ABCMdb

ABCC1 p.Trp653Cys

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, Y: D,

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[hide] Mutation of the aromatic amino acid interacting wi... J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7. 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 [PMID:15355964]

Abstract [show]
Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. 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. 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. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.

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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

[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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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

[hide] Replacement of the positively charged Walker A lys... Biochem J. 2006 Jul 1;397(1):121-30. Buyse F, Hou YX, Vigano C, Zhao Q, Ruysschaert JM, Chang XB
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.
Biochem J. 2006 Jul 1;397(1):121-30., 2006-07-01 [PMID:16551273]

Abstract [show]
MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. 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. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>>>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.

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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

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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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