ABCB1 p.Ile864Ala
| Predicted by SNAP2: | A: N (78%), C: N (93%), D: D (80%), E: D (75%), F: N (97%), G: D (71%), H: D (71%), K: D (75%), L: N (87%), M: N (93%), N: D (71%), P: D (71%), Q: D (66%), R: D (71%), S: N (53%), T: N (72%), V: N (97%), W: D (75%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: N, D: D, E: D, F: N, G: D, H: D, K: D, L: N, 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] Evidence for modulatory sites at the lipid-protein... Biochemistry. 2012 Apr 3;51(13):2852-66. Epub 2012 Mar 22. Mandal D, Moitra K, Ghosh D, Xia D, Dey S
Evidence for modulatory sites at the lipid-protein interface of the human multidrug transporter P-glycoprotein.
Biochemistry. 2012 Apr 3;51(13):2852-66. Epub 2012 Mar 22., [PMID:22360349]
Abstract [show]
The human multidrug transporter P-glycoprotein (Pgp or ABCB1) sets up pharmacological barriers to many clinically important drugs, a therapeutic remedy for which has yet to be formulated. For the rational design of mechanism-based inhibitors (or modulators), it is necessary to map the potential sites for modulator interaction and understand their modes of communication with the other functional domains of Pgp. In this study, combining directed mutagenesis with homology modeling, we provide evidence of two modulator-specific sites at the lipid protein interface of Pgp. Targeting 21 variant positions in the COOH-terminal transmembrane (TM) regions, we find residues M948 (in TM11) and F983, M986, V988, and Q990 (all four in TM12) critically involved in substrate-site modulation by a thioxanthene-based allosteric modulator cis-(Z)-flupentixol. Interestingly, for ATP-site modulation by the same modulator, only two (M948 and Q990) of those four residues appear indispensable, together with two additional residues, T837 and I864 in TM9 and TM10, respectively, suggesting independent modes of communication linking the allosteric site with the substrate binding and ATPase domains. None of the seven residues identified prove to be critical for modulation of the substrate or ATP sites by Pgp modulators that are transported by the pump, such as cyclosporin A or verapamil, indicating their specificity for cis-(Z)-flupentixol. On the other hand, ATP-site modulation by verapamil proves to be highly sensitive to replacement at positions F716 (in TM7) and I765 (in TM8), and to a more moderate extent at I764 and L772 (both in TM8). Homology modeling based on the known crystal structures of the bacterial multidrug transporter SAV1866 and the mouse Pgp homologue maps the identified residues primarily at the lipid-protein interface of Pgp, in two spatially distinct modulator-specific clusters. The two modulatory sites demonstrate negative synergism in influencing ATP hydrolysis, consolidating their spatial distinctness. Because Pgp is known to recruit drug molecules directly from the lipid bilayer, identification of modulatory sites at the lipid-protein interface and at the same time outside the conventional central drug binding cavity is mechanistically revealing.
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No. Sentence Comment
28 The desired nucleotide replacements were confirmed by Big-Dye version 3.1 (BD Sciences) nucleotide sequencing of the MDR1 open reading frame in recombinant plasmids pKM2-MDR1- V715A, -F716A, -I719A, -G723A, -I764A, -I765A, -I768A, -F770A, -L772A, -T837A, -I840A, -I864A, -I867A, -A871F, -T945A, -M948A, -F983A, -M986A, -V988A, -Q990A, and -V991A.
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ABCB1 p.Ile864Ala 22360349:28:264
status: NEW110 Stimulation of ATP Hydrolysis by cis-(Z)-Flupentixol and Verapamil cis-(Z)-flupentixol-mediated stimulation verapamil-mediated stimulation maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark WT 7.3 ± 0.4 6.8 ± 1.5 5.6 ± 0.19 6.6 ± 1.04 V715A 6.2 ± 0.2 5.0 ± 0.9 6.4 ± 0.37 16.3 ± 3.3 V716A 5.1 ± 0.2 4.7 ± 1.1 NDb NDb a I719A 4.6 ± 0.08 4.7 ± 0.4 5.8 ± 0.26 6.5 ± 1.31 G723A 5.3 ± 0.3 10.93 ± 2.7 5.4 ± 0.1 11.6 ± 0.88 I764A 4.5 ± 0.4 4.1 ± 2 3.2 ± 0.26 27.9 ± 8.4 I765A 4.96 ± 0.2 2.3 ± 0.47 NDb NDb a I768A 4.8 ± 0.2 1.2 ± 0.43 4.8 ± 0.19 8.5 ± 1.4 F770A 6.6 ± 0.6 10.9 ± 3.9 8.6 ± 0.59 13.2 ± 3.2 L772A 3.6 ± 0.2 2.96 ± 0.9 3.2 ± 0.11 9.5 ± 1.6 T837A 1.1 ± 0.06 NDb a 2.6 ± 0.1 3.1 ± 0.9 I840A 4.0 ± 0.3 25.6 ± 6.5 6.9 ± 0.2 13.5 ± 0.5 I864A 0.6 ± 0.05 NDb a 4.1 ± 0.25 0.9 ± 1.04 I867A 10.2 ± 1.3 72.3 ± 18.5 10.2 ± 0.52 30.7 ± 3.1 A871F NDb NDb NDb NDb a T945A 5.0 ± 0.3 13.7 ± 3.1 6.4 ± 0.15 6.9 ± 0.71 M948A NDb NDb a 6.8 ± 1.5 44.4 ± 12.4 F983A 4.95 ± 0.36 19.59 ± 5.0 6.1 ± 0.38 6.6 ± 1.8 M986A 3.8 ± 0.3 2.6 ± 1.5 7.7 ± 0.76 30.0 ± 8.6 V988A 3.0 ± 0.2 21.4 ± 7.6 6.5 ± 0.31 16.9 ± 2.9 Q990A 1.8 ± 0.4 18.9 ± 2.6 a 7.7 ± 0.58 12.9 ± 3.7 V991A 3.9 ± 0.2 21.1 ± 5.0 5.9 ± 0.59 21.9 ± 7.2 a Mutants with <2-fold maximal stimulation (<25% of the wild-type level).
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ABCB1 p.Ile864Ala 22360349:110:1094
status: NEW200 As one can see in the top panels (middle and right) of Figure 8, the negative synergistic effect of cis-(Z)-flupentixol on verapamil-stimulated ATP hydrolysis was considerably compromised in I864A and Q990A, Pgp mutants defective with respect to ATP-site stimulation by cis-(Z)-flupentixol.
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ABCB1 p.Ile864Ala 22360349:200:191
status: NEW205 ATP hydrolysis by the wild type and Pgp mutants I864A, Q990A, and I765A, in isolated membrane vesicles, was conducted either after preincubation with 50 μM verapamil (top) or with 50 μM cis-(Z)-flupentixol (bottom) followed by incubation with increasing concentrations of cis-(Z)-flupentixol (top) or verapamil (bottom), using the vanadate-sensitive phosphate release assay.
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ABCB1 p.Ile864Ala 22360349:205:48
status: NEW225 Similarly, a reciprocal effect was evident in T837A and I864A, where a 34-fold stimulation of substrate binding was retained (Figure 2) even though ATP-site stimulation was almost abrogated (Figure 4).
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ABCB1 p.Ile864Ala 22360349:225:56
status: NEW