Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers
Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers
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DOI：
10.1074/jbc.c100467200
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发表时间：
2001-10-05
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;Clarke, DM
人多药耐药p -糖蛋白（P-gp）与多种结构和大小不同的化合物相互作用。大量证据表明，跨膜4-6段和10-12段的残基形成了药物结合位点。我们试图通过使用含有2到17个原子间隔臂的硫醇特异性甲乙硫磺酸（MTS）交联剂来测量药物结合位点的大小。这些交联剂中的大多数也是P-gp的底物，因为它们刺激atp酶活性（2.5- 10.1倍）。将半胱氨酸残基对引入跨膜4-6段和10-12段的36个P-gp突变体与0.2 mm MTS交联剂在4℃下反应后进行了分析。交联产物在SDS凝胶中的迁移率比天然P-gp低。13个P-gp突变体用MTS交联剂交联，交联剂的间隔臂为9-25埃长春花碱，环孢素A抑制交联。从这些结果和其他研究中得出的结论是，药物结合区域足够大，可以容纳不同大小的化合物，并且药物结合区域呈“漏斗”状，在细胞质一侧狭窄，中间至少有9-25埃，在细胞外表面更宽。
The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4-6 and 10-12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4-6 and 10-12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 degreesC. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9-25 Angstrom Vinblastine and cyclosporin A inhibited crosslinking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is "funnel" shaped, narrow at the cytoplasmic side, at least 9-25 Angstrom in the middle, and wider still at the extracellular surface.