ABCB1 p.Ile1050Cys

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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
10 It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold.
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ABCB1 p.Ile1050Cys 22700974:10:95
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93 To address whether trapping the NBDs in close proximity (closed conformation) would activate or inhibit ATPase activity, cysteines were introduced at locations in the central part of NBD1 (P517C) and the N-terminal region of NBD2 (I1050C) of Cys-less P-gp predicted to be outside the catalytic sites but close to the LSGGQ (residues 531-535) and Walker A sites (residues 1070-1077) of NBD1 and NBD2, respectively.
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ABCB1 p.Ile1050Cys 22700974:93:35
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ABCB1 p.Ile1050Cys 22700974:93:231
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95 In the open conformation P517C and I1050C are predicted to be far apart (22.9 Å; measured from the ␣ carbons) but close (7.8 Å) in the closed conformation.
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ABCB1 p.Ile1050Cys 22700974:95:35
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96 The P517C/I1050C mutant was expressed in HEK 293 cells.
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ABCB1 p.Ile1050Cys 22700974:96:10
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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.
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ABCB1 p.Ile1050Cys 22700974:106:23
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ABCB1 p.Ile1050Cys 22700974:106:124
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107 Linkage of the NBDs Activates P-gp ATPase Activity 26808 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 32•AUGUST 3, activity mutant P517C/I1050C was highly activated (11.5-fold) by verapamil and exhibited about 80% (1.5 ϩ 0.2 ␮mol Pi/min/mg P-gp) of the verapamil-stimulated ATPase activity of Cys-less P-gp (1.9 ϩ 0.3 ␮mol Pi/min/mg P-gp).
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ABCB1 p.Ile1050Cys 22700974:107:157
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108 Cross-linking of P517C/I1050C with M4M increased its basal ATPase activity (14-fold higher than the basal ATPase activity of untreated P-gp) (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:108:23
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ABCB1 p.Ile1050Cys 22700974:108:48
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110 The enhanced activity of M4M cross-linked P517C/I1050C was not due to carry over of M4M during purification because M4M had little effect on Cys-less P-gp ATPase activity (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:110:48
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ABCB1 p.Ile1050Cys 22700974:110:71
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111 Reduction of M4M cross-linked P517C/I1050C with dithiothreitol decreased its activity back to basal levels (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:111:36
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112 The large increase in basal ATPase activity observed when mutant P517C/I1050C was cross-linked with the short M4M cross-linker appeared to be caused by trapping of the NBDs in close proximity rather than nonspecific cross-linking effects because treatment with the long M17M cross-linker caused a much smaller increase (about 2-fold) in basal ATPase activity (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:112:54
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ABCB1 p.Ile1050Cys 22700974:112:71
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114 The cross-linking results observed with mutant P517C/ I1050C suggest that covalently linking NBD1 and NBD2 in close proximity with M4M mimics activation of ATPase activity with compounds that highly activate P-gp ATPase activity.
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ABCB1 p.Ile1050Cys 22700974:114:54
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120 Membranes prepared from cells expressing mutant D177C/ N820C were treated with M4M and M17M.
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ABCB1 p.Ile1050Cys 22700974:120:80
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123 A, membranes prepared from HEK 293 cells expressing the NBD1/ NBD2 mutant P517C/I1050C were treated without (Control) or with cross-linkers M4M or M17M.
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ABCB1 p.Ile1050Cys 22700974:123:80
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125 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Ile1050Cys 22700974:125:122
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136 It was found that the effect of cross-linking on ATPase of mutant D177C/N820C (Fig. 3C) was very similar to those observed for mutant P517C/I1050C (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:136:140
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158 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.Ile1050Cys 22700974:158:102
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163 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.Ile1050Cys 22700974:163:102
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194 Mutants that showed activation of ATPase after cross-linking with M4M (P517C/I1050C and D177C/N820C) were tested for inhibition of cross-linking.
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ABCB1 p.Ile1050Cys 22700974:194:77
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225 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (-) or presence (ϩ) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis.
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ABCB1 p.Ile1050Cys 22700974:225:60
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226 D, membranes prepared from cells expressing mutant P517C/I1050C were pretreated without (None) or with 0.03 mM tariquidar (Tar) followed by incubation in the absence (-) or presence (ϩ) of 0.05 mM M4M cross-linker. Samples were then subjected to immunoblot analysis.
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ABCB1 p.Ile1050Cys 22700974:226:20
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ABCB1 p.Ile1050Cys 22700974:226:57
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232 An explanation for the increased activity is that P517C and I1050C are located at the N-terminal end and central segments of the NBDs close to the LSGGQ and Walker A sites of NBD1 and NBD2, respectively.
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ABCB1 p.Ile1050Cys 22700974:232:60
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233 Cross-linking P517C/I1050C (or D177C/ N820C) with M4M would hold the Walker A and LSGGQ sites in close proximity (Fig. 7A) to increase the probability of generating the ATP-bound sandwich conformation and therefore increase the basal ATPase activity.
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ABCB1 p.Ile1050Cys 22700974:233:20
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236 The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps.
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ABCB1 p.Ile1050Cys 22700974:236:48
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264 We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis.
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ABCB1 p.Ile1050Cys 22700974:264:134
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91 To address whether trapping the NBDs in close proximity (closed conformation) would activate or inhibit ATPase activity, cysteines were introduced at locations in the central part of NBD1 (P517C) and the N-terminal region of NBD2 (I1050C) of Cys-less P-gp predicted to be outside the catalytic sites but close to the LSGGQ (residues 531-535) and Walker A sites (residues 1070-1077) of NBD1 and NBD2, respectively.
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ABCB1 p.Ile1050Cys 22700974:91:231
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94 The P517C/I1050C mutant was expressed in HEK 293 cells.
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ABCB1 p.Ile1050Cys 22700974:94:10
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104 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.
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ABCB1 p.Ile1050Cys 22700974:104:124
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105 Linkage of the NBDs Activates P-gp ATPase Activity 26808 activity mutant P517C/I1050C was highly activated (11.5-fold) by verapamil and exhibited about 80% (1.5 af9; 0.2 òe;mol Pi/min/mg P-gp) of the verapamil-stimulated ATPase activity of Cys-less P-gp (1.9 af9; 0.3 òe;mol Pi/min/mg P-gp).
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ABCB1 p.Ile1050Cys 22700974:105:80
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109 Reduction of M4M cross-linked P517C/I1050C with dithiothreitol decreased its activity back to basal levels (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:109:36
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122 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Ile1050Cys 22700974:122:122
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133 It was found that the effect of cross-linking on ATPase of mutant D177C/N820C (Fig. 3C) was very similar to those observed for mutant P517C/I1050C (Fig. 2B).
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ABCB1 p.Ile1050Cys 22700974:133:140
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189 Mutants that showed activation of ATPase after cross-linking with M4M (P517C/I1050C and D177C/N820C) were tested for inhibition of cross-linking.
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ABCB1 p.Ile1050Cys 22700974:189:77
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219 D, membranes prepared from cells expressing mutant P517C/I1050C were pretreated without (None) or with 0.03 mM tariquidar (Tar) followed by incubation in the absence (afa;) or presence (af9;) of 0.05 mM M4M cross-linker. Samples were then subjected to immunoblot analysis.
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ABCB1 p.Ile1050Cys 22700974:219:57
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229 The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps.
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ABCB1 p.Ile1050Cys 22700974:229:48
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257 We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis.
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ABCB1 p.Ile1050Cys 22700974:257:134
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PMID: 25053414 [PubMed] Loo TW et al: "Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity."
No. Sentence Comment
257 Direct cross-linking of the cysteines L175C and N820C (23) or cross-linking of mutant P571C/I1050C with the short 1,4-butanediyl bismethanethiosulfonate cross-linker (7.8 &#c5;) (24) highly activated P-gp ATPase activity over 10-fold in the absence of drug substrates.
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ABCB1 p.Ile1050Cys 25053414:257:92
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258 Cysteines L175C/N820C are located in the intracellular loops (Fig. 1, C and D), whereas cysteines P517C/I1050C are located in the NBDs (Fig. 1, C and D).
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ABCB1 p.Ile1050Cys 25053414:258:104
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259 An explanation for the difference is that linkage of the homologous halves at the L175C/N820C or P517C/I1050C sites would tend to favor the closed conformation (Fig. 1C) to FIGURE 9.
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ABCB1 p.Ile1050Cys 25053414:259:103
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PMID: 25600711 [PubMed] Pan L et al: "Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics."
No. Sentence Comment
203 We did not simulate cross-linking per se, but our structure showed direct side-chain contact between the crosslinked pair LSGGQ (P517C) and Walker A (I1050C) (mouse P513-I1046) showing agreement between our homology model and the biochemical data.
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ABCB1 p.Ile1050Cys 25600711:203:150
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PMID: 26507655 [PubMed] Loo TW et al: "Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein."
No. Sentence Comment
67 Mutant P517C/I1050C was cross-linked in membranes at 0 &#b0;C with 50 òe;M 1,4-butanediyl bismethanethiosulfonate (BMTS) in the absence or presence of 1 òe;M tariquidar (30).
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ABCB1 p.Ile1050Cys 26507655:67:13
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118 The conditions for cross-linking mutants T333C/L975C and P517C/I1050C, however, were different.
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ABCB1 p.Ile1050Cys 26507655:118:63
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120 Cross-linking of mutant P517C/I1050C was performed on membranes using BMTS cross-linker (30).
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ABCB1 p.Ile1050Cys 26507655:120:30
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