ABCMdb

PMID: 9733949

Takada Y, Yamada K, Taguchi Y, Kino K, Matsuo M, Tucker SJ, Komano T, Amachi T, Ueda K
Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.
Biochim Biophys Acta. 1998 Aug 14;1373(1):131-6., [PubMed]

Sentences

No. Mutations Sentence Comment

51 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala The C431A/C1074A double-mutant form of P-glycoprotein, in which the cysteine residues of the WA motif in both NBFs were replaced by alanine, trapped nucleotides even after treatment with 100 WM NEM (Fig. 1B). Login to comment 52 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala Also, these cysteine-to-alanine mutations did not a¡ect the function of P-glycoprotein, because the pattern and degree of multidrug resistance conferred by the C431A/ C1074A double-mutant form were similar to those conferred by the wild-type protein (data not shown). Login to comment 53 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala By contrast, vanadate-induced nucleotide trapping of the C431A and C1074A mutant forms, in which the cysteine residue of the WA motif in only one of the NBFs was replaced by alanine, was a¡ected by NEM (Fig. 1C,D). Login to comment 54 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala Nucleotide trapping with the C431A mutant form was inhibited by 10 WM NEM (Fig. 1C), and nucleotide trapping with the C1074A mutant form was inhibited by 50 WM NEM (Fig. 1D). Login to comment 56 ABCB1 p.Cys431Ala ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala ABCB1 p.Cys1074Ala E¡ects of NEM on vanadate-induced nucleotide trapping in P-glycoprotein. Plasma membrane proteins (about 20 Wg) from stable KB-3-1 transfectants expressing equivalent amounts of the wild-type human P-glycoprotein (A), the C431A/C1074A double-mutant form, in which the cysteine residues of Walker A in both NBFs were replaced by alanine (B), or the C431A (C) or C1074A (D) single-mutant form were treated with NEM at 1 WM (lane 2), 10 WM (lane 3), 50 WM (lane 4), or 100 WM (lane 5), or with NEM (lane 1). Login to comment 61 ABCB1 p.Cys431Ala ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala ABCB1 p.Cys1074Ala A, P-glycoprotein-S; B, C431A/C1074A; C, C431A; D, C1074A. Experiments were done in duplicate. Login to comment 64 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala P-Glycoprotein-S was labeled by 5 WM biotin maleimide and speci'cally inhibited by the presence of 100 WM NEM (Fig. 2A), but the C431A/C1074A mutant form was labeled little if at all (Fig. 2B). Login to comment 65 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala The C431A and C1074A mutant forms were also both labeled in an NEM-dependent manner by biotin maleimide (Fig. 2C,D), suggesting that biotin maleimide at a concentration of 5 WM specifically and uniformly labels the WA cysteines in both NBFs, as previously reported [16]. Login to comment 70 ABCB1 p.Cys431Ala ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala 8-Azido-ATP binding with the C431A/C1074A mutant form was not inhibited by 100 WM NEM, but possibly increased (Fig. 3B), whereas 8-azido-ATP binding of the C431A mutant form appeared not to be a¡ected (Fig. 3C). Login to comment 71 ABCB1 p.Cys1074Ala However, ATP binding to the C1074A mutant form was signi'cantly reduced by treatment with 100 WM NEM (Fig. 3D), similar to the wild-type P-glycoprotein. Login to comment 73 ABCB1 p.Lys1076Met ABCB1 p.Lys1076Met ABCB1 p.Lys1076Met ABCB1 p.Lys433Met ABCB1 p.Lys433Met ABCB1 p.Lys433Met Vanadate-induced nucleotide trapping and ATP binding in K433M and K1076M mutant P-glycoproteins To further examine the roles of the two NBFs in the function of P-glycoprotein, we constructed three other mutants, K433M, K1076M, and K433M/ K1076M, in which the lysine residue in the WA motif of either or both NBFs was replaced by methionine. Login to comment 77 ABCB1 p.Cys431Ala ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala ABCB1 p.Cys1074Ala A, P-glycoprotein-S; B, C431A/C1074A; C, C431A; D, C1074A. Experiments were done in triplicate. Fig. 4. Login to comment 78 ABCB1 p.Lys1076Met ABCB1 p.Lys1076Met ABCB1 p.Lys433Met ABCB1 p.Lys433Met Immunoblot analysis (A), vanadate-induced nucleotide trapping (B), and 8-azido-ATP binding (C) of wild-type protein and lysine-to-methionine mutants of P-glycoprotein-S. Lanes: 1, cells not transfected; 2, P-glycoprotein-S; 3, K433M; 4, K1076M; 5, K433M/K1076M. Login to comment 86 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met As previously reported all these mutants bound ATP, although the binding to the K433M/K1076M double-mutant form appeared substantially reduced (lane 5). Login to comment 94 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala The C431A/C1074A mutant form of P-glycoprotein was indistinguishable from the wild-type in its function. Login to comment 95 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala Also the C431A/C1074A mutant P-glycoprotein trapped nucleotides even after treatment with 100 WM NEM, indicating that the other 've cysteines were probably not accessible to NEM. Login to comment 97 ABCB1 p.Cys431Ala ABCB1 p.Cys1074Ala However, the C431A/C1074A mutant P-glycoprotein showed an increase in ATP binding after NEM treatment (Fig. 3B), indicating that NEM modi'cation of other cysteine residues outside NBFs may allosterically a¡ect ATP binding in a positive manner. Login to comment 110 ABCB1 p.Cys431Ala The C431A mutant P-glycoprotein showed no change in 8-azido-ATP binding after NEM treatment. Login to comment 111 ABCB1 p.Cys1074Ala However, NEM treatment signi'cantly reduced 8-azido-ATP binding in the C1074A mutant P-glycoprotein. Login to comment