Abstract
Small multidrug resistance (SMR) transporters provide an ideal system to study the minimal requirements for active transport across a membrane. EmrE is an E. coli SMR transporter that exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. As a secondary active antiporter, EmrE drives the uphill export of each substrate molecule by coupling it to the downhill import of 2 protons across the inner membrane. EmrE is proposed to function via a single-site alternating access model. In this well-established model, transporters are inherently dynamic proteins, converting between inward- and outward-facing conformations in order to move substrate molecules across a membrane barrier.
There is general agreement that the minimal functional unit is an EmrE homodimer, but a great deal of controversy remains regarding its structure, topology, and detailed mechanism. We have used a combination of NMR and FRET experiments to directly follow the kinetics and structural changes occurring during individual steps in the transport cycle. Our results reveal that EmrE forms an antiparallel homodimer and exchanges between inward- and outward-facing states at a rate of 5 s-1 when bound to the substrate tetraphenylphosphonium. Furthermore, the inward- and outward-facing states are identical except that they have opposite orientation. These findings reconcile the controversial asymmetric EmrE crystal structure with the functional symmetry of residues in the active site and have important implications for the energetics of proton-driven coupled antiport.