Structure and pH-induced structural rearrangements of the putative multidrug efflux pump EmrD in liposomes probed by site-directed spin labeling.
Structure and pH-induced structural rearrangements of the putative multidrug efflux pump EmrD in liposomes probed by site-directed spin labeling.
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DOI：
10.1021/bi4012385
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发表时间：
2013-11-12
期刊：
影响因子：
2.9
通讯作者：
Mchaourab, Hassane S.
中科院分区：
文献类型：
--
作者：
Steed, P. Ryan;Zou, Ping;Trone, Kristin E.;Mchaourab, Hassane S.
EmrD 是主要促进子超家族 (MFS) 中唯一具有结构特征的药物/H+ 逆向转运蛋白。它已以双闭塞构象结晶，该构象被认为代表了 MFS 转运蛋白转运周期中的中间状态。然而，晶体结构的意外特征以及可用于 EmrD 的功能信息的缺乏限制了结构数据的实用性。为了评估晶体结构是否代表天然环境中的稳定状态，我们使用电子顺磁共振 (EPR) 光谱来确定脂质体中重组的 EmrD 的 6 个跨膜 (TM) 螺旋中 76 个位置处的自旋标记的迁移率和可及性。虽然 EPR 数据与晶体结构基本一致，但它们也揭示了多个位置的 TM 螺旋的预测方向和拓扑结构存在显着偏差。此外，我们无法在体外和基于细胞的药物转运测定中重现 EmrD 依赖性多药耐药表型。尽管结构和功能存在差异，我们还是绘制了 pH 依赖性构象变化，其中 N 端一半的细胞质侧响应质子化而局部打开。这种构象转换与 MFS H+ 偶联反向转运蛋白的预期 pH 依赖性行为一致。
EmrD is the only structurally characterized drug/H+ antiporter of the major facilitator superfamily (MFS). It has been crystallized in a doubly-occluded conformation that is considered representative of an intermediate state in the transport cycle of MFS transporters. However, unexpected features of the crystal structure and the lack of functional information available for EmrD limit the utility of the structural data. To assess whether the crystal structure represents a stable state in a native-like environment, we used electron paramagnetic resonance (EPR) spectroscopy to determine the mobility and accessibility of spin labels at 76 positions in six transmembrane (TM) helices of EmrD reconstituted in liposomes. While the EPR data were mostly consistent with the crystal structure, they also revealed significant deviations from the predicted orientation and topology of TM helices at several locations. Additionally, we were unable to reproduce EmrD-dependent multidrug resistance phenotypes in vitro and in cell-based assays of drug transport. In spite of structural and functional discrepancies, we mapped a pH-dependent conformational change in which the cytoplasmic side of the N-terminal half opened locally in response to protonation. This conformational switch is consistent with the expected pH-dependent behavior of MFS H+-coupled antiporters.
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影响因子：
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