ABCMdb

PMID: 15355964

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 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr 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 4 ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys 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 13 ABCC7 p.Gly551Asp Additional evidence comes from the naturally occurring CFTR mutant G551D, a mutation of the Gly551 residue in the ABC signature motif of NBD1 causing cystic fibrosis (21-23). Login to comment 15 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The corresponding mutations in MRP1, G771D in NBD1 and G1433D in NBD2, almost completely abolished ATP-dependent LTC4 transport (20). Login to comment 31 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr 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 ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys 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 ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Tyr1302Cys Y1302W and Y1302C mutations were generated by using the same strategy as for W653Y. Login to comment 46 ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Trp653Tyr 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 ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr 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 ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr 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 ABCC1 p.Trp653Tyr 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 124 ABCC1 p.Tyr1302Trp 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Cys 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 ABCC1 p.Trp653Tyr ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Tyr ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Tyr1302Trp ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Tyr1302Cys 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 ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys ABCC1 p.Trp653Cys 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 174 ABCC1 p.Trp653Tyr ABCC1 p.Tyr1302Cys ABCC1 p.Tyr1302Cys 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