ABCMdb

ABCB1 p.Ala947Leu

Predicted by SNAP2:	C: N (87%), D: D (75%), E: D (75%), F: D (71%), G: N (66%), H: D (75%), I: D (71%), K: D (80%), L: D (75%), M: D (66%), N: D (66%), P: D (80%), Q: D (75%), R: D (75%), S: N (82%), T: D (53%), V: D (66%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	C: N, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: D, V: D, W: D, Y: D,

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Publications
[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
79 Wild-type P-gp had an apparent affinity of 24 ␮M verapamil, while mutants F942A, T945A, Q946A, A947L, and Y953A had decreased apparent affinities of 93, 100, 165, 156, and 110 ␮M, respectively. Login to comment
84 Moderate decreases in activity (40-50%) were observed for mutants G939V, Q946A, A947L, Y953A, and F957A. Login to comment
85 The mutant, A947L, also exhibited a decrease in the apparent affinity for vinblastine. Login to comment
102 Wild-type P-gp had an apparent affinity of 620 ␮M colchicine, while those of mutants A947L and G939V were 1870 and 260 ␮M colchicine, respectively. Login to 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. Login to comment
147 DISCUSSION Mutants G939V, F942A, T945A, Q946A, A947L, and Y953A in TM11 had altered apparent affinities for verapamil, vinblastine, or colchicine. Login to comment
179 Similarly, it is also likely that some of the mutations in TM11 in this study that resulted in altered drug-stimulated ATPase activity could also be due to structural perturbations, since relatively small residues were replaced with larger ones (G939V, A947L, A954L, and G955V). Login to comment