ABCB1 p.Pro726Cys
| Predicted by SNAP2: | A: D (53%), C: N (57%), D: D (66%), E: D (66%), F: D (71%), G: D (63%), H: D (59%), I: D (66%), K: D (63%), L: D (63%), M: D (53%), N: D (63%), Q: N (53%), R: D (66%), S: D (59%), T: D (59%), V: D (66%), W: D (63%), Y: D (71%), |
| 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, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Transmembrane segment 7 of human P-glycoprotein fo... Biochem J. 2006 Oct 15;399(2):351-9. Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket.
Biochem J. 2006 Oct 15;399(2):351-9., 2006-10-15 [PMID:16813563]
Abstract [show]
P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.
Comments [show]
None has been submitted yet.
No. Sentence Comment
74 Mutant Verapamil S50 (µM) Vinblastine S50 (µM) Colchicine S50 (µM) F711C 9.8 + - 1.1 2.3 + - 0.2 410 + - 20 V712C 10.0 + - 0.8 1.9 + - 0.2 320 + - 40 V713C 9.9 + - 1.3 1.8 + - 0.2 340 + - 40 G714C 14.1 + - 2.1 1.9 + - 0.3 410 + - 30 V715C 9.8 + - 1.3 1.8 + - 0.3 330 + - 40 F716C 10.1 + - 1.0 2.1 + - 0.1 340 + - 30 C717C 10.0 + - 1.5 2.3 + - 0.3 390 + - 20 A718C 10.5 + - 1.3 2.0 + - 0.1 330 + - 30 I719C 10.5 + - 1.5 1.9 + - 0.2 370 + - 30 I720C 12.4 + - 0.7 2.4 + - 0.2 390 + - 10 N721C 13.0 + - 1.2 1.7 + - 0.2 380 + - 40 G722C ND ND ND G723C 11.1 + - 0.4 2.0 + - 0.3 410 + - 20 L724C 12.0 + - 1.5 2.4 + - 0.3 340 + - 30 Q725C 11.7 + - 1.1 2.0 + - 0.1 390 + - 20 P726C 11.9 + - 1.0 1.9 + - 0.2 450 + - 30 A727C 21.2 + - 3.1 1.9 + - 0.2 480 + - 40 F728C 48.7 + - 2.2 2.7 + - 0.3 830 + - 90 A729C 34.3 + - 3.0 2.3 + - 0.2 760 + - 80 I730C 12.0 + - 1.1 2.0 + - 0.1 420 + - 30 I731C 13.5 + - 1.9 1.9 + - 0.2 410 + - 30 Cys-less 11.6 + - 1.3 2.1 + - 0.3 400 + - 40 into the endoplasmic reticulum during synthesis of the protein.
X
ABCB1 p.Pro726Cys 16813563:74:682
status: NEW97 Samples of the isolated P-gp were mixed with lipid and assayed for ATPase activity and compared with untreated P-gp. All of the mutants, except Q725C, P726C, F728C and A729C, were unaffected by treatment with MTS-verapamil (Figure 2A).
X
ABCB1 p.Pro726Cys 16813563:97:151
status: NEW98 Mutant P726C, however, showed about a 50% reduction in basal activity after treatment with MTS-verapamil.
X
ABCB1 p.Pro726Cys 16813563:98:7
status: NEW224 Labelling of one mutant, P726C, with MTS-verapamil resulted in inhibition of the basal activity of P-gp (Figure 2A).
X
ABCB1 p.Pro726Cys 16813563:224:25
status: NEW226 Therefore an explanation for inhibition of ATPase activity after MTS-verapamil treatment of mutant P726C is that the bound verapamil molecule interferes with TM conformational changes associated with ATP hydrolysis [42-44].
X
ABCB1 p.Pro726Cys 16813563:226:99
status: NEW