ABCMdb

ABCC1 p.Tyr1302Trp

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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
45 Y1302W and Y1302C mutations were generated by using the same strategy as for W653Y. Login to comment
46 The forward and reverse primers for Y1302W and Y1302C are: Y1302W/forward, 5Ј-CGG AAC TAC TGC CTG CGC TGG CGA GAG GAC CTG GAC TTC-3Ј; Y1302W/reverse, 5Ј-GAA GTC CAG GTC CTC TCG CCA GCG CAG GCA GTA GTT CCG-3Ј; Y1302C/forward, 5Ј-CGG AAC TAC TGC CTG CGC TGC CGA GAG GAC CTG GAC TTC-3Ј; and Y1302C/reverse, 5Ј-GAA GTC CAG GTC CTC TCG GCA GCG CAG GCA GTA GTT CCG-3Ј. 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
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
124 Substitution of the aromatic residue Tyr1302 in NBD2 with a different aromatic Trp residue, Y1302W, also slightly decreased Km (ATP) value; however, the Vmax (LTC4) value was not changed (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
127 Table I shows that the Km (ATP) values for W653C, W653C/Y1302W, and W653C/Y1302C are 4.5-, 4.2-, and 22.8-fold higher than that of wild-type MRP1. Login to comment
130 Their Km (ATP) values are even higher than those N-half mutants containing only one cysteine replacement, such as W653C and W653C/Y1302W (Table I). Login to comment
131 However, their Vmax (LTC4) values are less than those of the N-half mutants containing only one cysteine replacement, such as W653C and W653C/Y1302W (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
141 In contrast, the Kd value for W653C-mutated NBD1, co-expressed with Y1302W-mutated NBD2, could not be determined because of very weak labeling of this mutated fragment (Fig. 4D), presumably with a very high Kd value. Login to comment
142 Interestingly, the Kd value for Y1302W-mutated NBD2, co-expressed with W653C-mutated NBD1, increased from 33 (the Kd of wild-type NBD2) to 139 ␮M ATP (Table II), presumably because of the negative effect of W653C-mutated NBD1 on the Y1302W-mutated NBD2. 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
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
170 Interestingly, substitution of the aromatic residue Trp653 with a polar cysteine residue, such as W653C, W653C/Y1302W, and W653C/Y1302C, greatly decreased their affinity for ATP but did not abolish ATP binding completely and lead to very high Km (ATP) and Vmax (LTC4) values in ATP-dependent LTC4 transport (Table I). Login to comment
171 We have found that release of bound ATP, no matter whether it is hydrolyzed or not, from the NBD1 of MRP1 facilitates the protein to start a new cycle of ATP-dependent solute transport.2 The increased Vmax values of the W653C-mutated NBD1s, including W653C, W653C/Y1302W, and W653C/Y1302C, can also be explained by this hypothesis. Login to comment