ABCMdb

ABCC1 p.Tyr1302Cys

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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
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
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
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
145 Aromatic Residue Interacting with ATP Adenine Moiety in MRP1 48509 Y1302C-mutated (Fig. J) NBD2, indicates that substitution of the aromatic residue with a polar amino acid greatly decreases the affinity for ATP at this mutated NBD1. 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
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
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: 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

220 Considering the data accumulated from other NBD2 mutants, such as Y1302C, E1455Q, H1486F and H1486L, the conformational alterations caused by the K1333L mutation may not be the only reason preventing ATP hydrolysis at the mutated site. 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