ABCMdb

ABCB1 p.Trp803Cys

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Publications

PMID: 24275649 [PubMed] Loo TW et al: "Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein."

No. Sentence Comment

9 Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Login to comment
123 It was found that the yield of all the F804C double mutants was significantly lower than the W803C double cysteine mutants (data not shown). Login to comment
125 Therefore, the W803C double mutants were tested for cross-linking with cupric chloride oxidant. Login to comment
128 Cross-linked product was observed in all mutants (data not shown), with the greatest amount present in mutant A259C/W803C (about 75%, Fig. 4A). Login to comment
129 We then tested whether cross-linking of mutant A259C/ W803C could be inhibited by nucleotides. Login to comment
137 To test the effect of cross-linking on activity, histidine-tagged mutant A259C/W803C was expressed in HEK 293 cells in the presence of cyclosporine A and P-gp isolated by nickel-chelate chromatography. Login to comment
156 Cross-linking of A259C (IH2) to W803C (IH3) inhibits activity. Login to comment
157 A, membranes prepared from cells expressing histidine-tagged mutant A259C/ W803C were treated without (afa;) or with (af9;) 0.5 mM CuCl2 for 10 min at 0 &#b0;C in the absence (None) or presence of 5 mM ATP, adenosine 5b18;-(beta,ॹ- imino)triphosphate(AMP.PNP),orADP.Thereactionswerestoppedbyaddition of SDS sample buffer containing no reducing agent, and samples were then subjected to immunoblot analysis. Login to comment
158 The positions of the cross-linked (X-link) and mature (170-kDa) P-gps are indicated. B, the amount of cross-linked protein relative to total P-gp (170-kDa plus 150-kDa P-gp) was quantitated from three different transfections af9; S.D. C, the histidine-tagged mutant A259C/ W803C was isolated by nickel-chelate chromatography, reconstituted with lipid, and treated without (afa;) or with (af9;) 0.2 mM CuCl2 for 10 min at 20 &#b0;C. The reaction was stopped with 1 mM EDTA. Login to comment
199 In support of these predictions, we found that clamping IH2 to IH3 by oxidative cross-linking of mutant A259C/W803C or mutations to Tyr1087 inhibited activity. Login to comment
209 Cross-linking of A259C (blue) in ICL2 to W803C (green) in ICL3 inhibited activity. Login to comment

PMID: 24523403 [PubMed] Loo TW et al: "Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch."

No. Sentence Comment

81 P-gp ATPase Activity Is Highly Activated When Residues L175C and N820C Are Cross-linked in Close Proximity-In a previous study, we found that cross-linking the two homologous halves through formation of a direct disulfide bond between A259C in intracellular helix 2 (IH2) and W803C in IH3 inhibited activity (49). Login to comment
82 It is possible that the A259C/W803C cross-link inhibited activity because these residues are located within IH segments that are predicted to form critical TMD/ NBD contact points. Login to comment
207 The P-gp molecular dynamics simulation study (64) may also provide an explanation for the inhibition of ATPase activity previously observed when cysteines A295C (in IH2 that connects TM segments 4 and 5) and W803C (in IH3 that connects TM segments 9 and 10) were directly cross-linked (49). Login to comment
209 It is possible that direct cross-linking of A295C (in IH2 that connects TM segments 4 and 5) to W803C in IH3 would interfere with straightening of TM segments 4 and 5 to trap P-gp in an inactive conformation. Login to comment

PMID: 25068895 [PubMed] Prajapati R et al: "Translocation mechanism of P-glycoprotein and conformational changes occurring at drug-binding site: Insights from multi-targeted molecular dynamics."

No. Sentence Comment

462 In this experiment Loo and Clarke cross-linked A259C (ICH2) and W803C (ICH3) that resulted in loss of P-gp ATPase activity. Login to comment

PMID: 25987565 [PubMed] Loo TW et al: "The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein."

No. Sentence Comment

220 Cysteines were placed in the N-terminal regions of IH1 and IH4 as this was the strategy employed to identify disulfide cross-linking between IH2 (A259C) and IH3 (W803C). Login to comment

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

65 Membranes were prepared and samples were incubated at 0 &#b0;C (A80C/R741C, A259C/W803C, I299C/F770C, and A266C/ F1086C) or 20 &#b0;C (L175C/N820C, C431/L1176C, and L521C/C1074) for 10 min in the presence or absence of 0.5 Mapping the P-glycoprotein Tariquidar-binding Site 29390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 49ߦDECEMBER 4, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 11, mM copper phenanthroline (oxidant to promote disulfide bond formation) in the presence or absence of 0.25 òe;M (for TM segment cysteine mutants) or 1 òe;M tariquidar (for the ICL and NBD cysteine mutants). Login to comment
112 Accordingly, a Cys-less P-gp (35) was used to generate mutants that contained double cysteines at the interfaces involving TM segments of TMD1 and TMD2 (A80C(TM1)/ R741C(TM7) (29), I299C(TM5)/F770C(TM8) (36), and T333C(TM6/L975C(TM12) (30), or ICL2 and ICL3 (L175C(ICL1)/N820C(ICL3) (23), A259C(ICL2)/W803C- (ICL3) (37), and A266C(ICL2/F1086C(NBD2) (38) (Fig. 1A). Login to comment