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  • Title: Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.
    Author: Loo TW, Clarke DM.
    Journal: J Biol Chem; 1997 Aug 22; 272(34):20986-9. PubMed ID: 9261097.
    Abstract:
    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.
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