ABCB1 p.Leu443Cys
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PMID: 22700974
[PubMed]
Loo TW et al: "The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together."
No.
Sentence
Comment
106
The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
X
ABCB1 p.Leu443Cys 22700974:106:148
status: NEW152 The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil.
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ABCB1 p.Leu443Cys 22700974:152:7
status: NEW154 ATPase Activity Is Not Activated when the Homologous Halves Are Cross-linked at Locations Predicted Not to Undergo Large Distance Changes during the Open to Closed Conformational Change-Mutant C137/A935C contains natural Cys-137 in TM segment 2 (TMD1) and the A935C mutation in TM segment 11 (TMD2).
X
ABCB1 p.Leu443Cys 22700974:154:63
status: NEWX
ABCB1 p.Leu443Cys 22700974:154:214
status: NEW156 Mutant L443C/S909C contains a cysteine (L443) in NBD1 and a cysteine (S909C) in the ICL4 loop that connects the cytoplasmic extensions of TM segments 10 and 11.
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ABCB1 p.Leu443Cys 22700974:156:7
status: NEW158 In previous studies we showed that mutants C137/A935C (39) and L443C/ S909C (27) had verapamil-stimulated ATPase activities similar to Cys-less P-gp.
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ABCB1 p.Leu443Cys 22700974:158:63
status: NEW159 Membranes prepared from cells expressing mutants C137/ A935C or L443C/S909C were treated with M4M cross-linker.
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ABCB1 p.Leu443Cys 22700974:159:64
status: NEW163 These results indicate that stimulation of ATPase activity observed in M4M cross-linked mutants P517C/I1050C (Fig. 2B), D177C/N820C, or L175C/N820C (Fig. 3C) was likely a specific effect of trapping the protein in the closed conformation.
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ABCB1 p.Leu443Cys 22700974:163:117
status: NEW166 A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (ϩ) or without (-) the M4M cross-linker.
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ABCB1 p.Leu443Cys 22700974:166:73
status: NEW168 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutants L443C/ S909C or C137/A935C were treated with or without (None) M4M cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Leu443Cys 22700974:168:117
status: NEW287 One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle.
X
ABCB1 p.Leu443Cys 22700974:287:72
status: NEW301 Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 Å, the average distance between ␣ carbons in a disulfide bond (64).
X
ABCB1 p.Leu443Cys 22700974:301:74
status: NEW304 Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal.
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ABCB1 p.Leu443Cys 22700974:304:86
status: NEW104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
X
ABCB1 p.Leu443Cys 22700974:104:148
status: NEW161 A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (af9;) or without (afa;) the M4M cross-linker.
X
ABCB1 p.Leu443Cys 22700974:161:73
status: NEW280 One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle.
X
ABCB1 p.Leu443Cys 22700974:280:72
status: NEW294 Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 &#c5;, the average distance between ॷ carbons in a disulfide bond (64).
X
ABCB1 p.Leu443Cys 22700974:294:74
status: NEW297 Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal.
X
ABCB1 p.Leu443Cys 22700974:297:86
status: NEW
PMID: 23733192
[PubMed]
Loo TW et al: "Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface."
No.
Sentence
Comment
76
Effect of F1086A on Vanadate Trapping-The double cysteine mutant L443C/S909C was used to test whether the F1086A mutation inhibited vanadate trapping of nucleotide.
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ABCB1 p.Leu443Cys 23733192:76:65
status: NEW77 Vanadate trapping inhibits cross-linking of L443C/S909C P-gp (20).
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ABCB1 p.Leu443Cys 23733192:77:44
status: NEW78 Membranes prepared from cells expressing L443C/S909C or L443C/S909C/F1086A were incubated in the presence or absence of 5 mM MgATP plus 0.2 mM vanadate for 5 min at 37 &#b0;C. Samples were then treated with 1 mM copper phenanthroline for 15 min at 0 &#b0;C. The reactions were performed using a protein concentration of 0.4 mg/ml.
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ABCB1 p.Leu443Cys 23733192:78:41
status: NEWX
ABCB1 p.Leu443Cys 23733192:78:56
status: NEW111 By contrast, introduction of cysteine mutations at the equivalent sites between IH4 (S909C) and NBD1 (L443C) (Fig. 3, A and B) had no effect on activity (21).
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ABCB1 p.Leu443Cys 23733192:111:102
status: NEW141 E, membranes prepared from cells expressing L443C/S909C af9; F1086A were first treated with (af9;VO4) or without (afa;VO4) MgATP plus vanadate.
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ABCB1 p.Leu443Cys 23733192:141:44
status: NEW146 To test whether mutant F1086A retained the ability to bind and hydrolyze ATP, we tested whether vanadate trapping of ATP would still inhibit cross-linking of mutant F1086A/L443C/S909C.
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ABCB1 p.Leu443Cys 23733192:146:172
status: NEW
PMID: 25987565
[PubMed]
Loo TW et al: "The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein."
No.
Sentence
Comment
286
Both mutants L443(NBD1)/S909C(IH4) and A266C(IH2)/ F1086C(NBD2) could be cross-linked with copper phenanthroline but only the L443C/S909C mutant was active.
X
ABCB1 p.Leu443Cys 25987565:286:126
status: NEW