Substrate-induced conformational, changes in the transmembrane segments of human P-glycoprotein - Direct evidence for the substrate-induced fit mechanism for drug binding
Substrate-induced conformational, changes in the transmembrane segments of human P-glycoprotein - Direct evidence for the substrate-induced fit mechanism for drug binding
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DOI：
10.1074/jbc.c300073200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Clarke, DM
中科院分区：
文献类型：
--
作者：
Loo, TW;Bartlett, MC;Clarke, DM
人类多药耐药p -糖蛋白（Pgp, ABCB1）是相当混杂的，因为它可以将多种结构多样的化合物运输出细胞。我们假设构成药物结合位点的跨膜（TM）片段具有很强的移动性，因此药物结合通过“底物诱导的配合”机制发生。在这里，我们使用半胱氨酸扫描诱变和氧化交联来测试底物诱导的TM片段的变化。将半胱氨酸对引入到不含cys的P-gp中，并用氧化剂（菲罗啉铜）处理突变体，在存在或不存在各种药物、底物的情况下。我们发现环孢素A促进了残基P350C(TM6)/G939C（TM11）之间的交联，而秋水仙碱和去水仙碱促进了残基P350C(TM6)/V991C（TM12）之间的交联。孕激素提升。残基P350C(TM6)/A935C（TM11）、P350C(TM6)/G939C（TM11）以及残基P350C(TM6)/V991C（TM12）之间存在交联。其他底物如长春花碱、维拉帕米、顺式-(Z)-氟苯硫醇或反式-(E)-氟苯硫醇在这些位点上没有诱导交联。这些结果提供了直接证据，表明当P-gp与特定底物结合时，药物结合位点的TM片段的包装会发生变化。诱导拟合机制解释了P-gp如何适应广泛的化合物。
The human multidrug resistance P-glycoprotein (Pgp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug,substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted. cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds.