ABCMdb

ABCB1 p.Leu531Cys

Predicted by SNAP2:	A: D (91%), C: D (80%), D: D (95%), E: D (95%), F: D (85%), G: D (95%), H: D (95%), I: D (75%), K: D (95%), M: N (93%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (91%), T: D (91%), V: D (80%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] The "LSGGQ" motif in each nucleotide-binding domai... J Biol Chem. 2002 Nov 1;277(44):41303-6. Epub 2002 Sep 10. Loo TW, Bartlett MC, Clarke DM
The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence.
J Biol Chem. 2002 Nov 1;277(44):41303-6. Epub 2002 Sep 10., 2002-11-01 [PMID:12226074]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) family of transport proteins, actively transports many cytotoxic compounds out of the cell. ABC transporters have two nucleotide-binding domains (NBD) and two transmembrane domains. The presence of the conserved "signature" sequence (LSGGQ) in each NBD is a unique feature in these transporters. The function of the signature sequences is unknown. In this study, we tested whether the signature sequences ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) in P-gp are in close proximity to the opposing Walker A consensus nucleotide-binding sequences ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1). Pairs of cysteines were introduced into a Cys-less P-gp at the signature and "Walker A" sites and the mutant P-gps were subjected to oxidative cross-linking. At 4 degrees C, when thermal motion is low, P-gp mutants (L531C(Signature)/C1074(Walker A) and C431(Walker A)/L1176C(Signature) were cross-linked. Cross-linking inhibited the drug-stimulated ATPase activities of these two mutants. Their activities were restored, however, after addition of the reducing agent, dithiothreitol. Vanadate trapping of nucleotide at the ATP-binding sites prevented cross-linking of the mutants. These results indicate that the signature sequences are adjacent to the opposing Walker A site. They likely participate in forming the ATP-binding sites and are displaced upon ATP hydrolysis. The resulting conformational change may be the signal responsible for coupling ATP hydrolysis to drug transport by inducing conformational changes in the transmembrane segments.

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No. Sentence Comment
50 The cross-linking results of three mutants, L531C/G1070C, L531C/S1072C, and L531C/C1074, are shown in Fig. 1. Login to comment
51 No cross-linked product was detected in mutant L531C/G1070C at any temperature. Login to comment
52 In mutant L531C/S1072C, no cross-linked product was detected when treated with oxidant at 4 °C. Login to comment
56 In mutant L531C/ C1074, cross-linked product was detected at all three temperatures. Login to comment
58 The complete cross-linking results between the cysteines in the NBD1 terminal signature sequence (531 LSGGQ535 ) and the cysteines in the NBD2 Walker A site (1070 GSSGCGKS1077 ) are shown in Table I. Residues G1073C and C1074 in the NBD2 Walker A site and L531C and S532C in the NBD1 signature sequence appear to be closest, since mutants L531C/G1073C, L531C/C1074, and S532C/G1073C were cross-linked when treated with oxidant at 4 °C. Login to comment
59 The cysteines in other mutants (e.g. L531C/S1072C, S532/S1072C, and G533C/G1073C) must be further apart, since cross-linked product was only observed at either 21 or 37 °C. Login to comment
66 Representative cross-linking results of two (NBD1 signature/NBD2 Walker A) mutants (L531C/G1073C and L531C/C1074) are shown in Fig. 2. Login to comment
69 Cross-linking of mutant L531C/C1074 was almost completely inhibited by vanadate trapping of nucleotide. Login to comment
70 Similar results were observed in mutants L531C/ S1071C, S532C/C1074, G533C/C1074C, and L531C/K1076C (Table I). Login to comment
71 Mutants L531C/C1074, L531C/S1071C, S532C/ C1074, G533C/C1074, and L531C/K1076C had verapamil-stimulated ATPase activities of 22, 22, 30, 90, and 11% respectively, relative to Cys-less P-gp. Login to comment
72 The other mutants (Table I) such as L531C/G1073C (Fig. 2) showed no detectable inhibition of cross-linking when preincubated with ATP plus vanadate. Login to comment
74 This appears to be the case for mutants such as L531C/G1073C that showed no inhibition of cross-linking after treatment with ATP plus vanadate. Login to comment
84 Membranes were prepared from HEK 293 cells expressing mutants L531C/G1070C, L531C/ S1072C, or L531C/C1074. Login to comment
89 CP, copper phenanthroline TABLE I Cross-linking between residues in the NBD1 signature sequence and in the NBD2 Walker A site L531C (21%)a S532C (38%) G533C (91%) G534C (4%) Q535C (0%) 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C G1070C (0%) -b - - - - - - - - - - - - - - S1071C (85%) - - *c - - - - - - - - ϩ - - ϩ S1072C (23%) - ϩc ϩϩd - ϩϩ ϩϩ - - ϩ - - ϩ - - - G1073C (4%) ϩ ϩϩ ϩϩ ϩ ϩϩ ϩϩ - ϩ ϩϩ - - - - - - C1074 (103%) ** ** **d,e - * * - - * - - - - - - G1075C (0%) - - ϩϩ - - ϩ - - - - - - - - - K1076C (12%) - - * - - - - - - - - - - - - S1077C (0%) - - - - - - - - - - - - - - - a Activity of the single cysteine mutant relative to Cys-less P-gp. b No cross-linked product detected in SDS-PAGE. c Relatively weak cross-linking (Ͻ50% of P-gp cross-linked). Login to comment
105 Two mutants, L531C/C1074 and L1176C/C431, were selected for analysis; because these residues are found at identical positions when the two halves of P-gp are aligned, both mutants can be cross-linked with oxidant at 4 °C and both retained ATPase activity (22 and 41%, respectively, relative to Cys-less P-gp). Login to comment
106 Fig. 3 shows that the activities of mutants L531C/C1074 and L1176C/C431 after treatment with oxidant were inhibited 82 and 72%, respectively. Login to comment
112 Membranes prepared from HEK 293 cells expressing mutants L531C/ G1073C, L531C/C1074, or L1176C/C431 were preincubated for 10 min at 37 °C in the presence (ϩ) or absence (-) of ATP plus vanadate. Login to comment
118 Mutants L531C/C1074 and L1176C/C431 were isolated by nickel-chelate chromatography and treated with (ϩCP) or without (-CP) oxidant, copper phenanthroline (CP), for 15 min at 21 °C. Login to comment
131 Hydrolysis of ATP was essential, since inhibition of cross-linking was not observed in the presence of the non-hydrolyzable ATP analog AMP.PNP (data not shown) or in the inactive mutants (e.g. mutant L531C/G1073C; Fig. 2). Login to comment

[hide] Drug binding in human P-glycoprotein causes confor... J Biol Chem. 2003 Jan 17;278(3):1575-8. Epub 2002 Nov 5. Loo TW, Bartlett MC, Clarke DM
Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains.
J Biol Chem. 2003 Jan 17;278(3):1575-8. Epub 2002 Nov 5., 2003-01-17 [PMID:12421806]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.

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Sentences [show]
No. Sentence Comment
27 One mutant (L531C/Cys1074 ) contained a cysteine in the NH2-terminal 531 LSGGQ535 site and an endogenous cysteine (Cys1074 ) in the COOH-terminal Walker A site (1070 GSSGCGKC1077 ). Login to comment
56 Fig. 1 shows that that cross-linking was almost complete in mutants L531C/Cys1074 and Cys431 /L1176C when treated with 0.5 mM copper phenanthroline for 15 min at 21 °C. Login to comment
63 Fig. 1 shows that inhibition of cross-linking in mutants L531C/Cys1074 and Cys431 /L1176C was observed only after treatment with vanadate plus Mg-ATP. Login to comment
70 Therefore, it is possible that the conformational changes in the TMDs may be monitored by changes in the cross-linking patterns in mutants L531C/Cys1074 and Cys431 /L1176C. Login to comment
73 These drug substrates were then tested on mutants L531C/Cys1074 and Cys431 / L1176C. Login to comment
74 Fig. 2 shows that the ATPase activity of mutant L531C/Cys1074 was stimulated by calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil (6.4-, 6.8-, 3.5-, and 4-fold, respectively). Login to comment
76 Cyclosporin A, Hoechst 33342, and trans(E)-flupentixol inhibited the activity of mutant L531C/Cys1074 with 50% inhibition occurring at concentrations of about 0.12, 0.67, and 1.1 mM, respectively. Login to comment
79 Accordingly, membranes from mutant L531C/ Cys1074 were preincubated for 10 min at 21 °C with 1 mM calcein-AM, 2 mM demecolcine, 1 mM cis(Z)-flupentixol, 0.2 mM verapamil, 0.5 mM cyclosporin A, 0.5 mM Hoechst 33342, or 1 mM trans(E)-flupentixol. Login to comment
83 Membranes were prepared from HEK 293 cells expressing mutants L531C/Cys1074 (L531C/C1074) or Cys431 /L1176C (C431/L1176C). Login to comment
90 Effect of drug substrates on the ATPase activity of P-gp mutant L531C/Cys1074 . Login to comment
91 Histidine-tagged mutant L531C/Cys1074 was isolated by nickel-chelate chromatography, mixed with E. coli lipids, and sonicated. Login to comment
96 Fig. 3 shows the effects of substrates on cross-linking of mutant L531C/Cys1074 . Login to comment
106 The effect of substrates on mutants L531C/Cys1074 and Cys431 /L1176C were very similar. Login to comment
117 Effect of drug substrate on cross-linking of mutant L531C/Cys1074 . Login to comment
118 Membranes containing P-gp mutant L531C/Cys1074 were pre-incubated with no drug, calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, or trans(E)-flupentixol and then treated with oxidant at 4 °C for the indicated times. Login to comment

[hide] Methanethiosulfonate derivatives of rhodamine and ... J Biol Chem. 2003 Dec 12;278(50):50136-41. Epub 2003 Oct 1. Loo TW, Bartlett MC, Clarke DM
Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites.
J Biol Chem. 2003 Dec 12;278(50):50136-41. Epub 2003 Oct 1., 2003-12-12 [PMID:14522974]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.

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No. Sentence Comment
61 Effect of Rhodamine on Disulfide Cross-linking Analysis-The cDNA for mutant L531C/C1074 was expressed in HEK 293 cells in the absence of cyclosporin A (16). Login to comment
136 For example, verapamil-induced conformational change can be monitored by the rate of cross-linking between a cysteine introduced into NBD1 "LSGGQ" site (L531C) and the endogenous cysteine in the NBD2 Walker A site (C1074). Login to comment
139 Rhodamine B, instead of MTS-rhodamine, was used to test the effect on cross-linking of mutant L531C/C1074 because MTS-rhodamine could potentially react with the cysteines in the NBDs. Login to comment
140 Accordingly, membranes were prepared from cells expressing mutant L531C/C1074 and incubated with or without 1 mM rhodamine B at 4 °C in the presence of oxidant (1 mM copper phenanthroline). Login to comment
143 Fig. 5 shows that rhodamine B promoted cross-linking of mutant L531C/C1074. Login to comment
172 FIG. 5. Effect of rhodamine B on cross-linking of mutant L531C/C1074. Login to comment
173 Membranes were prepared from HEK 293 cells expressing mutant L531C/C1074 and incubated with no drug substrate or with 1 mM rhodamine B (ϩ Rhodamine B) for 15 min at 4 °C. The samples were then treated with 1 mM copper phenanthroline at 4 °C for various intervals. Login to comment

[hide] The drug-binding pocket of the human multidrug res... Biochemistry. 2004 Sep 28;43(38):12081-9. Loo TW, Bartlett MC, Clarke DM
The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium.
Biochemistry. 2004 Sep 28;43(38):12081-9., 2004-09-28 [PMID:15379547]

Abstract [show]
P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates. Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the drug-binding pocket is accessible to water.

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No. Sentence Comment
197 Mutant L531C- (NBD1)/C1074C(NBD2) can be used as a control since the cysteines are in the nucleotide-binding domain (NBD) can be cross-linked (17) and should be accessible to membrane-permeant MTSEA but not to membrane-impermeant MTSES or MTSET (58). Login to comment