ABCB1 p.Tyr1087Ala
| 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] Human P-glycoprotein contains a greasy ball-and-so... J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.
J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3., [PMID:23733192]
Abstract [show]
The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.
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No. Sentence Comment
126 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.Tyr1087Ala 23733192:126:107
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.Tyr1087Ala 23733192:127:23
status: NEW[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.Tyr1087Ala 24275649:11:8
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.Tyr1087Ala 24275649:148:124
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.Tyr1087Ala 24275649:149:335
status: NEW150 Both mutants Y1087A and Y1087L, however, could be rescued when they were expressed in the presence of cyclosporine A (Fig. 5A).
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ABCB1 p.Tyr1087Ala 24275649:150:13
status: NEW153 It was found that the Y1087A and Y1087L mutations reduced activity by over 90% (Fig. 5B).
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ABCB1 p.Tyr1087Ala 24275649:153:22
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.Tyr1087Ala 24275649:164:14
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.Tyr1087Ala 24275649:166:102
status: NEW167 Mutants Y1087A and Y1087L were expressed in the presence of cyclosporine A to promote maturation.
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ABCB1 p.Tyr1087Ala 24275649:167:8
status: NEW171 Mutations of Tyr1087 to Ala or Leu severely reduced both maturation and activity of P-gp, and even the small change of replacing Tyr with Phe caused about a 70% reduction in activity.
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ABCB1 p.Tyr1087Ala 24275649:171:13
status: NEW