ABCMdb

ABCB1 p.Cys1074Ala

Predicted by SNAP2:	A: D (71%), D: D (91%), E: D (91%), F: D (85%), G: D (80%), H: D (91%), I: D (85%), K: D (91%), L: D (85%), M: D (85%), N: D (85%), P: D (91%), Q: D (91%), R: D (91%), S: D (71%), T: D (85%), V: D (80%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, 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: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Comparative aspects of the function and mechanism ... Biochim Biophys Acta. 1999 Dec 6;1461(2):305-13. Ueda K, Matsuo M, Tanabe K, Morita K, Kioka N, Amachi T
Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins.
Biochim Biophys Acta. 1999 Dec 6;1461(2):305-13., [PMID:10581363]

Abstract [show]
ATP-binding cassette (ABC) superfamily proteins have divergent functions and can be classified as transporters, channels, and receptors, although their predicted secondary structures are very much alike. Prominent members include the sulfonylurea receptor (SUR1) and the multidrug transporter (MDR1). SUR1 is a subunit of the pancreatic beta-cell K(ATP) channel and plays a key role in the regulation of glucose-induced insulin secretion. SUR1 binds ATP at NBF1, and ADP at NBF2 and the two NBFs work cooperatively. The pore-forming subunit of the pancreatic beta-cell K(ATP) channel, Kir6.2, is a member of the inwardly rectifying K(+) channel family, and also binds ATP. In this article, we present a model in which the activity of the K(ATP) channel is determined by the balance of the action of ADP, which activates the channel through SUR1, and the action of ATP, which stabilizes the long closed state by binding to Kir6.2. The concentration of ATP could also affect the channel activity through binding to NBF1 of SUR1. MDR1, on the other hand, is an ATP-dependent efflux pump which extrudes cytotoxic drugs from cells before they can reach their intracellular targets, and in this way confers multidrug resistance to cancer cells. Both NBFs of MDR1 can hydrolyze nucleotides, and their ATPase activity is necessary for drug transport. The interaction of SUR1 with nucleotides is quite different from that of MDR1. Variations in the interactions with nucleotides of ABC proteins may account for the differences in their functions.

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Sentences [show]
No. Sentence Comment
467 8-Azido-ATP binding with the wild-type MDR1 was inhibited by 100 WM NEM, while 8-azido-ATP binding with the C431A/C1074A mutant form was not, suggesting that the cysteines of Walker A motifs in both NBFs are responsible for the e¡ects of NEM on ATP binding. Login to comment
469 However, ATP-binding to the C1074A mutant form was signi'cantly reduced by the same treatment, similar to the wild-type MDR1. Login to comment
465 8-Azido-ATP binding with the wild-type MDR1 was inhibited by 100 WM NEM, while 8-azido-ATP binding with the C431A/C1074A mutant form was not, suggesting that the cysteines of Walker A motifs in both NBFs are responsible for the e&#a1;ects of NEM on ATP binding. Login to comment

[hide] Non-equivalent cooperation between the two nucleot... Biochim Biophys Acta. 1998 Aug 14;1373(1):131-6. 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., [PMID:9733949]

Abstract [show]
To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

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Sentences [show]
No. Sentence Comment
51 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 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 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 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 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 A, P-glycoprotein-S; B, C431A/C1074A; C, C431A; D, C1074A. Experiments were done in duplicate. Login to comment
64 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 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 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 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
77 A, P-glycoprotein-S; B, C431A/C1074A; C, C431A; D, C1074A. Experiments were done in triplicate. Fig. 4. Login to comment
94 The C431A/C1074A mutant form of P-glycoprotein was indistinguishable from the wild-type in its function. Login to comment
95 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 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
111 However, NEM treatment signi'cantly reduced 8-azido-ATP binding in the C1074A mutant P-glycoprotein. Login to comment