ABCMdb

ABCB1 p.Ser993Cys

Predicted by SNAP2:	A: D (59%), C: D (80%), D: D (85%), E: D (85%), F: D (85%), G: D (75%), H: D (80%), I: D (85%), K: D (91%), L: D (91%), M: D (80%), N: D (53%), P: D (91%), Q: D (80%), R: D (85%), T: D (53%), V: D (71%), W: D (91%), Y: D (85%),
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] The packing of the transmembrane segments of human... J Biol Chem. 2000 Feb 25;275(8):5253-6. Loo TW, Clarke DM
The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis.
J Biol Chem. 2000 Feb 25;275(8):5253-6., 2000-02-25 [PMID:10681495]

Abstract [show]
Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments.

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35 If the TM segments of P-gp possess enough flexibility to accommodate substrates of varying sizes, then it was reasonable to assume that residues in other TMs could be cross-linked to P350C or S993C. Login to comment
51 Printed in U.S.A. This paper is available on line at http://www.jbc.org atUniversityofNorthCarolinaatChapelHill,onSeptember29,2011www.jbc.orgDownloadedfrom ation was that P350C (TM6) and S993C (TM12) are predicted to be close to the cytoplasmic side of the membrane. Login to comment
53 Accordingly, mutants were constructed that had either P350C (TM6) and a cysteine residue introduced into predicted TMs 7 to 11, or between S993C (TM12) and a cysteine introduced into predicted TMs 1 to 5. Login to comment
58 All of the mutants, except mutant G54C/S993C, had greater than 70% of the activity of Cys-less P-gp. Login to comment
59 The level of expression of mutant G54C/S993C was adequate for cross-linking analysis, but was too low for measurement of ATPase activity. Login to comment
64 No cross-linked product was detected between S993C (TM12) and cysteines introduced into TMs 1, 2, or 3 or between P350C (TM6) and cysteines introduced into TMs 7, 8, or 9 (Table I). Login to comment
65 Twelve of the 125 P-gp mutants (TM4/TM12 constructs L227C/S993C, V231C/S993C, W232C/S993C, A233C/S993C, I235C/S993C, and L236C/S993C; TM5/TM12 constructs A295C/S993C and I299C/S993C; TM10/TM6 constructs V874C/P350C, E875C/ P350C, and M876C/P350C; and TM11/TM6 construct G939C/ P350C), however, had slower mobilities in SDS-PAGE after treatment with oxidant. Login to comment
74 Table II shows the minimum temperature required for cross-linking was 4 °C for mutants A233C(TM4)/ S993C(TM12), I235C(TM4)/S993C(TM12), L236C(TM4)/ S993C(TM12), and I299C(TM5)/S993(TM12); 21 °C for mutants V231C(TM4)/S993C(TM12), W232C(TM4)/S993C(TM12), A295C(TM5)/S993C(TM12), V874C (TM10)/P350C (TM6), M876C(TM10)/P350C(TM6), and G939C(TM11)/P350C(TM6); and 37 °C for mutants L227C (TM4)/S993C/(TM12) and E875C(TM10)/P350C(TM6). Login to comment
77 In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE. Login to comment
86 Vinblastine enhanced the cross-linking of mutant L227C(TM4)/S993C(TM12) and inhibited that of mutants I299C(TM5)/S993C (TM12), E875C(TM10)/P350C(TM6), and G939C(TM11)/P350C(TM6). Login to comment
99 Mutants L227C/S993C, V231C/ S993C, W232C/S993C, A233C/S993C, I235C/S993C, L236C/ S993C, A295C/S993C, I299C/S993C, V874C/P350C, E875C/ P350C, M876C/P350C, and G939C/P350C were inhibited by 81, 88, 90, 89, 93, 81, 78, 87, 87, 77, 70, and 78%, respectively. Login to comment
118 TABLE II Minimum temperature required for cross-linking Residues TM segments 4 °C 21 °C 37 °C L227C/S993C 4/12 -a - ϩ V231C/S993C 4/12 - ϩ ϩ W232C/S993C 4/12 - ϩ ϩ A233C/S993C 4/12 ϩb ϩ ϩ I235C/S993C 4/12 ϩ ϩ ϩ L236C/S993C 4/12 ϩ ϩ ϩ A295C/S993C 5/12 - ϩ ϩ I299C/S993C 5/12 ϩ ϩ ϩ V874C/P350C 10/6 - ϩ ϩ E875C/P350C 10/6 - - ϩ M876C/P350C 10/6 - ϩ ϩ G939C/P350C 11/6 - ϩ ϩ a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE. Login to comment

[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
51 These mutants contained P350C(TM6) and another cysteine on the cytoplasmic half of TMs 7-12 (i.e. P350C ϩ another cysteine at positions: 711-723 (TM7), 770 -783 (TM8), 828 - 840 (TM9), 867-879 (TM10), 935-947 (TM11), and 986-994 (TM12)) or S993C(TM12) with another cysteine in the cytoplasmic half of TMs 1-6 (i.e. S993C ϩ another cysteine at positions: 51-61 (TM1), 130-141 (TM2), 185-196 (TM3), 226-237 (TM4), 293-304 (TM5), and 343-351 (TM6)). Login to comment
123 In the absence of drug substrate, disulfide cross-linking occurs between Pro350 (yellow ball) and S993C (purple ball). Login to comment
134 In the absence of drug substrate, residue P350C in TM6 can be cross-linked to S993C in TM12. Login to comment

[hide] Disulfide cross-linking analysis shows that transm... J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10. Loo TW, Bartlett MC, Clarke DM
Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane.
J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10., 2004-02-27 [PMID:14670948]

Abstract [show]
Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.

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No. Sentence Comment
221 In previous cross-linking studies on the packing of the TM segments, we showed that a cysteine residue in TM6 (P350C) could be cross-linked to a cysteine at the cytoplasmic ends in TMs 10, 11, and 12, whereas a cysteine in TM12 (S993C) could be cross-linked to cysteines at the cytoplasmic ends of TMs 4, 5, and 6 (33). Login to comment

[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. Login to comment
76 Fig. 1D shows that a product with reduced mobility on SDS-PAGE gels was present when mutants F343C/M986C, G346C/G989C, and P350C/ S993C were treated with oxidant. Login to comment
77 The highest level of cross-linking occurred in mutant P350C/S993C, since most of the protein migrated with reduced mobility after treatment with oxidant (Fig. 1D, lane 14). Login to comment
100 The effect of nucleotides on cross-linking was also tested on mutants F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
103 To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine. Login to comment
105 By contrast, all the drug substrates were effective in blocking cross-linking of mutants F343C/M986C and G346C/G989C (Fig. 3, B and C), but were less effective in preventing cross-linking of mutant P350C/S993C (Fig. 3D). Login to comment
106 In mutant P350C/S993C, verapamil, cyclosporin A, and vinblastine were more effective than colchicine in inhibiting cross-linking. Login to comment
108 Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein. Login to comment
109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A). Login to comment
111 Mutant P350C/S993C was tested because it exhibited the greatest degree of cross-linking (Fig. 1D). Login to comment
112 In addition, mutant P350C/S993C had the highest activity of those mutants whose cross-linked products were sensitive to dithiothreitol. Login to comment
113 Membranes prepared from HEK 293 cells expressing Cys-less or mutant P350C/S993C P-glycoprotein- (His)10 were treated with or without 2 mM copper phenanthroline for 10 min at 37 °C. Login to comment
116 Fig. 4B (lane 4) shows that the cross-linked mutant P350C/S993C was also efficiently recovered, indicating that the histidine tag remained accessible after cross-linking. Login to comment
119 Mutant P350C/S993C, however, showed approximately 75% reduction of the verapamil-stimulated ATPase activity after cross-linking. Login to comment
121 This result suggested that dithiothreitol broke the disulfide bond between residues P350C and S993C and allowed movement to occur between TM6 and TM12 during drug-stimulated ATPase activity. Login to comment
126 Membranes prepared from HEK 293 cells expressing mutants L332C/L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 5 mM ATP, 5 mM ATP plus 0.2 mM sodium vanadate, 5 mM ADP, 5 mM ADP plus 0.2 mM sodium vanadate, or 5 mM AMP-PNP. Login to comment
132 Membranes prepared from HEK 293 cells expressing mutants L332C/ L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 1 mM verapamil, 0.1 mM vinblastine, 50 ␮M cyclosporin A, or 5 mM colchicine. Login to comment
144 Drug substrates inhibited cross-linking of mutants F343C/ M986C, G346C/G989C, and P350C/S993C. Login to comment

[hide] Identification of residues in the drug-binding sit... J Biol Chem. 1997 Dec 19;272(51):31945-8. Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PMID:9405384]

Abstract [show]
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.

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No. Sentence Comment
141 Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment

[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
204 Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules. Login to comment
207 Cross-linking of P350C to S993C severely impaired drug-stimulated ATPase activity, suggesting that the two regions of which they are components must be free to move independently for proper function. Login to comment