ABCB1 p.Val991Cys
| Predicted by SNAP2: | A: N (87%), C: N (57%), D: D (80%), E: D (75%), F: D (59%), G: D (71%), H: D (75%), I: N (72%), K: D (80%), L: D (71%), M: N (53%), N: D (53%), P: D (85%), Q: D (75%), R: D (75%), S: N (72%), T: N (87%), W: D (80%), Y: D (80%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, W: D, Y: N, |
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[hide] Substrate-induced conformational changes in the tr... J Biol Chem. 2003 Apr 18;278(16):13603-6. Epub 2003 Feb 27. Loo TW, Bartlett MC, Clarke DM
Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding.
J Biol Chem. 2003 Apr 18;278(16):13603-6. Epub 2003 Feb 27., 2003-04-18 [PMID:12609990]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, 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.
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No. Sentence Comment
85 Membranes prepared from HEK 293 cells expressing mutants P350C(TM6)/ A935C(TM11), P350C(TM6)/G939C(TM11), or P350C(TM6)/V991C- (TM12) were preincubated for 10 min at 21 °C with no drug, 1 mM progesterone, 0.1 mM cyclosporin A, 5 mM colchicine, or 1 mM demecolcine.
X
ABCB1 p.Val991Cys 12609990:85:120
status: NEW92 In mutant P350C(TM6)/V991C- (TM12), the drug substrates colchicine, demecolcine, and progesterone were equally effective in promoting cross-linking.
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ABCB1 p.Val991Cys 12609990:92:21
status: NEW107 Accordingly, we tested whether the drug substrates colchicine, demecolcine, progesterone, cis-(Z)-flupenthixol, verapamil, or vinblastine stimulated the ATPase activities of histidine-tagged mutants P350C(TM6)/A935C- (TM11), P350C(TM6)/G939C(TM11), and P350C(TM6)/V991C- (TM12).
X
ABCB1 p.Val991Cys 12609990:107:264
status: NEW124 In the presence of drug substrate (progesterone), TM segments 11 and 12 undergo rotational and/or lateral movements so that cross-linking can occur between P350C and V991C (black ball) in TM12 and with A935C (red ball) and G939C (turquoise ball) in TM11.
X
ABCB1 p.Val991Cys 12609990:124:166
status: NEW135 The presence of progesterone, however, promoted cross-linking of residue P350C(TM6) with two residues in TM 11 (A935C and G939C) and to residue V991C in TM12.
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ABCB1 p.Val991Cys 12609990:135:144
status: NEW139 Therefore, the presence of drug substrate (progesterone) likely causes a slight rotation/rearrangement of TM12 relative to TM6 such that residue V991C comes closer to P350C.
X
ABCB1 p.Val991Cys 12609990:139:145
status: NEW[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
80 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.
X
ABCB1 p.Val991Cys 9261097:80:86
status: NEW