve a number of additional intermediates that arise during the transition between the major states, which due to their transient nature have not yet been structurally characterized by experiments, but can account for the uncoupled water and ion transport during the transport cycle.