Hanson Leanne, May Leopold, Tuma Pamela, Keeven James, Mehl Patrick, Ferenz Michelle, Ambudkar Suresh V, Golin John
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
Biochemistry. 2005 Jul 19;44(28):9703-13. doi: 10.1021/bi0502994.
The yeast ABC (ATP-binding cassette protein) multidrug transporter Pdr5p transports a broad spectrum of xenobiotic compounds, including antifungal and antitumor agents. Previously, we demonstrated that substrate size is an important factor in substrate-transporter interaction and that Pdr5p has at least three substrate-binding sites. In this study, we use a combination of whole cell transport assays and photoaffinity labeling of Pdr5p with [(125)I]iodoarylazidoprazosin in purified plasma membrane vesicles to study the behavior of two series of novel substrates: trityl (triphenylmethyl) and carbazole derivatives. The results indicate that site 2, defined initially by tritylimidazole efflux, requires at least a single hydrogen bond acceptor group (electron pair donor). In contrast, complete inhibition of rhodamine 6G efflux and [(125)I]iodoarylazidoprazosin binding at site 1 requires substrates with three electronegative groups. Carbazole and trityl substrates with two groups show saturating, incomplete inhibition at this site. This type of inhibition is frequently observed in bacterial multidrug-binding proteins that use a pocket with multiple binding sites. The presence of multiple sites with different requirements for substrate-Pdr5p interaction may explain the broad specificity of xenobiotic compounds transported by this protein.
酵母ABC（ATP结合盒蛋白）多药转运蛋白Pdr5p可转运多种外源性化合物，包括抗真菌和抗肿瘤药物。此前，我们证明底物大小是底物与转运蛋白相互作用的一个重要因素，且Pdr5p至少有三个底物结合位点。在本研究中，我们结合全细胞转运分析以及在纯化的质膜囊泡中用[(125)I]碘芳基叠氮基哌唑嗪对Pdr5p进行光亲和标记，来研究两个系列新型底物：三苯甲基和咔唑衍生物的行为。结果表明，最初由三苯甲基咪唑外排定义的位点2至少需要一个氢键受体基团（电子对供体）。相比之下，若要完全抑制若丹明6G外排以及位点1处[(125)I]碘芳基叠氮基哌唑嗪的结合，则需要具有三个电负性基团的底物。具有两个基团的咔唑和三苯甲基底物在此位点表现出饱和性、不完全抑制。这种抑制类型在使用具有多个结合位点口袋的细菌多药结合蛋白中经常观察到。底物与Pdr5p相互作用时对多个具有不同要求的位点的存在，可能解释了该蛋白转运外源性化合物的广泛特异性。
