ABCB1 p.Tyr1087Phe
| Predicted by SNAP2: | A: D (80%), C: D (59%), D: D (91%), E: D (91%), F: D (71%), G: D (85%), H: D (85%), I: D (75%), K: D (91%), L: D (80%), M: D (80%), N: D (91%), P: D (91%), Q: D (85%), R: D (85%), S: D (85%), T: D (85%), V: D (75%), W: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: 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
11 Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity.
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ABCB1 p.Tyr1087Phe 24275649:11:35
status: NEW148 To test whether Tyr1087 was important for folding or mediating drug stimulation of ATPase activity, we tested the effect of Y1087A, Y1087L, and Y1087F mutations.
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ABCB1 p.Tyr1087Phe 24275649:148:144
status: NEW149 The A52-tagged mutants were expressed in HEK 293 cells in the absence or presence of cyclosporine A, and samples of whole cell SDS extracts were subjected to immunoblot analysis. Fig. 5A shows that the mature 170-kDa protein was the major product in mutant Y1087F, whereas the 150-kDa immature protein was the major product in mutants Y1087A and Y1087L.
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ABCB1 p.Tyr1087Phe 24275649:149:257
status: NEW154 Mutant Y1087F showed about one-third of wild-type P-gp ATPase activity.
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ABCB1 p.Tyr1087Phe 24275649:154:7
status: NEW164 A, A52-tagged Y1087A (A), Y1087F (F), and Y1087L (L) mutants were expressed in the absence (afa;) or presence (af9;) of 5 òe;M cyclosporine A (Cyclo).
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ABCB1 p.Tyr1087Phe 24275649:164:26
status: NEW166 The positions of mature (170-kDa) and immature (150-kDa) P-gps are indicated. B, histidine-tagged WT, Y1087A (A), Y1087F (F), and Y1087L (L) P-gp were expressed in HEK 293 cells and isolated by nickel-chelate chromatography.
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ABCB1 p.Tyr1087Phe 24275649:166:114
status: NEW203 Mutation of Tyr1087 to Leu severely inhibited activity, whereas the conservative Y1087F change caused about a 70% reduction in activity.
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ABCB1 p.Tyr1087Phe 24275649:203:81
status: NEW