Loo T W, Clarke D M
Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Biol Chem. 1997 Aug 22;272(34):20986-9. doi: 10.1074/jbc.272.34.20986.
Transmembrane segments (TM) 6 and 12 are directly connected to the ATP-binding domain in each homologous half of P-glycoprotein and are postulated to be important for drug-protein interactions. Cysteines introduced into TM6 (L332C, F343C, G346C, and P350C) were oxidatively cross-linked to cysteines introduced into TM12 (L975C, M986C, G989C, and S993C, respectively). The pattern of cross-linking was consistent with a left-handed coiled coil arrangement of the two helices. To detect conformational changes between the helices during drug-stimulated ATPase activity, we tested the effects of substrates and ATP on cross-linking. Cyclosporin A, verapamil, vinblastine, and colchicine inhibited cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C. By contrast, ATP promoted cross-linking between only L332C/L975C. Enhanced cross-linking between L332C/L975C was due to ATP hydrolysis, since cross-linked product was not observed in the presence of ATP and vanadate, ADP, ADP and vanadate, or AMP-PNP. Cross-linking between P350C/S993C inhibited verapamil-stimulated ATPase activity by about 75%. Drug-stimulated ATPase activity, however, was fully restored in the presence of dithiothreitol. These results show that TM6 and TM12 undergo different conformational changes upon drug binding or during ATP hydrolysis, and that movement between these two helices is essential for drug-stimulated ATPase activity.
跨膜片段（TM）6和12直接连接到P-糖蛋白每个同源半体中的ATP结合结构域，据推测对药物与蛋白质的相互作用很重要。引入TM6的半胱氨酸（L332C、F343C、G346C和P350C）分别与引入TM12的半胱氨酸（L975C、M986C、G989C和S993C）发生氧化交联。交联模式与两个螺旋的左手卷曲螺旋排列一致。为了检测药物刺激的ATP酶活性过程中螺旋之间的构象变化，我们测试了底物和ATP对交联的影响。环孢素A、维拉帕米、长春碱和秋水仙碱抑制了突变体F343C/M986C、G346C/G989C和P350C/S993C的交联。相比之下，ATP仅促进L332C/L975C之间的交联。L332C/L975C之间交联增强是由于ATP水解，因为在ATP和钒酸盐、ADP、ADP和钒酸盐或AMP-PNP存在下未观察到交联产物。P350C/S993C之间的交联使维拉帕米刺激的ATP酶活性抑制约75%。然而，在二硫苏糖醇存在下，药物刺激的ATP酶活性完全恢复。这些结果表明，TM6和TM12在药物结合或ATP水解过程中经历不同的构象变化，并且这两个螺旋之间的移动对于药物刺激的ATP酶活性至关重要。
