P-glycoprotein is fully active after multiple tryptophan substitutions.
P-glycoprotein is fully active after multiple tryptophan substitutions.
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P-糖蛋白在多次色氨酸取代后具有完全活性。
DOI：
10.1016/j.bbamem.2012.12.005
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Urbatsch,InaL
中科院分区：
文献类型：
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作者：
Swartz,DouglasJ;Weber,Joachim;Urbatsch,InaL
P-糖蛋白（Pgp）是癌症多药耐药性的重要贡献者。 Pgp 含有十一种天然色氨酸 (Trps)，它们在直向同源物中高度保守。我们用保守取代取代了每个色氨酸，以确定哪些色氨酸对功能很重要。位于膜表面的各个Trp突变体W44R、W208Y、W132Y、W704Y和W851Y显示，酿酒酵母中Pgp诱导的针对一种或多种杀菌剂的耐药性显着降低和/或交配效率降低。位于细胞内偶联螺旋的 W158F 和 W799F 消除了交配，但保留了对大多数药物的耐药性。相比之下，NBD2中位于膜内的W228F和W311Y、位于细胞质膜界面处的W694L和W1104Y保留了与野生型Pgp相似的高水平的耐药性和交配效率。这些被组合成对（W228F/W311Y和W694L/W1104Y）和四重（W228F/W311Y/W694L/W1104Y）突变体，它们在酵母中完全活跃，并且可以纯化至均质。纯化的对和四突变体表现出药物刺激的 ATP 酶活性，其结合亲和力与野生型 Pgp 非常相似。组合突变使 Trp 荧光降低了 35%，但药物诱导的荧光猝灭与野生型 Pgp 相比没有变化，这表明几种膜结合的 Trp 对药物结合敏感。总的来说，我们得出的结论是，膜表面的色氨酸对于维持药物结合位点的完整性至关重要，而耦合螺旋中的色氨酸对于正确的域间通信很重要。我们还证明，功能性单一 Trp 突变体可以组合形成完全活性的 Pgp，保持药物多特异性，同时显着降低内在荧光。
P-glycoprotein (Pgp) is an important contributor to multidrug resistance of cancer. Pgp contains eleven native tryptophans (Trps) that are highly conserved among orthologs. We replaced each Trp by a conservative substitution to determine which Trps are important for function. Individual Trp mutants W44R, W208Y, W132Y, W704Y and W851Y, situated at the membrane surface, revealed significantly reduced Pgp induced drug resistance against one or more fungicides and/or reduced mating efficiencies in Saccharomyces cerevisiae. W158F and W799F, located in the intracellular coupling helices, abolished mating but retained resistance against most drugs. In contrast, W228F and W311Y, located within the membrane, W694L, at the cytoplasmic membrane interface, and W1104Y in NBD2 retained high levels of drug resistance and mating efficiencies similar to wild-type Pgp. Those were combined into pair (W228F/W311Y and W694L/W1104Y) and quadruple (W228F/W311Y/W694L/W1104Y) mutants that were fully active in yeast, and could be purified to homogeneity. Purified pair and quad mutants exhibited drug-stimulated ATPase activity with binding affinities very similar to wild-type Pgp. The combined mutations reduced Trp fluorescence by 35%, but drug induced fluorescence quenching was unchanged from wild-type Pgp suggesting that several membrane-bound Trps are sensitive to drug binding. Overall, we conclude that Trps at the membrane surface are critical for maintaining the integrity of the drug binding sites, while Trps in the coupling helices are important for proper interdomain communication. We also demonstrate that functional single Trp mutants can be combined to form a fully active Pgp that maintains drug polyspecificity, while significantly reducing intrinsic fluorescence.