Protonation-dependent conformational dynamics of the multidrug transporter EmrE
Protonation-dependent conformational dynamics of the multidrug transporter EmrE
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DOI：
10.1073/pnas.1520431113
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发表时间：
2016-02-02
影响因子：
11.1
通讯作者：
Mchaourab, Hassane S.
中科院分区：
文献类型：
--
作者：
Dastvan, Reza;Fischer, Axel W.;Mchaourab, Hassane S.
来自大肠杆菌的小的多药物转运蛋白EmrE将疏水阳离子从细胞中的大力向上挤出耦合到两个质子沿其电化学梯度的运输。虽然质子/基板反向传输的主要机械元素已被描述，结构记录仅限于基板结合状态的构象，这已被证明是经历等能交替访问。结构/机制关系中的一个核心但缺失的环节是对质子结合态的描述，这是运输循环中的一个强制性中间体。在这里，我们报告了一个系统的自旋标记和双电子电子共振（DEER）的研究，揭示了EmrE的构象变化后，质子化的关键酸性残基的配体诱导的结构重排的全球性描述的背景下。我们发现，质子化的E14导致广泛的旋转和倾斜的跨膜螺旋1-3结合重新包装的循环，构象变化，改变了结合底物的协调和调节其访问的结合位点从脂质双层。从我们的数据中出现的传输模型假定了一个质子束缚的，但闭塞的，静止状态。来自双层内小叶的底物结合释放质子并触发装载底物的转运蛋白的向内和向外构象之间的交替进入，从而使得能够反向转运而不耗散质子梯度。
The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.