The Multidrug Transporter MdfA Deviates from the Canonical Model of Alternating Access of MFS Transporters.
The Multidrug Transporter MdfA Deviates from the Canonical Model of Alternating Access of MFS Transporters.
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DOI：
10.1016/j.jmb.2020.08.017
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发表时间：
2020-09-18
影响因子：
5.6
通讯作者：
Mchaourab HS
中科院分区：
文献类型：
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作者：
Yardeni EH;Mishra S;Stein RA;Bibi E;Mchaourab HS
多药转运蛋白MdfA是大肠杆菌（E.大肠杆菌流出化学上不同的底物以交换质子。与其他转运蛋白类似，MdfA据称通过交替进入中央底物结合口袋到膜的任一侧来发挥作用。因此，MdfA应该在细胞质侧和/或侧向朝向膜打开，以使药物能够进入其口袋。在周期结束时，预期周质侧打开以释放药物。MdfA的两种不同构象已被X射线晶体学捕获：向外开放（Oo）构象，由Fab片段稳定，和配体结合向内（If）构象，可能由突变（Q131 R）稳定。在这里，我们研究了这些结构如何与配体依赖的构象动力学的MdfA在脂质双层。为此，我们结合了距离测量的双电子电子共振（DEER）之间的自旋标记MdfA与半胱氨酸交联天然表达的膜嵌入的MdfA变体的纳米盘重建。我们的研究结果表明，在膜环境中，MdfA假设一个相对灵活的，向外关闭/向内关闭（OC/IC）的构象。出乎意料的是，我们的数据表明，无论是底物TPP，也没有质子化诱导大规模的构象变化。相反，我们确定了一个底物响应性的侧门，它是开放的内部小叶的膜，但关闭药物结合。总之，我们的研究结果表明，MdfA的功能构象周期的修改后的模型，不调用交替访问的典型元素。
The prototypic multidrug (Mdr) transporter MdfA from E. coli efflux chemically-dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (Oo) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (If) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distance measurements by Double Electron Electron Resonance (DEER) between pairs of spin labels in MdfA reconstituted in nanodiscs with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (Oc/Ic) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large scale conformational changes. Rather, we identified a substrate-responsive lateral gate which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access.