Bahrenberg Thorsten, Yardeni Eliane Hadas, Feintuch Akiva, Bibi Eitan, Goldfarb Daniella
Departments of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel.
Departments of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.
Biophys J. 2021 May 18;120(10):1984-1993. doi: 10.1016/j.bpj.2021.03.014. Epub 2021 Mar 24.
MdfA from Escherichia coli is a prototypical secondary multi-drug (Mdr) transporter that exchanges drugs for protons. MdfA-mediated drug efflux is driven by the proton gradient and enabled by conformational changes that accompany the recruitment of drugs and their release. In this work, we applied distance measurements by W-band double electron-electron resonance (DEER) spectroscopy to explore the binding of mito-TEMPO, a nitroxide-labeled substrate analog, to Gd(III)-labeled MdfA. The choice of Gd(III)-nitroxide DEER enabled measurements in the presence of excess of mito-TEMPO, which has a relatively low affinity to MdfA. Distance measurements between mito-TEMPO and MdfA labeled at the periplasmic edges of either of three selected transmembrane helices (TM3, TM5, and TM9) revealed rather similar distance distributions in detergent micelles (n-dodecyl-β-d-maltopyranoside, DDM)) and in lipid nanodiscs (ND). By grafting the predicted positions of the Gd(III) tag on the inward-facing (I) crystal structure, we looked for binding positions that reproduced the maxima of the distance distributions. The results show that the location of the mito-TEMPO nitroxide in DDM-solubilized or ND-reconstituted MdfA is similar (only 0.4 nm apart). In both cases, we located the nitroxide moiety near the ligand binding pocket in the I structure. However, according to the DEER-derived position, the substrate clashes with TM11, suggesting that for mito-TEMPO-bound MdfA, TM11 should move relative to the I structure. Additional DEER studies with MdfA labeled with Gd(III) at two sites revealed that TM9 also dislocates upon substrate binding. Together with our previous reports, this study demonstrates the utility of Gd(III)-Gd(III) and Gd(III)-nitroxide DEER measurements for studying the conformational behavior of transporters.
来自大肠杆菌的MdfA是一种典型的二级多药（Mdr）转运蛋白，它能将药物与质子进行交换。MdfA介导的药物外排由质子梯度驱动，并通过伴随药物募集及其释放的构象变化来实现。在这项工作中，我们应用W波段双电子-电子共振（DEER）光谱进行距离测量，以探究线粒体靶向性TEMPO（一种氮氧化物标记的底物类似物）与钆（III）标记的MdfA的结合情况。钆（III）-氮氧化物DEER的选择使得能够在过量线粒体靶向性TEMPO存在的情况下进行测量，线粒体靶向性TEMPO对MdfA的亲和力相对较低。对线粒体靶向性TEMPO与在三个选定跨膜螺旋（TM3、TM5和TM9）中任一个的周质边缘标记的MdfA之间的距离测量显示，在去污剂胶束（正十二烷基-β-D-麦芽糖苷，DDM）和脂质纳米盘（ND）中，距离分布相当相似。通过将钆（III）标签的预测位置嫁接到向内朝向（I）的晶体结构上，我们寻找能够重现距离分布最大值的结合位置。结果表明，线粒体靶向性TEMPO氮氧化物在DDM溶解或ND重构的MdfA中的位置相似（仅相差0.4纳米）。在这两种情况下，我们都将氮氧化物部分定位在I结构中的配体结合口袋附近。然而，根据DEER得出的位置，底物与TM11发生冲突，这表明对于结合了线粒体靶向性TEMPO的MdfA，TM11应相对于I结构移动。对在两个位点用钆（III）标记的MdfA进行的额外DEER研究表明，TM9在底物结合时也会发生位移。与我们之前的报告一起，这项研究证明了钆（III）-钆（III）和钆（III）-氮氧化物DEER测量在研究转运蛋白构象行为方面的实用性。
