ABCMdb

ABCB1 p.Phe804Tyr

Predicted by SNAP2:	A: D (85%), C: D (80%), D: D (95%), E: D (91%), G: D (91%), H: D (71%), I: D (85%), K: D (95%), L: D (85%), M: D (71%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (85%), W: D (75%), Y: N (82%),
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: N,

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Publications
[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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Sentences [show]
No. Sentence Comment
104 The results are derived from three different transfections af9; S.D. Clamping IH2 to IH3 Inhibits P-gp JANUARY 3, 2014ߦVOLUME 289ߦNUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 231 at SEMMELWEIS UNIV OF MEDICINE on December 12, We then tested whether mutating Trp803 to Phe (hydrophobic), Leu (hydrophobic), Ser (hydrophilic), Tyr (aromatic), or Asp (charged) and Phe804 to Leu, Ser, Tyr, Asp, or Trp affected maturation of the protein. Login to comment
108 Mature 170-kDa P-gp was the major product in mutants F804Y and F804W (Fig. 3, C and D), although the amount of mature protein in the absence of cyclosporine A in mutant F804W was lower than in F804Y (65% versus 85%, respectively). Login to comment