ABCMdb

ABCB1 p.Arg276Glu

Predicted by SNAP2:	A: D (59%), C: D (59%), D: D (75%), E: D (66%), F: D (71%), G: D (66%), H: N (53%), I: D (59%), K: N (87%), L: D (63%), M: N (53%), N: D (53%), P: D (75%), Q: N (57%), S: N (53%), T: N (53%), V: D (63%), W: D (75%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] A salt bridge in intracellular loop 2 is essential... Biochemistry. 2013 May 14;52(19):3194-6. doi: 10.1021/bi400425k. Epub 2013 May 3. Loo TW, Clarke DM
A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein.
Biochemistry. 2013 May 14;52(19):3194-6. doi: 10.1021/bi400425k. Epub 2013 May 3., [PMID:23634976]

Abstract [show]
There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.

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No. Sentence Comment
33 If a salt bridge between Glu256 and Arg276 were important for the maturation of human P-gp, it would be expected that an E256R/R276E mutant would still mature. Login to comment
34 Accordingly, we reversed the positions of the glutamic acid and arginine residues (E256R/R276E) and tested the mutant`s ability to undergo maturation. Login to comment
35 The mature 170 kDa P-gp protein was the major product in mutant E256R/R276E (Figure 2A). Login to comment
37 The E256R or R276E mutation, however, inhibited maturation of P-gp (Figure 2A). Login to comment
38 For example, Figure 2B shows that the 150 kDa E256R protein was sensitive to treatment with endoglycosidase H or PNGase F. Similar results were obtained when mutant R276E was treated with endoglycosidase H or PNGase F (data not shown). Login to comment
40 To test if mutant E256R/R276E yielded an active protein, the histidine-tagged mutant was expressed in HEK293 cells, isolated by nickel chelate chromatography, and assayed for verapamil-stimulated ATPase activity. Login to comment
41 Verapamil was used because it is transported by P-gp14 and strongly stimulates (>10-fold) the ATPase activity of human P-gp.15 The E256R/ R276E mutant showed verapamil-stimulated ATPase activity that was similar to that of the wild-type protein (Figure 2C). Login to comment
61 (A) Whole cell sodium dodecyl sulfate (SDS) extracts of wild-type P-gp and mutants E256R, R276E, and E256R/R276E. Login to comment