ABCB1 p.Ala259Cys
| Predicted by SNAP2: | C: D (59%), D: D (80%), E: D (75%), F: D (75%), G: N (78%), H: D (63%), I: D (66%), K: D (75%), L: D (71%), M: D (66%), N: D (59%), P: D (80%), Q: D (71%), R: D (71%), S: N (93%), T: N (72%), V: D (63%), W: D (80%), Y: D (75%), |
| Predicted by PROVEAN: | C: N, D: N, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D, |
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[hide] Locking intracellular helices 2 and 3 together ina... J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25. Loo TW, Clarke DM
Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.
J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25., [PMID:24275649]
Abstract [show]
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(beta,gamma-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.
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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.
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ABCB1 p.Ala259Cys 24275649:9:24
status: NEW128 Cross-linked product was observed in all mutants (data not shown), with the greatest amount present in mutant A259C/W803C (about 75%, Fig. 4A).
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ABCB1 p.Ala259Cys 24275649:128:110
status: NEW129 We then tested whether cross-linking of mutant A259C/ W803C could be inhibited by nucleotides.
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ABCB1 p.Ala259Cys 24275649:129:47
status: NEW137 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.
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ABCB1 p.Ala259Cys 24275649:137:73
status: NEW156 Cross-linking of A259C (IH2) to W803C (IH3) inhibits activity.
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ABCB1 p.Ala259Cys 24275649:156:17
status: NEW157 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.
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ABCB1 p.Ala259Cys 24275649:157:68
status: NEW158 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.
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ABCB1 p.Ala259Cys 24275649:158:269
status: NEW199 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.
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ABCB1 p.Ala259Cys 24275649:199:104
status: NEW209 Cross-linking of A259C (blue) in ICL2 to W803C (green) in ICL3 inhibited activity.
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ABCB1 p.Ala259Cys 24275649:209:17
status: NEW[hide] Identification of the distance between the homolog... J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12. Loo TW, Clarke DM
Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.
J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12., [PMID:24523403]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 A). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 A (alpha-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 A, although cross-linking at distances greater than 20 A yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 A, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141).
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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).
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ABCB1 p.Ala259Cys 24523403:81:235
status: NEW82 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.
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ABCB1 p.Ala259Cys 24523403:82:24
status: NEW