ABCB1 p.Leu1176Cys
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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
79
One mutant, L1176C/C431, was cross-linked at 4, 21, and 37 °C.
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ABCB1 p.Leu1176Cys 12226074:79:12
status: NEW80 Mutants L1176C/S429C and L1176C/G432C were cross-linked only at 21 and 37 °C, while cross-linking of mutants L1176C/G430C, S1177C/S429C, S1177C/G430C, S1177C/ C431, S1177C/G432C, and G1179C/S429C was only observed at 37 °C.
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ABCB1 p.Leu1176Cys 12226074:80:8
status: NEWX
ABCB1 p.Leu1176Cys 12226074:80:25
status: NEWX
ABCB1 p.Leu1176Cys 12226074:80:114
status: NEW82 Cross-linking of four mutants, L1176C/ FIG. 1.
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ABCB1 p.Leu1176Cys 12226074:82:31
status: NEW92 S429C, S1177C/S429C, L1176C/C431, and S1177C/C431, was inhibited by preincubation with ATP plus vanadate (Table II).
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ABCB1 p.Leu1176Cys 12226074:92:21
status: NEW93 A representative blot of mutant L1176C/C431 is shown in Fig. 2.
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ABCB1 p.Leu1176Cys 12226074:93:32
status: NEW94 No inhibition of cross-linking by vanadate trapping was observed in mutants such as L1176C/G430C, L1176C/G432C, S1177C/G430C, or S1177C/G432C.
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ABCB1 p.Leu1176Cys 12226074:94:84
status: NEWX
ABCB1 p.Leu1176Cys 12226074:94:98
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.Leu1176Cys 12226074:105:29
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.Leu1176Cys 12226074:106:60
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.Leu1176Cys 12226074:112:88
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.Leu1176Cys 12226074:118:24
status: NEW124 Lines indicate residues cross-linked at 4 °C. TABLE II Cross-linking between residues in the NBD2 signature sequence and in the NBD1 Walker A site L1176C (53%)a S1177C (35%) G1178C (78%) G1179C (14%) Q1180C (23%) 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 S429C (38%) -b *c ** - - * - - - - - ϩ - - - G430C (12%) - - ϩϩd - - ϩ - - - - - - - - - C431 (100%) * ** **d,e - - * - - - - - - - - - G432C (0%) - ϩc ϩϩ - - ϩ - - - - - - - - - K433C (12%) - - - - - - - - - - - - - - - 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.Leu1176Cys 12226074:124:152
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
28
The other mutant (Cys431 / L1176C) contained the endogenous Cys431 in the NH2-terminal Walker A site (427 GNSGCGKS434 ) and another cysteine in the COOH-terminal 1176 LSGGQ1180 site.
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ABCB1 p.Leu1176Cys 12421806:28:27
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.Leu1176Cys 12421806:56:94
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.Leu1176Cys 12421806:63:83
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.Leu1176Cys 12421806:70:165
status: NEW73 These drug substrates were then tested on mutants L531C/Cys1074 and Cys431 / L1176C.
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ABCB1 p.Leu1176Cys 12421806:73:77
status: NEW77 Similar results were obtained with mutant Cys431 / L1176C (data not shown).
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ABCB1 p.Leu1176Cys 12421806:77:51
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.Leu1176Cys 12421806:83:101
status: NEWX
ABCB1 p.Leu1176Cys 12421806:83:114
status: NEW101 We then tested the effect of drug substrates on cross-linking of mutant Cys431 /L1176C.
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ABCB1 p.Leu1176Cys 12421806:101:80
status: NEW106 The effect of substrates on mutants L531C/Cys1074 and Cys431 /L1176C were very similar.
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ABCB1 p.Leu1176Cys 12421806:106:62
status: NEW122 Effect of drug substrates on cross-linking of mutant Cys431 /L1176C.
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ABCB1 p.Leu1176Cys 12421806:122:61
status: NEW123 Cross-linking on membranes containing P-gp mutant Cys431 /L1176C were done as described in the legend to Fig. 3.
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ABCB1 p.Leu1176Cys 12421806:123:58
status: NEW
PMID: 18035039
[PubMed]
Lawson J et al: "Structure-based interpretation of the mutagenesis database for the nucleotide binding domains of P-glycoprotein."
No.
Sentence
Comment
681
More recently, in the light of the NBD sandwich dimer, Loo et al. have demonstrated that the Walker-A cysteine can be cross-linked to a cysteine residue introduced within the opposite signature motif (L1176C, [19]), 10 Å apart (Cα-Cα, Table S1).
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ABCB1 p.Leu1176Cys 18035039:681:201
status: NEW676 More recently, in the light of the NBD sandwich dimer, Loo et al. have demonstrated that the Walker-A cysteine can be cross-linked to a cysteine residue introduced within the opposite signature motif (L1176C, [19]), 10 &#c5; apart (Cb1;-Cb1;, Table S1).
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ABCB1 p.Leu1176Cys 18035039:676:201
status: NEW