ABCB1 p.Ser979Cys
| Predicted by SNAP2: | A: D (53%), C: D (63%), D: D (75%), E: N (53%), F: N (57%), G: N (57%), H: D (75%), I: D (71%), K: D (66%), L: D (53%), M: N (66%), N: N (66%), P: D (80%), Q: D (63%), R: D (80%), T: N (82%), V: D (71%), W: D (85%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D, |
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[hide] Drug-stimulated ATPase activity of human P-glycopr... J Biol Chem. 1997 Aug 22;272(34):20986-9. Loo TW, Clarke DM
Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.
J Biol Chem. 1997 Aug 22;272(34):20986-9., 1997-08-22 [PMID:9261097]
Abstract [show]
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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No. Sentence Comment
68 To test these predictions, we introduced pairs of cysteines into a Cys-less mutant of P-glycoprotein to create the mutants F336C/S979C, L339C/V982C, F343C/M986C, G346C/G989C, and P350C/S993C.
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ABCB1 p.Ser979Cys 9261097:68:129
status: NEW78 No cross-linked product was observed for mutants F336C/S979C and L339C/V982C.
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ABCB1 p.Ser979Cys 9261097:78:55
status: NEW80 We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil.
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ABCB1 p.Ser979Cys 9261097:80:42
status: NEW107 Mutants S979C/F336C or L339C/V982C did not yield any cross-linked product even in the presence of ATP or drug substrates (data not shown).
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ABCB1 p.Ser979Cys 9261097:107:8
status: NEW124 Cross-linking was not observed between F336C/S979C or L339C/V982C, even in the presence of ATP or drug substrates FIG. 2.
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ABCB1 p.Ser979Cys 9261097:124:45
status: NEW[hide] A new structural model for P-glycoprotein. J Membr Biol. 1998 Nov 15;166(2):133-47. Jones PM, George AM
A new structural model for P-glycoprotein.
J Membr Biol. 1998 Nov 15;166(2):133-47., 1998-11-15 [PMID:9841738]
Abstract [show]
Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane alpha-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand beta-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 beta-strands and 1 alpha-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.
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No. Sentence Comment
211 In contrast, two other potential pairs that lie between the first and second of the four cross-linked pairs within TMs 6 and 12 (Loo & Clarke, 1997), namely F336C/S979C and L339C/V982C, failed to form cross-links.
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ABCB1 p.Ser979Cys 9841738:211:163
status: NEW