The C-terminus of the multi-drug efflux pump EmrE prevents proton leak by gating transport

The model multi-drug efflux pump from Escherichia coli, EmrE, can perform multiple types of transport leading to different biological outcomes. While transporters have traditionally been classified as antiporters, symporters, or uniporters, there is growing recognition that some transporters may exhibit mixed transport modalities. This raises new questions about the underlying regulation and mechanisms of these transporters. Here we show that the C-terminal tail of EmrE acts as a secondary gate in EmrE, preventing proton leak in the absence of drug. Substrate binding unlocks this gate, allowing proton leak or coupled antiport, depending on the substrate. Truncation of the C-terminal tail (∆107-EmrE) leads to altered pH regulation of alternating access, the key step in moving molecules across the membrane, as measured by NMR. Solid-supported membrane electrophysiology shows that ∆107-EmrE also has increased proton leak in the presence of a pH gradient. MD simulations of ∆107-EmrE show formation of a water wire from the open face of the transporter to the primary binding site in the core. In WT-EmrE, the C-terminal tail folds over the open face, forming specific interactions that alter the orientation of the TM helices and block formation of the water wire. These phenomena are pH dependent and agree with experimental observations. Together these data strongly support the C-terminus of EmrE acting as a secondary gate that regulates access to the primary binding site in the core of the transporter. These results show that even small transporters such as EmrE can have complex transport mechanisms with multiple gates.