ABCB1 p.Phe1086Asp
| Predicted by SNAP2: | A: D (75%), C: D (59%), D: D (91%), E: D (85%), G: D (80%), H: D (80%), I: D (71%), K: D (91%), L: D (59%), M: D (59%), N: D (80%), P: D (85%), Q: D (71%), R: D (85%), S: D (71%), T: D (75%), V: D (71%), W: D (59%), Y: N (53%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: 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, Y: 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
175 Fig. 5A shows that the F1086D or F1086R mutations inhibited maturation of P-gp.
X
ABCB1 p.Phe1086Asp 23733192:175:23
status: NEW177 Mutants F1086D and F1086R could still be rescued if they were expressed in the presence of a drug substrate (Fig. 5B).
X
ABCB1 p.Phe1086Asp 23733192:177:8
status: NEW179 Mutants F1086D and F1086R were first expressed in the presence of cyclosporine A to promote maturation before isolating the histidine-tagged protein.
X
ABCB1 p.Phe1086Asp 23733192:179:8
status: NEW212 A, F1086X mutants were expressed in the absence of drug substrates. B, processing mutants F1086D and F1086R were expressed in the presence (af9;) or absence (afa;) of cyclosporine A (Cyclo), and whole cell SDS extracts were subjected to immunoblot analysis.
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ABCB1 p.Phe1086Asp 23733192:212:90
status: NEW214 Histidine-tagged mutants F1086D and F1086R were first expressed in the presence of cyclosporine A to promote maturation of P-gp before isolation of the protein by nickel-chelate chromatography. Each value is the mean afe; S.D. (n afd; 3).
X
ABCB1 p.Phe1086Asp 23733192:214:25
status: NEW