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.

来自大肠杆菌（Escherichia coli）的多药外排泵（multi-drug efflux pump）EmrE，可介导多种类型的转运过程，进而产生不同的生物学效应。传统上，转运体被归类为反向转运体（antiporters）、同向转运体（symporters）或单向转运体（uniporters），但如今学界逐渐认识到，部分转运体可能兼具多种转运模式。这为这类转运体的潜在调控机制与运作原理带来了新的科学问题。本研究证实，EmrE的C末端尾区（C-terminal tail）可作为其次级门控结构，在无药物存在时阻止质子泄漏。底物结合可解锁该门控结构，进而根据底物类型允许质子泄漏或偶联反向转运。对C末端尾区进行截短（∆107-EmrE）后，核磁共振波谱法（NMR）检测结果显示，介导分子跨膜转运的核心步骤——交替穿梭调控的pH依赖性发生了改变。固相支撑膜电生理学实验表明，在存在pH梯度的条件下，∆107-EmrE的质子泄漏水平也有所升高。针对∆107-EmrE的分子动力学（Molecular Dynamics）模拟显示，该截短体的转运体开放面可形成一条连接至核心区初级结合位点的水丝。而在野生型EmrE（WT-EmrE）中，C末端尾区会折叠覆盖至开放面，通过形成特异性相互作用改变跨膜螺旋的取向，并阻断水丝的形成。上述现象均具有pH依赖性，且与实验观测结果一致。综上，本研究数据充分支持EmrE的C末端可作为次级门控结构，调控转运体核心区初级结合位点的可及性。该结果表明，即便像EmrE这样的小型转运体，也可拥有包含多重门控结构的复杂转运机制。