ABCB1 p.Leu797Ala
| Predicted by SNAP2: | A: D (71%), C: N (53%), D: D (85%), E: D (80%), F: D (66%), G: D (80%), H: D (75%), I: N (57%), K: D (80%), M: N (93%), N: D (80%), P: D (85%), Q: D (63%), R: D (75%), S: D (75%), T: D (66%), V: N (72%), W: D (75%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, 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
12 Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation.
X
ABCB1 p.Leu797Ala 24275649:12:41
status: NEW81 Point mutations to the residues flanking IH3 (L797A, Fig. 1B) along with F793A, L814A, and L818A (data not shown) also inhibited maturation so that the 150-kDa protein was the major product.
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ABCB1 p.Leu797Ala 24275649:81:46
status: NEW86 Because the mutations F793A, L797A, L814A, and L818A in the flanking regions of IH3 also inhibited maturation, these mutants were also expressed in the presence of cyclosporine A to test for rescue.
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ABCB1 p.Leu797Ala 24275649:86:29
status: NEW88 Mutants F793A, L797A, and L818A were less efficiently rescued as they yielded about equivalent levels of mature 170-kDa and immature 150-kDa P-gp in the presence of cyclosporine A (Fig. 2B).
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ABCB1 p.Leu797Ala 24275649:88:15
status: NEW102 A52-tagged WT P-gp, mutants W803A and F804A (A), or IH3 flanking mutants (F793A, L797A, L814A, and L818A) (B) were expressed in HEK293cellsintheabsence(afa;)orpresence(af9;)of5òe;M cyclosporineA(Cyclo) for 18 h, and whole cell extracts were subjected to immunoblot analysis.
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ABCB1 p.Leu797Ala 24275649:102:81
status: NEW185 In support of the prediction that Trp803 and Phe804 form a hydrophobic pocket with these residues, alanine-scanning mutagenesis of the 793-797 and 814-818 regions showed that only the F793A, L797A, L814A, and L818A mutations inhibited maturation (Fig. 2B).
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ABCB1 p.Leu797Ala 24275649:185:191
status: NEW