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首页> 外文期刊>Biochemistry >Proximity of the nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface: A resonance energy transfer study
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Proximity of the nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface: A resonance energy transfer study

机译：P-糖蛋白多药转运蛋白核苷酸结合结构域与膜表面的邻近性：共振能转移研究

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摘要

Very little structural information is available for P-glycoprotein (Pgp), which has been implicated in the multidrug resistance of human tumors because of its ability to act as an ATP-driven efflux pump for hydrophobic compounds. Highly purified Pgp has been labeled on two cysteine residues with the fluorescence probe NBD-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). We show that NBD labels the same cysteine residues as MIANS [2-(4-maleimidoanilino)naphthalene-6-sulfonic acid]; they are located within the Walker A motif of the nucleotide binding domain, close to the site where ATP binds. NBD-and MIANS-labeled Pgps were reconstituted by detergent dilution into phospholipid vesicles containing increasing mole fractions of rhodamine- or NBD-labeled phosphatidylethanolamine (PE), respectivery. The fluorescence of the NBD-Pgp and MIANS-Pgp donors was quenched in a concentration-dependent manner by the rhodamine-PE and NBD-PE accepters. Using two different methods to analyze Forster resonance energy transfer, the distance of the Pgp-bound probes from the lipid-water interfacial region of the bilayer was estimated to be 31-35 Angstrom. This distance is compatible with the low-resolution structure of Pgp determined by electron microscopy, and indicates that the nucleotide binding domains lie close to the membrane surface. The experimental data fitted very well to theoretical quench curves for a single protein-bound fluor, suggesting that the two nucleotide binding domains are located equidistant from the bilayer. Following the addition of ATP to MIANS-Pgp, the NBD-PE quench curve no longer conformed to the models. These results imply that Pgp interacts differently with PE when it is in the ATP-bound form. [References: 42]
机译：关于P-糖蛋白（Pgp）的结构信息很少，由于P-糖蛋白具有作为ATP驱动的疏水化合物外排泵的功能，因而与人肿瘤的多药耐药性有关。用荧光探针NBD-Cl（7-氯-4-硝基-2,1,3-苯并恶二唑）在两个半胱氨酸残基上标记了高度纯化的Pgp。我们显示NBD标记与MIANS [2-（4-maleimidoanilino）萘-6磺酸]相同的半胱氨酸残基;它们位于核苷酸结合结构域的Walker A基序内，靠近ATP结合的位点。通过去污剂稀释将NBD和MIANS标记的Pgps重构为分别含有罗丹明或NBD标记的磷脂酰乙醇胺（PE）摩尔分数增加的磷脂囊泡。 NBD-Pgp和MIANS-Pgp供体的荧光被罗丹明-PE和NBD-PE受体以浓度依赖的方式猝灭。使用两种不同的方法分析Forster共振能量转移，结合Pgp的探针与双层脂质-水界面区域的距离估计为31-35埃。该距离与通过电子显微镜确定的Pgp的低分辨率结构是相容的，并且表明核苷酸结合结构域位于膜表面附近。实验数据与单个蛋白结合的氟的理论猝灭曲线非常吻合，表明两个核苷酸结合结构域与双层的距离相等。在MIANS-Pgp中添加ATP之后，NBD-PE淬灭曲线不再符合模型。这些结果表明，当Pgp呈ATP结合形式时，它与PE的相互作用不同。 [参考：42]

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