ABCMdb

ABCC1 p.Trp653Cys

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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

4 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. 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
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
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
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
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
157 Because of the very weak labeling of the W653C-mutated NBD1 (D and J), there is no plotting shown in E and K. Aromatic Residue Interacting with ATP Adenine Moiety in MRP148510 the W653C-mutated NBD1 and Y1302C-mutated NBD2 lead to a very high Km (ATP) value (1573 ␮M ATP in Table I) of the double mutated MRP1 in ATP-dependent LTC4 transport. 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
172 The rationales are: 1) although the binding of ATP to the W653C-mutated NBD1 requires higher nucleotide concentration than that of the wild type because of lower affinity of W653C-mutated NBD1 for that nucleotide, ATP still can easily bind to the W653C-mutated NBD1 because of the ATP concentration in mM range; 2) the much higher Kd value of the W653C-mutated NBD1 also indicates that the release rate of the bound ATP from the W653C-mutated NBD1 is much higher than that of wild type; and 3) release of the bound ATP from the W653C-mutated NBD1 resets the MRP1 protein back to its original conformational state so that the molecule can start a new cycle of ATP-dependent solute transport, leading to a higher Vmax (LTC4) value than that of wild-type MRP1. Login to comment

PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."

No. Sentence Comment

324 [145] HsMRP1 W653C, Y1302C Mutations decreased affinity for ATP and increased Kd values for ATP binding or Km values for ATP in ATP-dependent LT C4 transport. Login to comment

PMID: 16551273 [PubMed] Buyse F et al: "Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis."

No. Sentence Comment

2 Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. Login to comment

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

266 Interestingly, substitution of the aromatic residue W653, which was predicted [150] and proved [151] to interact with the adenine ring of the bound ATP, with a polar C 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-dependent LTC4 transport, but significantly increased the rate of ATP-dependent LTC4 transport [152]. Login to comment