The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter
The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter
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DOI：
10.1021/bi0502994
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发表时间：
2005-07-19
期刊：
影响因子：
2.9
通讯作者：
Golin, J
中科院分区：
文献类型：
--
作者：
Hanson, L;May, L;Golin, J
酵母ABC(三磷酸腺苷结合盒蛋白)多药转运体Pdr5p可转运多种异源化合物，包括抗真菌和抗肿瘤药物。此前，我们证明了底物大小是底物-转运蛋白相互作用的重要因素，并且Pdr5p至少有三个底物结合位点。在这项研究中，我们使用全细胞转运分析和光亲和标记Pdr5p与[I-125]I-芳基氮杂咪唑在纯化的质膜囊泡中相结合的方法来研究两种新型底物：三苯甲基和咔唑衍生物的行为。结果表明，最初由三咪唑外排定义的第二位点需要至少一个氢键受体基团(电子对供体)。相反，要完全抑制罗丹明6G外排和[I-125]碘代芳基氮唑结合，需要有三个电负性基团的底物。带有两个基团的咔唑和三丁基底物在该位置表现出饱和的、不完全的抑制作用。这种类型的抑制经常在使用具有多个结合位点的口袋的细菌多药物结合蛋白中观察到。对于底物-Pdr5p相互作用具有不同要求的多个位点的存在，可能解释了该蛋白运输的外源化合物的广泛特异性。
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 [I-125]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 [I-125]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.