ABCMdb

ABCC1 p.Trp653Tyr

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Publications

PMID: 15355964 [PubMed] Zhao Q et al: "Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1."

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. Login to comment
31 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. Login to comment
42 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). Login to comment
43 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. Login to comment
45 Y1302W and Y1302C mutations were generated by using the same strategy as for W653Y. Login to comment
59 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. Login to comment
65 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. Login to comment
66 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. Login to comment
67 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. Login to comment
101 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. Login to comment
116 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. Login to comment
117 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. Login to comment
121 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. Login to comment
123 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). Login to comment
125 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). Login to comment
134 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). Login to comment
144 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. . Login to comment
146 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. Login to comment
147 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. Login to comment
152 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. Login to comment
174 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). Login to comment

PMID: 19063607 [PubMed] Qin L et al: "Residues responsible for the asymmetric function of the nucleotide binding domains of multidrug resistance protein 1."

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. Login to comment
37 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. Login to comment
55 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). Login to comment
56 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. Login to comment
58 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. Login to comment
147 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. Login to comment
149 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. Login to comment
151 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. Login to comment
156 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. Login to comment
172 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. Login to comment
180 (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. Login to comment
181 (C) Immunoblots of the wt and mutant dual halves of MRP1 (dh D793E and dh W653Y/D793E/P794A). Login to comment
192 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. Login to comment
195 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). Login to comment
224 Introduction of a W653Y mutation in wt MRP1 has been reported to have no detectable effect on ATP binding and hydrolysis (49). Login to comment
294 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). Login to comment
295 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). Login to comment
298 Consistent with this suggestion, the W653Y and P794A mutations increased VO4-dependent nucleotide trapping at NBS1 of MRP1D793E. Login to comment
301 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). Login to comment
328 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. Login to comment