ABCB1 p.Phe1086Ala
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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.Phe1086Ala 23733192:76:10
status: NEWX
ABCB1 p.Phe1086Ala 23733192:76:106
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.Phe1086Ala 23733192:78:68
status: NEW83 The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells.
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ABCB1 p.Phe1086Ala 23733192:83:65
status: NEW106 The F1086A NBD1 Mutation Blocks ATP-dependent Extracellular Conformation Changes between the TMDs-Models of P-gp in the open and closed conformations are shown (Fig. 3, A and B).
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ABCB1 p.Phe1086Ala 23733192:106:4
status: NEW126 To test whether replacement of Phe-1086 with a small amino acid would affect maturation, mutants F1086A or Y1087A in the wild-type background were constructed and expressed in HEK 293 cells.
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ABCB1 p.Phe1086Ala 23733192:126:97
status: NEW127 Fig. 3D shows that the Y1087A but not the F1086A mutation inhibited P-gp maturation (Fig. 3D).
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ABCB1 p.Phe1086Ala 23733192:127:42
status: NEW128 Although the F1086A mutation did not inhibit folding, it still inhibited verapamil-stimulated ATPase activity (Fig. 3C).
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ABCB1 p.Phe1086Ala 23733192:128:13
status: NEW130 To address this question, we first tested whether the F1086A mutation blocked interactions with ATP or verapamil.
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ABCB1 p.Phe1086Ala 23733192:130:54
status: NEW138 A and B, structures of P-gp in the open (A) (17) or closed (B) (24) conformations are shown. C, histidine-tagged Cys-less P-gp or mutants A266C/F1086C, A266C, and F1086C (in Cys-less background) as well as wild-type P-gp and mutant F1086A ( in wild-type background) were isolated, and ATPase activities were measured in the presence of verapamil.
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ABCB1 p.Phe1086Ala 23733192:138:232
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.Phe1086Ala 23733192:141:64
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.Phe1086Ala 23733192:146:23
status: NEWX
ABCB1 p.Phe1086Ala 23733192:146:165
status: NEW151 Accordingly, membranes prepared from cells expressing mutant F1086A/ L443C(NBD1)/S909C(IH4) were preincubated with or without ATP plus vanadate for 5 min at 37 &#b0;C. Samples were then cooled on ice, treated with copper phenanthroline, and subjected to immunoblot analysis.
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ABCB1 p.Phe1086Ala 23733192:151:61
status: NEW152 Fig. 3E shows that vanadate trapping of nucleotide blocked cross-linking in both mutants L443C(NBD1)/S909C(IH4) and L443C(NBD1)/S909C(IH4)/ F1086A.
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ABCB1 p.Phe1086Ala 23733192:152:140
status: NEW153 These results show that the F1086A mutation did not inhibit ATP hydrolysis.
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ABCB1 p.Phe1086Ala 23733192:153:28
status: NEW154 A drug rescue assay was then used to test whether the F1086A mutation disrupted verapamil interactions with the TMDs.
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ABCB1 p.Phe1086Ala 23733192:154:54
status: NEW157 Accordingly, the F1086A mutation was introduced into the P709A processing mutant.
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ABCB1 p.Phe1086Ala 23733192:157:17
status: NEW159 Expression of mutant P709A/ F1086A yielded the 150-kDa immature form of P-gp as the major product (Fig. 3F).
X
ABCB1 p.Phe1086Ala 23733192:159:28
status: NEW161 It is possible that the F1086A mutation inhibited activity by disrupting coupling between the NBDs and TMDs.
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ABCB1 p.Phe1086Ala 23733192:161:24
status: NEW162 Accordingly, cross-linking of mutant T333C/L975C was used to test the effect of F1086A on coupling.
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ABCB1 p.Phe1086Ala 23733192:162:80
status: NEW170 To determine whether the F1086A mutation affected NBD/ TMD coupling, it was introduced into mutant T333C/L975C and subjected to cross-linking.
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ABCB1 p.Phe1086Ala 23733192:170:25
status: NEW171 It was observed that the F1086A mutation inhibited ATP-dependent cross-linking of the mutant T333C/L975C (Fig. 4B).
X
ABCB1 p.Phe1086Ala 23733192:171:25
status: NEW172 These results suggest that F1086A disrupted NBD/TMD coupling.
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ABCB1 p.Phe1086Ala 23733192:172:27
status: NEW173 Replacement of Phe-1086 with Bulky Hydrophobic Amino Acids Yields Active Mutants-Mutant F1086A was inactive (Fig. 3C).
X
ABCB1 p.Phe1086Ala 23733192:173:88
status: NEW182 Replacement of Ala-266 with Hydrophobic Residues Restores Activity of Mutant F1086A-Models (Fig. 3, A and B) and cross-linking results (21) predict that Ala-266 in IH2 is close to Phe-1086 in NBD2.
X
ABCB1 p.Phe1086Ala 23733192:182:77
status: NEW183 Because a large hydrophobic amino acid might be required at the Ala-266/Phe-1086 interface for activity, we tested whether replacement of Ala-266 with the aromatic amino acids Tyr, Phe, or Trp could act as suppressor mutations to restore activity of the F1086A.
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ABCB1 p.Phe1086Ala 23733192:183:254
status: NEW184 We also predicted that replacement of Ala-266 with a small charged amino acid (Asp) would not restore activity to F1086A.
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ABCB1 p.Phe1086Ala 23733192:184:114
status: NEW185 It was found that replacement of Ala-266 with aromatic amino acids (A266Y, A266F or A266W) in the F1086A background yielded mature 170-kDa FIGURE 4.
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ABCB1 p.Phe1086Ala 23733192:185:98
status: NEW186 F1086A inhibits ATP-dependent cross-linking at the extracellular surface.
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ABCB1 p.Phe1086Ala 23733192:186:0
status: NEW187 A and B, membranes prepared from cells expressing mutant T333C/L975C (A) or T333C/L975C/F1086A (B) were treated with BMOE in the absence (None) or presence of nucleotides (ATP, AMP-PNP, or ADP).
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ABCB1 p.Phe1086Ala 23733192:187:88
status: NEW190 By contrast, mutant A266D/F1086A did not mature, although it could be rescued when expressed in the presence of cyclosporine A (Fig. 6B).
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ABCB1 p.Phe1086Ala 23733192:190:26
status: NEW192 Mutant A266D/ F1086A was first expressed in the presence of cyclosporine A to promote maturation before isolating the histidine-tagged protein.
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ABCB1 p.Phe1086Ala 23733192:192:14
status: NEW193 Fig. 6C shows that the verapamil-stimulated ATPase activities of mutants A266Y/F1086A, A266F/F1086A, and A266W/ F1086A were similar to that of wild-type P-gp.
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ABCB1 p.Phe1086Ala 23733192:193:79
status: NEWX
ABCB1 p.Phe1086Ala 23733192:193:93
status: NEWX
ABCB1 p.Phe1086Ala 23733192:193:112
status: NEW194 Mutant A266D/ F1086A showed little activity (Fig. 6C).
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ABCB1 p.Phe1086Ala 23733192:194:14
status: NEW195 The results show that the aromatic replacements to Ala-266 acted as second-site suppressor mutations to restore activity of F1086A.
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ABCB1 p.Phe1086Ala 23733192:195:124
status: NEW196 Replacement of Ala-266 with an aromatic amino acid might rescue defective TMD/NBD coupling and thereby restore activity to F1086A.
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ABCB1 p.Phe1086Ala 23733192:196:123
status: NEW197 Accordingly, the A266F mutation was introduced into mutant F1086A/T333C/L975C to test whether it affected cross-linking between the TMDs.
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ABCB1 p.Phe1086Ala 23733192:197:59
status: NEW216 Replacement of Ala-266 with aromatic residues restores F1086A activity.
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ABCB1 p.Phe1086Ala 23733192:216:55
status: NEW217 A, wild-type P-gp or A266X mutants containing the F1086A mutation were expressed in the absence of drug substrates. B, mutant A266D/F1086A was expressed in the presence (af9;) or absence (afa;) of cyclosporine A (Cyclo).
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ABCB1 p.Phe1086Ala 23733192:217:50
status: NEWX
ABCB1 p.Phe1086Ala 23733192:217:132
status: NEW219 The positions of mature (170 kDa) and immature (150 kDa) P-gps are indicated. C, ATPase activities of wild-type P-gp and F1086A mutants containing replacements to Ala-266 were measured in the presence of verapamil.
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ABCB1 p.Phe1086Ala 23733192:219:121
status: NEW221 D, membranes prepared from cells expressing mutants T333C/L975C (None), T333C/L975C/F1086A, or T333C/L975C/F1086A/A266F were treated with BMOE in the presence (af9;) or absence (afa;) of ATP.
X
ABCB1 p.Phe1086Ala 23733192:221:84
status: NEWX
ABCB1 p.Phe1086Ala 23733192:221:107
status: NEW239 In addition, activity of the F1086A mutant could be restored if Ala-266 was replaced with an aromatic amino acid.
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ABCB1 p.Phe1086Ala 23733192:239:29
status: NEW
PMID: 24275649
[PubMed]
Loo TW et al: "Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein."
No.
Sentence
Comment
201
Although we showed previously that mutation of the adjacent Phe1086 to Ala also inhibited activity (16), the activity of P-gp was much more sensitive to changes to Tyr1087 .
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ABCB1 p.Phe1086Ala 24275649:201:60
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
170
For example, the F1086A mutation abolished activity.
X
ABCB1 p.Phe1086Ala 25987565:170:17
status: NEW171 Activity of the F1086A could be restored if the opposing Ala-266 residue in IH2 was replaced with an aromatic residue (23).
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ABCB1 p.Phe1086Ala 25987565:171:16
status: NEW178 Mutants F1086A, F1086L, and F1086W yielded mature 170 kDa protein as the major product, whereas F1086R yielded little mature P-gp (b0d;5%) (Fig. 4A).
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ABCB1 p.Phe1086Ala 25987565:178:8
status: NEW185 While the F1086A and F1086R mutations FIGURE 3.
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ABCB1 p.Phe1086Ala 25987565:185:10
status: NEW