CLC secondary active transporters exchange Cl - for H +. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Glu ex) upon its protonation. Using 19F NMR, we show that as [H +] is increased to protonate Glu ex and enrich the outward-facing state, a residue ~20 Å away from Glu ex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
Cells have transporter proteins on their surface to carry molecules in and out of the cell. For example, the CLC family of transporters move two chloride ions in one direction at the same time as moving one hydrogen ion in the opposite direction.
To be able to move these ions in opposite directions, transporters have to cycle through a series of shapes in which the ions can only access alternate sides of the membrane. First, the transporter adopts an 'outward-facing' shape when the ions first bind to the transporter, then it switches into the 'occluded' shape to move the ions through the membrane. Finally, the transporter takes on the 'inward-facing' shape to release the ions on the other side of the membrane. However, structural studies of CLCs suggest that the structures of these proteins do not change much while they are moving ions, which suggests that they might work in a different way.
Khantwal, Abraham et al. have now used techniques called “nuclear magnetic resonance” and "double electron-electron resonance" to investigate how a CLC from a bacterium moves ions. The experiments suggest that when the transporter adopts the outward-facing shape, points on the protein known as Y419 and D417 shift their positions. Chemically linking two regions of the CLC prevented this movement and inhibited the transport of chloride ions across the membrane.
Khantwal, Abraham et al. then used a computer simulation to model how the protein changes shape in more detail. This model predicts that two regions of the transporter undergo major rearrangements resulting in a gate-opening motion that widens a passage to allow the chloride ions to bind to the protein. Khantwal, Abraham et al.’s findings will prompt future studies to reveal the other shapes and how CLCs transition between them.