PT  - JOURNAL ARTICLE
AU  - Brousseau, Merissa
AU  - Teng, Da
AU  - Thomas, Nathan E.
AU  - Voth, Gregory A.
AU  - Henzler-Wildman, Katherine A.
TI  - The C-terminus of the multi-drug efflux pump EmrE prevents proton leak by gating transport
AID  - 10.1101/2024.11.21.624706
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.11.21.624706
4099  - http://biorxiv.org/content/early/2024/11/22/2024.11.21.624706.short
4100  - http://biorxiv.org/content/early/2024/11/22/2024.11.21.624706.full
AB  - The model multi-drug efflux pump from Escherichia coli, EmrE, can perform multiple types of transport leading to different biological outcomes, conferring resistance to some drug substrates and enhancing susceptibility to others. While transporters have traditionally been classified as antiporters, symporters, or uniporters, there is growing recognition that some transporters may exhibit mixed modalities. This raises new questions about the regulation and mechanisms of these transporters. Here we show that the C-terminal tail of EmrE acts as a secondary gate, preventing proton leak in the absence of drug. Substrate binding unlocks this gate, allowing transport to proceed. Truncation of the C-terminal tail (Δ107-EmrE) leads to altered pH regulation of alternating access, an important kinetic step in the transport cycle, as measured by NMR. Δ107-EmrE has increased proton leak in proteoliposome assays and bacteria expressing this mutant have reduced growth. MD simulations of Δ107-EmrE show formation of a water wire from the open face of the transporter to the primary binding site in the core, facilitating proton leak. In WT-EmrE, the C-terminal tail forms specific interactions that block formation of the water wire. 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.Competing Interest StatementThe authors have declared no competing interest.