Lewinson O, Bibi E
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Biochemistry. 2001 Oct 23;40(42):12612-8. doi: 10.1021/bi011040y.
The mechanism by which multidrug transporters interact with structurally unrelated substrates remains enigmatic. Based on transport competition experiments, photoaffinity labeling, and effects on enzymatic activities, it was proposed in the past that multidrug transporters can interact simultaneously with a number of dissimilar substrate molecules. To study this phenomenon, we applied a direct binding approach and transport assays using the Escherichia coli multidrug transporter MdfA, which exports both positively charged (e.g., tetraphenylphosphonium, TPP(+)), zwitterionic (e.g., ciprofloxacin), and neutral (e.g., chloramphenicol) drugs. The interaction of MdfA with various substrates was examined by direct binding assays with the purified transporter. The immobilized MdfA binds TPP(+) in a specific manner, and all the tested positively charged substrates inhibit TPP(+) binding. Surprisingly, although TPP(+) binding is not affected by zwitterionic substrates, the neutral substrate chloramphenicol stimulates TPP(+) binding by enhancing its affinity to MdfA. In contrast, transport competition assays show inhibition of TPP(+) transport by chloramphenicol. We suggest that MdfA binds TPP(+) and chloramphenicol simultaneously to distinct but interacting binding sites, and the interaction between these two substrates during transport is discussed.
多药转运蛋白与结构不相关底物相互作用的机制仍然是个谜。基于转运竞争实验、光亲和标记以及对酶活性的影响，过去有人提出多药转运蛋白可以同时与多种不同的底物分子相互作用。为了研究这一现象，我们采用了直接结合方法和转运测定，使用了大肠杆菌多药转运蛋白MdfA，它能输出带正电荷的（如四苯基鏻，TPP(+)）、两性离子的（如环丙沙星）和中性的（如氯霉素）药物。通过与纯化的转运蛋白进行直接结合测定来检查MdfA与各种底物的相互作用。固定化的MdfA以特定方式结合TPP(+)，并且所有测试的带正电荷底物都抑制TPP(+)的结合。令人惊讶的是，虽然TPP(+)的结合不受两性离子底物的影响，但中性底物氯霉素通过增强其对MdfA的亲和力来刺激TPP(+)的结合。相反，转运竞争测定显示氯霉素抑制TPP(+)的转运。我们认为MdfA同时将TPP(+)和氯霉素结合到不同但相互作用的结合位点，并讨论了这两种底物在转运过程中的相互作用。
