ABCB1 p.Met948Ala
| Predicted by SNAP2: | A: N (53%), C: N (61%), D: D (85%), E: D (85%), F: N (72%), G: D (75%), H: D (75%), I: N (82%), K: D (85%), L: N (57%), N: D (85%), P: D (91%), Q: D (80%), R: D (85%), S: D (71%), T: N (53%), V: N (72%), W: D (85%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: N, |
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[hide] Identification of residues in the drug-binding dom... J Biol Chem. 1999 Dec 10;274(50):35388-92. Loo TW, Clarke DM
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
J Biol Chem. 1999 Dec 10;274(50):35388-92., 1999-12-10 [PMID:10585407]
Abstract [show]
The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.
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No. Sentence Comment
128 TABLE I Drug-stimulated ATPase activity Mutant Drug Verapamil Vinblastine Colchicine Vmax Km Vmax Km Vmax Km % of WTa M % of WT M % of WT mM WT 100 24 100 5.4 100 0.62 I937S 94 22 93 6.1 100 0.69 F938A 106 32 96 5.1 96 0.68 G939V 62 8 45 4.0 165 0.26 I940S 93 32 93 5.6 93 0.65 T941A 100 25 104 5.5 100 0.66 F942A 88 93 30 5.1 24 0.80 S943A 92 26 100 5.2 85 0.62 F944A 93 14 105 5.3 101 0.64 T945A 140 100 165 8.3 56 0.65 Q946A 101 165 57 8.5 18 0.64 A947L 105 156 60 13.0 51 1.87 M948A 103 23 101 5.9 103 0.62 M949A 82 40 96 5.5 61 0.60 Y950A 109 37 119 5.1 99 0.62 F951A 94 31 99 5.2 101 0.64 S952A 108 36 123 5.1 91 0.69 Y953A 205 110 59 8.5 131 0.67 A954L 108 44 13 NDb 8 ND G955V 143 10 104 3.5 220 0.47 C956A 97 24 95 5.3 145 0.63 F957A 126 21 47 4.8 32 1.0 a WT, wild type. b ND, not determined due to low activity.
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ABCB1 p.Met948Ala 10585407:128:497
status: NEW[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]
Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.
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No. Sentence Comment
219 The presence of mutations Q946A, M948A, M949A, F951A, or S952A did not appear to affect the maturation of the M68R/ G251V mutant (Fig. 8A, lanes 2-5, 7, and 8).
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ABCB1 p.Met948Ala 18596043:219:33
status: NEW[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.Met948Ala 22360349:28:296
status: NEW102 A comparable level of basal binding of [125 I]IAAP to wild-type Pgp and Pgp mutants M948A, F983A, V988A, M986A, and Q990A (Figure 2B) rules out the possibility that the reduced level of stimulation in the mutants is due to loss of [125 I]IAAP binding and not stimulation per se. To investigate whether M948, Q990, F983, M986, and V988 are part of a general module involved in the regulation of substrate binding or are molecular components specific to cis- (Z)-flupentixol, we studied the effect of a structurally unrelated Pgp modulator, cyclosporin A, on binding of [125 I]IAAP to all 21 mutants.
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ABCB1 p.Met948Ala 22360349:102:84
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.Met948Ala 22360349:110:1319
status: NEW