Na⁺-substrate coupling in the multidrug antiporter norm probed with a spin-labeled substrate.
Na⁺-substrate coupling in the multidrug antiporter norm probed with a spin-labeled substrate.
复制标题
用自旋标记底物探测多药反向转运蛋白范数中的 Na 底物偶联。
DOI：
10.1021/bi4008935
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
McHaourab,HassaneS
中科院分区：
文献类型：
--
作者：
Steed,PRyan;Stein,RichardA;Mishra,Smriti;Goodman,MichaelC;McHaourab,HassaneS
多药和毒性化合物外排（MATE）转运蛋白家族的NorM将广泛的疏水分子外排与跨细胞膜的内向Na+梯度偶联。MATE转运蛋白的几种晶体结构揭示了不同的底物结合位点，导致离子偶联底物挤出机制的不同模型。在本文报道的实验中，我们观察到柔红霉素的自旋标记衍生物Ruboxyl由来自霍乱弧菌的NorM转运。因此，它是表征与NorM的机械相关结合相互作用和直接解决离子和药物结合的耦合的理想选择。荧光和电子顺磁共振实验表明，Ruboxyl结合到NorM的微摩尔亲和力，并成为固定后结合，即使在Na+的存在下。使用双电子-电子共振光谱，我们确定Ruboxyl结合到蛋白质周质侧的单个位点。Na+的存在并没有像以前提出的那样将底物转运到第二个位点。这些实验令人惊讶地表明，Na+不影响去污剂溶解的NorM上的底物结合位点的亲和力或位置，从而表明除了结合的简单互斥性之外的其他因素，例如跨天然膜的Na+梯度的存在，在反向转运期间控制Na+-药物偶联。
NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM fromVibrio cholerae. It is therefore ideal for characterizing mechanistically relevant binding interactions with NorM and directly addressing the coupling of ion and drug binding. Fluorescence and electron paramagnetic resonance experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron–electron resonance spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+gradient across the native membrane, govern Na+–drug coupling during antiport.