ABCMdb

ABCB1 p.Cys431Ala

None has been submitted yet.

Publications

PMID: 10820025 [PubMed] Booth CL et al: "Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein."

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

PMID: 20623213 [PubMed] Crouthamel MH et al: "A novel MDR1 GT1292-3TG (Cys431Leu) genetic variation and its effect on P-glycoprotein biologic functions."

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

PMID: 10581363 [PubMed] Ueda K et al: "Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins."

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
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

PMID: 9733949 [PubMed] Takada Y et al: "Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein."

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
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