ABCMdb

ABCB1 p.Cys431Ala

Predicted by SNAP2:	A: N (53%), 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 (59%), N: D (85%), P: D (91%), Q: D (91%), R: D (91%), S: N (57%), T: D (80%), 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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[hide] Analysis of the properties of the N-terminal nucle... Biochemistry. 2000 May 9;39(18):5518-26. Booth CL, Pulaski L, Gottesman MM, Pastan I
Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.
Biochemistry. 2000 May 9;39(18):5518-26., 2000-05-09 [PMID:10820025]

Abstract [show]
Human P-glycoprotein, the MDR1 gene product, requires both Mg(2+)-ATP binding and hydrolysis to function as a drug transporter; however, the mechanism(s) defining these events is not understood. In the present study, we explored the nature of Mg(2+)-ATP binding in the N-terminal nucleotide-binding domain of human P-glycoprotein and identified the minimal functional unit required for specific ATP binding. Recombinant proteins encompassing amino acids within the region beginning at 348 and ending at 707 were expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions, and renatured by rapid dilution. The ability of ATP to interact with these proteins was examined by use of the photoactive ATP analogue [alpha-(32)P]-8-azido-ATP. Photoaffinity labeling of recombinant proteins identified the region between amino acids 375 and 635 as the region necessary to obtain specific ATP-binding properties. Specific protein labeling was saturable, enhanced by Mg(2+), and inhibited by ATP. Recombinant proteins confined within the region beginning at amino acid 392 and ending at amino acid 590 demonstrated nonspecific [alpha-(32)P]-8-azido-ATP labeling. Nonspecific labeling was not enhanced by Mg(2+) and was inhibited only by high concentrations of ATP. Using a D555N mutated protein, we found that the conserved aspartate residue in the Walker B motif plays a role in magnesium-enhanced ATP-binding. Taken together, these data define the region of the N-terminal nucleotide-binding domain of P-glycoprotein that is required for specific ATP binding and suggest that magnesium may play a role in stabilizing the ATP-binding site.

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No. Sentence Comment
55 The resulting expression vector was used as a template for site-directed mutagenesis to introduce a C431A mutation. Login to comment
58 The coding sequence for the C431A mutant primer was 5'-GTTGGAAACAGTG- GCGCTGGGAAGAGCACA-3'. Login to comment
61 The plasmid obtained contained a sequence encoding MDR1 amino acids 358-707 with a C431A mutation and was labeled pET- NBD1MDR1-C431A (358-707). Login to comment
62 A plasmid, containing a Table 1: Cloning Summary for Expression Vectors plasmid name cloning vector PCR primers (5' f 3')a restriction enzymesb pET-NBD1MDR1-C431A (358-707) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: AAAGGATCCTCATTCAGTTAAATTTAGCTTCATAATCCT B: BamHI pET-NBD1MDR1-C431A/D555N (358-707) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: AAAGGATCCTCATTCAGTTAAATTTAGCTTCATAATCCT B: BamHI pET-NBD1MDR1-C431A (412-574) pET23a A: GTTAAGATGCATATGGGCCTGAACCTGAAGGTGCAGAGT A: NdeI B: ACCTTTGAATTCTCAATCCAGAGCCACCTGAACCACTGC B: EcoRI pET-NBD1MDR1-C431A (358-590) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: ATTGGATCCTCAAGACAAACGATGAGCTATCACAATGGT B: BamHI pET-NBD1MDR1-C431A (358-635) pET23a A: GAGATATACATATGGCAAGAGGAGCA A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI pET-NBD1MDR1-C431A (409-635) pET23a A: GAGACATATGATCTTGAAGGGCCTGAACCTG A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI pET-NBD1MDR1-C431A (375-707) pET23a A: GAGACATATGATTGACAGCTATTCGAAGAGT A: NdeI B: TCTCCTCGAGCTATTCAGTTAAATTTAGCTTCAT B: XhoI pET-NBD1MDR1-C431A (392-707) pET23a A: GAGACATATGTTGGAATTCAGAAATGTTCAC A: NdeI B: TCTCCTCGAGCTATTCAGTTAAATTTAGCTTCAT B: XhoI pET-NBD1MDR1-C431A (348-635) pET23a A: GAGACATATGGCATCTCCAAGCATTGAAGCATTTGCAAAT- GCAAGAGGAGCAGCT A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI a A, forward primer; B, reverse primer. Login to comment
64 sequence encoding MDR1 amino acids 358-707 with both C431A and D555N mutations, was prepared as described above, with the pTM1-MDR1-D555N vector bearing the MDR1 coding sequence with a D555N mutation as the primary template, and was labeled pET-NBD1MDR1-C431A/ D555N (358-707). Login to comment
65 pET-NBD1MDR1-C431A was used as the template for construction of subsequent expression vectors as indicated in Table 1. Login to comment
104 Recombinant proteins, encompassing amino acids 412-574, 358-590, and 358-707 of human P-glycoprotein each with a C431A mutation, encoded by pET-NBD1MDR1-C431A-based plasmids (Table 1) were expressed and purified from E. coli, purified from inclusion bodies under denaturing conditions, and renatured by rapid dilution. Login to comment
105 The C431A mutant was used to prevent disulfide interactions. Login to comment
206 In the present work, human P-glycoprotein cDNAs with a C431A mutation were utilized to express a series of recombinant proteins containing NBD1 in E. coli. Login to comment
249 The C431A and D555N mutations within the Walker A and Walker B motifs, respectively, are identified by vertical bars. Login to comment

[hide] A novel MDR1 GT1292-3TG (Cys431Leu) genetic variat... AAPS J. 2010 Dec;12(4):548-55. Epub 2010 Jul 10. Crouthamel MH, Wu D, Yang Z, Ho RJ
A novel MDR1 GT1292-3TG (Cys431Leu) genetic variation and its effect on P-glycoprotein biologic functions.
AAPS J. 2010 Dec;12(4):548-55. Epub 2010 Jul 10., [PMID:20623213]

Abstract [show]
P-glycoprotein (P-gp) is a membrane-bound transporter protein that is encoded by the human multidrug resistance gene MDR1 (ABCB1). P-gp recognizes a wide range of xenobiotics, is pivotal in mediating cancer drug resistance, and plays an important role in limiting drug penetration across the blood-brain barrier. MDR1 genetic variation can lead to changes in P-gp function and may have implications on drug pharmacokinetics. We have identified a novel MDR1 (GT1292-3TG) (Cys431Leu) genetic variation through systematic profiling of subjects with leukemia. The cellular and transport function of this variation was investigated with recombinant human embryonic kidney cells expressing MDR1. Compared with the wild type, MDR1 (GT1292-3TG) recombinant cells exhibited a lower drug resistance phenotype for a panel of chemotherapeutic agents. When compared with wild type, MDR1 (GT1292-3TG) recombinant cells exposed exhibited a 75% decrease in IC for doxorubicin (162.6 +/- 17.4 to 37.9 +/- 2.6 nM) and a 50% decrease in IC(50) for paclitaxel (155.7 +/- 27.5 to 87.7 +/- 9.2 nM), vinblastine (128.0 +/- 15.9 to 65.9 +/- 5.1 nM), and vincristine (593.7 +/- 61.8 to 307.3 +/- 17.0 nM). The effects of the Cys431Leu variation, due to MDR1 (GT1292-3TG) nucleotide transition, on P-gp-dependent intracellular substrate accumulation appeared to be substrate dependent where doxorubicin, vinblastine, and paclitaxel exhibit an increased accumulation (p < 0.05), while verapamil and Hoechst33342 exhibit a decreased intracellular concentration compared with wild type (p < 0.05). Collectively, these data suggest MDR1 (GT1292-3TG) variation of P-gp may reduce drug resistance and that subjects with this genotype undergoing chemotherapy with drugs that are transported by P-gp could potentially be more responsive to therapy than those with MDR1 wild-type genotype.

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No. Sentence Comment
146 In addition, the Cys431Ala mutation did not impact ATPase activity. Login to comment
147 When the cysteine was re-inserted at amino acid 431 as the only cysteine within the Cys-less (Cys431Ala) P-gp molecule, covalent modification of the thiol side chain with N-ethylmaleimide inhibited activity of P-gp, suggesting that both NBDs must be active for function (22). Login to comment
156 Although a direct comparison of Cys431Ala and Cys431Leu with WT P-gp in HEK recombinant cells remained, such a study is beyond the scope of this report. Login to comment

[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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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
468 8-Azido-ATP binding of the C431A mutant form appeared not to be a¡ected by treatment with 100 WM NEM. 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
466 8-Azido-ATP binding of the C431A mutant form appeared not to be a&#a1;ected by treatment with 100 WM NEM. 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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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
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
110 The C431A mutant P-glycoprotein showed no change in 8-azido-ATP binding after NEM treatment. Login to comment