ABCB1 p.Leu531Cys
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PMID: 12226074
[PubMed]
Loo TW et al: "The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence."
No.
Sentence
Comment
50
The cross-linking results of three mutants, L531C/G1070C, L531C/S1072C, and L531C/C1074, are shown in Fig. 1.
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ABCB1 p.Leu531Cys 12226074:50:44
status: NEWX
ABCB1 p.Leu531Cys 12226074:50:58
status: NEWX
ABCB1 p.Leu531Cys 12226074:50:76
status: NEW51 No cross-linked product was detected in mutant L531C/G1070C at any temperature.
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ABCB1 p.Leu531Cys 12226074:51:47
status: NEW52 In mutant L531C/S1072C, no cross-linked product was detected when treated with oxidant at 4 °C.
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ABCB1 p.Leu531Cys 12226074:52:10
status: NEW56 In mutant L531C/ C1074, cross-linked product was detected at all three temperatures.
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ABCB1 p.Leu531Cys 12226074:56:10
status: NEW58 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.
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ABCB1 p.Leu531Cys 12226074:58:256
status: NEWX
ABCB1 p.Leu531Cys 12226074:58:339
status: NEWX
ABCB1 p.Leu531Cys 12226074:58:353
status: NEW59 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.
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ABCB1 p.Leu531Cys 12226074:59:37
status: NEW66 Representative cross-linking results of two (NBD1 signature/NBD2 Walker A) mutants (L531C/G1073C and L531C/C1074) are shown in Fig. 2.
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ABCB1 p.Leu531Cys 12226074:66:84
status: NEWX
ABCB1 p.Leu531Cys 12226074:66:101
status: NEW69 Cross-linking of mutant L531C/C1074 was almost completely inhibited by vanadate trapping of nucleotide.
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ABCB1 p.Leu531Cys 12226074:69:24
status: NEW70 Similar results were observed in mutants L531C/ S1071C, S532C/C1074, G533C/C1074C, and L531C/K1076C (Table I).
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ABCB1 p.Leu531Cys 12226074:70:41
status: NEWX
ABCB1 p.Leu531Cys 12226074:70:87
status: NEW71 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.
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ABCB1 p.Leu531Cys 12226074:71:8
status: NEWX
ABCB1 p.Leu531Cys 12226074:71:21
status: NEWX
ABCB1 p.Leu531Cys 12226074:71:66
status: NEW72 The other mutants (Table I) such as L531C/G1073C (Fig. 2) showed no detectable inhibition of cross-linking when preincubated with ATP plus vanadate.
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ABCB1 p.Leu531Cys 12226074:72:36
status: NEW74 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.
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ABCB1 p.Leu531Cys 12226074:74:48
status: NEW84 Membranes were prepared from HEK 293 cells expressing mutants L531C/G1070C, L531C/ S1072C, or L531C/C1074.
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ABCB1 p.Leu531Cys 12226074:84:62
status: NEWX
ABCB1 p.Leu531Cys 12226074:84:76
status: NEWX
ABCB1 p.Leu531Cys 12226074:84:94
status: NEW89 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).
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ABCB1 p.Leu531Cys 12226074:89:126
status: NEW105 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).
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ABCB1 p.Leu531Cys 12226074:105:13
status: NEW106 Fig. 3 shows that the activities of mutants L531C/C1074 and L1176C/C431 after treatment with oxidant were inhibited 82 and 72%, respectively.
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ABCB1 p.Leu531Cys 12226074:106:44
status: NEW112 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.
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ABCB1 p.Leu531Cys 12226074:112:57
status: NEWX
ABCB1 p.Leu531Cys 12226074:112:72
status: NEW118 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.
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ABCB1 p.Leu531Cys 12226074:118:8
status: NEW131 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).
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ABCB1 p.Leu531Cys 12226074:131:200
status: NEW
PMID: 12421806
[PubMed]
Loo TW et al: "Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains."
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 ).
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ABCB1 p.Leu531Cys 12421806:27:12
status: NEW56 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.
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ABCB1 p.Leu531Cys 12421806:56:68
status: NEW63 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.
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ABCB1 p.Leu531Cys 12421806:63:57
status: NEW70 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.
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ABCB1 p.Leu531Cys 12421806:70:139
status: NEW73 These drug substrates were then tested on mutants L531C/Cys1074 and Cys431 / L1176C.
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ABCB1 p.Leu531Cys 12421806:73:50
status: NEW74 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).
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ABCB1 p.Leu531Cys 12421806:74:48
status: NEW76 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.
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ABCB1 p.Leu531Cys 12421806:76:88
status: NEW79 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.
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ABCB1 p.Leu531Cys 12421806:79:35
status: NEW83 Membranes were prepared from HEK 293 cells expressing mutants L531C/Cys1074 (L531C/C1074) or Cys431 /L1176C (C431/L1176C).
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ABCB1 p.Leu531Cys 12421806:83:62
status: NEWX
ABCB1 p.Leu531Cys 12421806:83:77
status: NEW90 Effect of drug substrates on the ATPase activity of P-gp mutant L531C/Cys1074 .
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ABCB1 p.Leu531Cys 12421806:90:64
status: NEW91 Histidine-tagged mutant L531C/Cys1074 was isolated by nickel-chelate chromatography, mixed with E. coli lipids, and sonicated.
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ABCB1 p.Leu531Cys 12421806:91:24
status: NEW96 Fig. 3 shows the effects of substrates on cross-linking of mutant L531C/Cys1074 .
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ABCB1 p.Leu531Cys 12421806:96:66
status: NEW106 The effect of substrates on mutants L531C/Cys1074 and Cys431 /L1176C were very similar.
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ABCB1 p.Leu531Cys 12421806:106:36
status: NEW117 Effect of drug substrate on cross-linking of mutant L531C/Cys1074 .
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ABCB1 p.Leu531Cys 12421806:117:52
status: NEW118 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.
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ABCB1 p.Leu531Cys 12421806:118:33
status: NEW
PMID: 14522974
[PubMed]
Loo TW et al: "Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites."
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).
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ABCB1 p.Leu531Cys 14522974:61:76
status: NEW136 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).
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ABCB1 p.Leu531Cys 14522974:136:153
status: NEW139 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.
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ABCB1 p.Leu531Cys 14522974:139:94
status: NEW140 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).
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ABCB1 p.Leu531Cys 14522974:140:66
status: NEW143 Fig. 5 shows that rhodamine B promoted cross-linking of mutant L531C/C1074.
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ABCB1 p.Leu531Cys 14522974:143:63
status: NEW172 FIG. 5. Effect of rhodamine B on cross-linking of mutant L531C/C1074.
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ABCB1 p.Leu531Cys 14522974:172:57
status: NEW173 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.
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ABCB1 p.Leu531Cys 14522974:173:61
status: NEW
PMID: 15379547
[PubMed]
Loo TW et al: "The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium."
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).
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ABCB1 p.Leu531Cys 15379547:197:7
status: NEW