ABCC1 p.Trp653Tyr
Predicted by SNAP2: | A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), Y: D (85%), |
Predicted by PROVEAN: | A: D, C: 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, Y: D, |
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[hide] Mutation of the aromatic amino acid interacting wi... J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7. Zhao Q, Chang XB
Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1.
J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7., 2004-11-19 [PMID:15355964]
Abstract [show]
Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.
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No. Sentence Comment
3 Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport.
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ABCC1 p.Trp653Tyr 15355964:3:83
status: NEW31 The substitution of Trp653 or Tyr1302 with a different aromatic amino acid, such as W653Y or Y1302W, has little effect on the Km values for ATP in ATP-dependent LTC4 transport.
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ABCC1 p.Trp653Tyr 15355964:31:84
status: NEW42 The tryptophan residue at position 653 was mutated to either tyrosine or cysteine (see Fig. 1A, W653Y or W653C) by using the forward/reverse primers and the QuikChange site-directed mutagenesis kit from Stratagene (19).
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ABCC1 p.Trp653Tyr 15355964:42:96
status: NEW43 The forward and reverse primers for W653Y and W653C are: W653Y/forward, 5Ј-AGG AAT GCC ACA TTC ACC TAT GCC AGG AGC GAC CCT CCC-3Ј; W653Y/reverse, 5Ј-GGG AGG GTC GCT CCT GGC ATA GGT GAA TGT GGC ATT CCT-3Ј; W653C/forward, 5Ј-AGG AAT GCC ACA TTC ACC TGT GCC AGG AGC GAC CCT CCC-3Ј; and W653C/reverse: 5Ј-GGG AGG GTC GCT CCT GGC ACA GGT GAA TGT GGC ATT CCT-3Ј.
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ABCC1 p.Trp653Tyr 15355964:43:36
status: NEWX
ABCC1 p.Trp653Tyr 15355964:43:57
status: NEWX
ABCC1 p.Trp653Tyr 15355964:43:143
status: NEW45 Y1302W and Y1302C mutations were generated by using the same strategy as for W653Y.
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ABCC1 p.Trp653Tyr 15355964:45:77
status: NEW59 The samples are: Wild-type MRP1, wild-type N-half co-expressed with wild-type C-half; W653Y, W653Y-mutated N-half ϩ wild-type C-half; Y1302W, wild-type N-half ϩ Y1302W-mutated C-half; W653Y/Y1302W, W653Y-mutated N-half ϩ Y1302W-mutated C-half; W653C, W653C-mutated N-half ϩ wild-type C-half; W653C/Y1302W, W653C-mutated N-half ϩ Y1302W-mutated C-half; Y1302C, wild-type N-half ϩ Y1302C-mutated C-half; W653Y/Y1302C, W653Y-mutated N-half ϩ Y1302C-mutated C-half; and W653C/Y1302C, W653C-mutated N-half ϩ Y1302C-mutated C-half.
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ABCC1 p.Trp653Tyr 15355964:59:86
status: NEWX
ABCC1 p.Trp653Tyr 15355964:59:93
status: NEWX
ABCC1 p.Trp653Tyr 15355964:59:196
status: NEWX
ABCC1 p.Trp653Tyr 15355964:59:210
status: NEWX
ABCC1 p.Trp653Tyr 15355964:59:438
status: NEWX
ABCC1 p.Trp653Tyr 15355964:59:452
status: NEW65 The ratios of the band intensities with the same amount of total membrane proteins, for example, 0.4 g of wild-type N-half (co-expressed with wild-type C-half) versus 0.4 g of the W653Y-mutated N-half (co-expressed with wild-type C-half) were determined.
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ABCC1 p.Trp653Tyr 15355964:65:196
status: NEW66 Because the ratio of N-half, for example, W653Y-mutated N-half, is similar to that of the wild-type C-half co-expressed with W653Y-mutated N-half, the mean ratio of the protein expression includes N-half and C-half.
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ABCC1 p.Trp653Tyr 15355964:66:42
status: NEWX
ABCC1 p.Trp653Tyr 15355964:66:125
status: NEW67 Aromatic Residue Interacting with ATP Adenine Moiety in MRP148506 binations, a KpnI-RsrII fragment containing C-half, for example, with a Y1302W mutation, was cloned into a KpnI-RsrII fragment containing pDual vector DNA and N-half, for example, with a W653Y mutation, to generate W653Y/Y1302W.
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ABCC1 p.Trp653Tyr 15355964:67:254
status: NEWX
ABCC1 p.Trp653Tyr 15355964:67:282
status: NEW101 To test this possibility, membrane vesicles containing wild-type and mutated MRP1s (Fig. 1B) were utilized to do ATP-dependent LTC4 transport in the presence of 50 M ATP, which is within the range of the Km values for ATP (Km (ATP)) of wild-type N-half ϩ wild-type C-half mediated LTC4 transport (34).2 Fig. 2 shows that the transport activity of W653Y-mutated N-half ϩ wild-type C-half is ϳ2-fold of wild type, whereas the transport activity of wild-type N-half ϩ Y1302W- FIG. 2.
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ABCC1 p.Trp653Tyr 15355964:101:361
status: NEW116 Aromatic Residue Interacting with ATP Adenine Moiety in MRP148508 mutated C-half or W653Y-mutated N-half ϩ Y1302W-mutated C-half is similar to that of wild type, indicating that substitution of an aromatic residue with a different aromatic amino acid, such as Trp to Tyr or Tyr to Trp, does not have a significant negative effect on the ATP binding and hydrolysis.
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ABCC1 p.Trp653Tyr 15355964:116:85
status: NEW117 In contrast, the substitution of the aromatic residue, no matter whether it is in NBD1 or NBD2, with a nucleophilic cysteine residue, such as W653C-mutated N-half ϩ wild-type C-half, W653C-mutated N-half ϩ Y1302W-mutated C-half, wild-type N-half ϩ Y1302C-mutated C-half, W653Y-mutated N-half ϩ Y1302C-mutated C-half, or W653C-mutated N-half ϩ Y1302C-mutated C-half, greatly decreased the ATP-dependent LTC4 transport activities (Fig. 2), implying that both aromatic residues, Trp653 in NBD1 and Tyr1302 in NBD2, are involved in ATP binding.
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ABCC1 p.Trp653Tyr 15355964:117:289
status: NEW121 The transport activity of W653Y-mutated N-half ϩ wild-type C-half is much higher than that of wild type, whereas the activities of wild-type N-half ϩ Y1302W-mutated C-half and W653Y-mutated N-half ϩ Y1302W-mutated C-half are similar to that of the wild type (Fig. 3A), which are consistent with the results in Fig. 2.
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ABCC1 p.Trp653Tyr 15355964:121:26
status: NEWX
ABCC1 p.Trp653Tyr 15355964:121:188
status: NEW123 Substitution of the aromatic Trp653 with a different aromatic amino acid (Tyr), i.e. W653Y, slightly decreased the Km (ATP) value but increased Vmax (LTC4) value 2-fold (Table I).
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ABCC1 p.Trp653Tyr 15355964:123:85
status: NEW125 Interestingly, the switch of those two aromatic residues in NBD1 and NBD2, W653Y-mutated N-half ϩ Y1302W-mutated C-half, did not alter the Km (ATP) value but slightly increased the Vmax (LTC4) value (Table I).
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ABCC1 p.Trp653Tyr 15355964:125:75
status: NEW134 To test whether these substitutions really alter their affinities for ATP, membrane vesicles containing wild-type N-half (Trp653 ) ϩ wild-type C-half (Tyr1302 ), W653C-mutated N-half ϩ Y1302W-mutated C-half, W653Y-mutated N-half ϩ Y1302C-mutated C-half, and W653C-mutated N-half ϩ Y1302C-mutated C-half were labeled with [␣-32 P]8-N3ATP on ice to determine their Kd values (Fig. 4).
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ABCC1 p.Trp653Tyr 15355964:134:220
status: NEW144 The very weak labeling of W653C-mutated NBD1, including the labeling of W653C-mutated NBD1 co-expressed with Y1302W-mutated (Fig. 4D) and TABLE I The mean Km (M ATP) and Vmax (pmol of LTC4/mg of protein/min) of wild-type and mutant MRP1s Protein Amino acid at position Km a Vmax a 653 (NBD1) 1302 (NBD2) M ATP pmol⅐mg-1 ⅐min-1 Wild-type MRP1 Trp Tyr 69.0 Ϯ 5.2 389.0 Ϯ 32.9 W653Y Tyr Tyr 46.5 Ϯ 0.7 820.0 Ϯ 21.2 Y1302W Trp Trp 47.7 Ϯ 2.1 386.7 Ϯ 60.3 W653Y/Y1302W Tyr Trp 65.5 Ϯ 0.7 499.0 Ϯ 5.66 W653C Cys Tyr 311.7 Ϯ 46.5 881.7 Ϯ 78.5 W653C/Y1302W Cys Trp 290.0 Ϯ 10.0 1353.3 Ϯ 203.4 Y1302C Trp Cys 340.0 Ϯ 42.4 700.0 Ϯ 70.7 W653Y/Y1302C Tyr Cys 395.0 Ϯ 15.0 380.3 Ϯ 66.9 W653C/Y1302C Cys Cys 1573.3 Ϯ 25.2 782.7 Ϯ 20.5 a The Km (n ϭ 3) and Vmax (n ϭ 3) values were derived from Fig. .
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ABCC1 p.Trp653Tyr 15355964:144:416
status: NEWX
ABCC1 p.Trp653Tyr 15355964:144:517
status: NEWX
ABCC1 p.Trp653Tyr 15355964:144:740
status: NEW146 The Kd value of W653Y-mutated NBD1, co-expressed with Y1302C-mutated NBD2, increased from 9 (the Kd of wild-type NBD1) to 29 (Table II), presumably because of the negative effect of Y1302C-mutated NBD2.
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ABCC1 p.Trp653Tyr 15355964:146:16
status: NEW147 The Kd values of the Y1302C-mutated NBD2 (Table II) increased from 33 (the Kd of wild-type NBD2) to 122 (the Kd of Y1302C-mutated NBD2 co-expressed with W653Y-mutated NBD1) and 160 M ATP (the Kd of Y1302C-mutated NBD2 co-expressed with W653C-mutated NBD1), indicating that substitution of this aromatic residue with a polar amino acid also decreased the affinity for ATP at the mutated NBD2.
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ABCC1 p.Trp653Tyr 15355964:147:153
status: NEW152 A, D, G, and J, autoradiograms of wild-type N-half ϩ wild-type C-half, W653C-mutated N-half ϩ Y1302W-mutated C-half, W653Y-mutated N-half ϩ Y1302C-mutated C-half, and W653C-mutated N-half ϩ Y1302C-mutated C-half.
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ABCC1 p.Trp653Tyr 15355964:152:129
status: NEW174 Whether this decreased affinity for nucleotide in NBD2 also facilitates the molecule to start a new cycle of ATP-dependent solute transport is not clear, because the Y1302C-mutated NBD2 co-expressed with wild-type NBD1 increased its Vmax (LTC4) 1.8-fold (Table I), whereas the Y1302C-mutated NBD2 co-expressed with W653Y-mutated NBD1 did not have a significant effect on its Vmax (LTC4) value (Table I).
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ABCC1 p.Trp653Tyr 15355964:174:315
status: NEW[hide] Residues responsible for the asymmetric function o... Biochemistry. 2008 Dec 30;47(52):13952-65. Qin L, Zheng J, Grant CE, Jia Z, Cole SP, Deeley RG
Residues responsible for the asymmetric function of the nucleotide binding domains of multidrug resistance protein 1.
Biochemistry. 2008 Dec 30;47(52):13952-65., 2008-12-30 [PMID:19063607]
Abstract [show]
The two nucleotide binding domains (NBDs) of ATP binding cassette (ABC) transporters dimerize to form composite nucleotide binding sites (NBSs) each containing Walker A and B motifs from one domain and the ABC "C" signature from the other. In many ABC proteins, the NBSs are thought to be functionally equivalent. However, this is not the case for ABCC proteins, such as MRP1, in which NBS1 containing the Walker A and B motifs from the N-proximal NBD1 typically binds ATP with high affinity but has low hydrolytic activity, while the reverse is true of NBS2. A notable feature of NBD1 of the ABCC proteins is the lack of a catalytic Glu residue following the core Walker B motif. In multidrug resistance protein (MRP) 1, this residue is Asp (D793). Previously, we demonstrated that mutation of D793 to Glu was sufficient to increase ATP hydrolysis at NBS1, but paradoxically, transport activity decreased by 50-70% as a result of tight binding of ADP at the mutated NBS1. Here, we identify two atypical amino acids in NBD1 that contribute to the retention of ADP. We found that conversion of Trp653 to Tyr and/or Pro794 to Ala enhanced transport activity of the D793E mutant and the release of ADP from NBS1. Moreover, introduction of the P794A mutation into wild-type MRP1 increased transport of leukotriene C(4) approximately 2-fold. Molecular dynamic simulations revealed that, while the D793E mutation increased hydrolysis of ATP, the presence of the adjacent Pro794, rather than the more typical Ala, decreased flexibility of the region linking Walker B and the D-loop, markedly diminishing the rate of release of Mg(2+) and ADP. Overall, these results suggest that the rate of release of ADP by NBD1 in the D793E background may be the rate-limiting step in the transport cycle of MRP1.
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No. Sentence Comment
7 We found that conversion of Trp653 to Tyr and/or Pro794 to Ala enhanced transport activity of the D793E mutant and the release of ADP from NBS1.
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ABCC1 p.Trp653Tyr 19063607:7:28
status: NEW37 These studies show that conversion of Trp653 to Tyr and/or Pro794 to Ala is sufficient to restore ADP release by the D793E mutant and to increase LTC4 transport activity to the level of the wild-type (wt) protein.
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ABCC1 p.Trp653Tyr 19063607:37:38
status: NEW55 Additional mutations (W653Y, V680T, P794A, V1432G, L1437R, and C1439S) were introduced into the dh D793E vector by site-directed mutagenesis using a QuikChange II kit (Stratagene, La Jolla, CA).
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ABCC1 p.Trp653Tyr 19063607:55:22
status: NEW56 The forward primers for W653Y, V680T, P794A, V1432G, L1437R, and C1439S were 5'-GCCACATTCACCTATGCCAGGAGC- GAC-3', 5'-GTGGTGGGCCAGACGGGCTGCGGAAAG-3', 5'-TTTA- CCTTCGATGAGGCCCTCTCAGCAGTGGA-3', 5'-AGAAC- CTCAGTGGCGGGCAGCGC-3', and 5'-CAGCGCCAGC- GTGTGAGCCTAGCCCG-3', respectively.
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ABCC1 p.Trp653Tyr 19063607:56:24
status: NEW58 Briefly, to make W653Y, V680T, and P794A mutations, a BamHI-XbaI fragment from the dh D793E construct was cloned into pBluescript II KS (+) vector (Fermentas International Inc., Burlington, Ontario, Canada), and mutagenesis PCR reactions were performed according to the manufacturer`s manual.
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ABCC1 p.Trp653Tyr 19063607:58:17
status: NEW147 Determination of the effects of the additional mutations revealed that two second mutations, P794A and W653Y, were each able to restore activity of the D793E mutant to levels that were comparable to or somewhat higher than wt MRP1.
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ABCC1 p.Trp653Tyr 19063607:147:103
status: NEW149 To investigate if the effects of the P794A and W653Y mutations on LTC4 transport activity were additive, both mutations were introduced into the D793E mutant.
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ABCC1 p.Trp653Tyr 19063607:149:47
status: NEW151 Thus the positive effect of the individual W653Y and P794A mutations on activity of the D793E mutant MRP1 was not additive to any detectable extent.
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ABCC1 p.Trp653Tyr 19063607:151:43
status: NEW156 Overall, the effects of the additional mutations on the labeling profile of the D793E mutant were minimal with the possible exception of the W653Y mutation which modestly decreased the photolabeling of NBD1 relative to NBD2.
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ABCC1 p.Trp653Tyr 19063607:156:141
status: NEW172 However, both second mutations that restored LTC4 transport by D793E MRP1, W653Y and P794A, decreased VO4-independent photolabeling of NBD1 and enhanced the extent of VO4-dependent trapping at this site.
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ABCC1 p.Trp653Tyr 19063607:172:75
status: NEW180 (A) Immunoblot of membrane vesicle proteins isolated from Sf21 cells expressing wt or mutant dual halves of MRP1 (dh D793E, dh W653Y/D793E, dh V680T/D793E, dh D793E/P794A, dh D793E/V1432G, and dh D793E/L1437R/C1439S) or Sf21 cells infected with a control vector expressing -gus.
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ABCC1 p.Trp653Tyr 19063607:180:127
status: NEW181 (C) Immunoblots of the wt and mutant dual halves of MRP1 (dh D793E and dh W653Y/D793E/P794A).
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ABCC1 p.Trp653Tyr 19063607:181:74
status: NEW192 In view of the effects of the W653Y and P794A mutations on VO4-dependent nucleotide trapping at NBS1, we examined their influence on the ability of MRP1 D793E to shift between high-and low-affinity conformations.
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ABCC1 p.Trp653Tyr 19063607:192:30
status: NEW195 Consistent with their effects on VO4-dependent nucleotide trapping by the D793E mutant protein (Figure 5), both the W653Y and P794D second mutations restored the ability of ATP plus VO4 to induce the shift from high- to low-affinity LTC4 binding (Figure 6A).
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ABCC1 p.Trp653Tyr 19063607:195:116
status: NEW224 Introduction of a W653Y mutation in wt MRP1 has been reported to have no detectable effect on ATP binding and hydrolysis (49).
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ABCC1 p.Trp653Tyr 19063607:224:18
status: NEW294 Among the mutants tested, two secondary mutations, W653Y and P794A, increased LTC4 transport activity of the D793E mutant to levels equal to or greater than that of the wt protein (Figure 3).
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ABCC1 p.Trp653Tyr 19063607:294:51
status: NEW295 Neither of the W653Y or P794A mutations altered azido-ATP binding by MRP1D793E under nonhydrolytic conditions, but both decreased VO4-independent tight binding of ADP at 37 °C by NBD1 (Figure 5).
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ABCC1 p.Trp653Tyr 19063607:295:15
status: NEW298 Consistent with this suggestion, the W653Y and P794A mutations increased VO4-dependent nucleotide trapping at NBS1 of MRP1D793E.
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ABCC1 p.Trp653Tyr 19063607:298:37
status: NEW301 However, the extent of VO4-dependent ADP trapping at NBS2 in the W653Y/D793E and P794A/D793E mutants was reduced relative to wt MRP1, as observed with the original D793E mutant (26).
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ABCC1 p.Trp653Tyr 19063607:301:65
status: NEW328 These effects on transport are consistent with our observations with MRP1D793E, but our results strongly suggest that the increase in activity is attributable to an increase in the release of ADP from the D793E/W653Y mutant protein, that may not have been apparent from studies of wt MRP1.
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ABCC1 p.Trp653Tyr 19063607:328:211
status: NEW