ABCMdb

ABCC1 p.His1486Leu

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (91%), E: D (91%), F: D (91%), G: D (85%), I: D (91%), K: D (85%), L: D (91%), M: D (85%), N: D (80%), P: D (91%), Q: D (80%), R: D (85%), S: D (80%), T: D (85%), V: D (85%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[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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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
231 Interestingly, although replacement of His1486 (H-loop in NBD2), which forms a hydrogen bond with the γ -phosphate of the bound ATP in NBD2, with an aromatic phenylalanine residue or a hydrophobic leucine residue has different effects on ATP binding, i.e. the H1486F mutation had no effect on ATP binding at the unmutated NBD1 and the H1486F-mutated NBD2, whereas the H1486L mutation increased the Kd value by approx. Login to comment
232 2-fold, the mutation of either H1486F or H1486L abolished the ATP-dependent LTC4 transport activity (R. Yang and X.-b. Chang, unpublished work). Login to comment

[hide] Hydrogen-bond formation of the residue in H-loop o... Biochim Biophys Acta. 2007 Feb;1768(2):324-35. Epub 2006 Nov 18. Yang R, Chang XB
Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport.
Biochim Biophys Acta. 2007 Feb;1768(2):324-35. Epub 2006 Nov 18., [PMID:17187755]

Abstract [show]
MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the gamma-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.

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No. Sentence Comment
40 The histidine residue at position of 827 or 1486 was mutated to either leucine or phenylalanine (Fig. 1A, H827L, H827F, H1486L or H1486F) by using the forward/reverse primers and the QuikChange site-directed mutagenesis kit from Stratagene [20]. Login to comment
41 The forward and reverse primers for these mutations are: H827L/ forward, 5'-CGG ATC TTG GTC ACG CTC AGC ATG AGC TAC TTG-3'; H827L/reverse, 5'-CAA GTA GCT CAT GCT GAG CGT GAC CAA GAT CCG-3'; H1486L/forward, 5'-GTC CTC ACC ATC GCC CTC CGG CTC AAC ACC ATC-3'; H1486L/reverse, 5'-GAT GGT GTT GAG CCG GAG GGC GAT GGT GAG GAC-3'; H827F/forward, 5'-CGG ATC TTG GTC ACG TTC AGC ATG AGC TAC TTG-3'; H827F/reverse, 5'-CAA GTA GCT CAT GCT GAA CGT GAC CAA GAT CCG-3'; H1486F/forward, 5'-GTC CTC ACC ATC GCC TTC CGG CTC AAC ACC ATC-3'; H1486F/reverse, 5'-GAT GGT GTT GAG CCG GAA GGC GAT GGT GAG GAC-3'. Login to comment
42 These mutations (Fig. 4A, H827L, H827F, H1486L, H1486F, H827L/H1486L or H827F/H1486F) were also introduced into full length MRP1 cDNA by using the QuikChange site-directed mutagenesis kit [20] with the plasmid DNA pNUT/MRP1/His [31] as a template and the forward/reverse oligonucleotides shown above as primers. Login to comment
90 Substitution of H827 in H-loop of NBD1 with an amino acid that prevents formation of the hydrogen-bond with the γ-phosphate of the bound ATP has no effect on the ATP-dependent LTC4 transport whereas corresponding mutation in NBD2 almost abolishes the ATP-dependent LTC4 transport activity completely Structural analyses of bacterial ABC transporter NBDs revealed that the histidine residue in H-loop contributes to Table 1 Mean Kd (8-N3ATP) values of wild-type and mutant MRP1 Samples Kd a of NBD1 (μM 8-N3ATP) Kd of NBD2 (μM 8-N3ATP) N-half C-half Wild-type Wild-type 11.7±2.7 32.4±2.5 H827L Wild-type 59.5±0.5b 60.0±3.0 Wild-type H1486L 31.0±0.8 58.7±1.3 H827L H1486L 59.7±1.3 53.0±0.8 H827F Wild-type 12.3±0.5 32.7±0.9 Wild-type H1486F 11.5±0.5 33.0±1.0 H827F H1486F 58.0±1.0 51.5±0.5 a The Kd (8-N3ATP) values (n=3) of wild-type and mutant MRP1s were derived from Fig. 2. Login to comment
93 b Statistical analysis indicated that the Kd values of NBD1 from H827L, H1486L, H827L/H1486L and H827F/H1486F or the Kd values of NBD2 from H827L, H1486L, H827L/H1486L and H827F/H1486F are significantly different from that of wild-type NBD1 (11.7 μM 8-N3ATP) or wild-type NBD2 (32.4 μM 8-N3ATP). Login to comment
99 These mutants made in N-half, including H827L or H827F in NBD1, or in C-half, including H1486L or H1486F in NBD2 or H827L/ H1486L and H827F/H1486F in N-half and C-half, were expressed simultaneously in Sf21 insect cells. Login to comment
102 Mutations at NBD1, including H827L and H827F, did not have a significant effect on the ATP-dependent LTC4 transport, whereas mutations at NBD2, including H1486L, H1486F, H827L/H1486L and H827F/H1486F, greatly decreased the ATP-dependent LTC4 transport activity (Fig. 1C). Login to comment
103 Table 2 Ratio of [γ-32 P]-8N3ATP/[α-32 P]-8N3ATP labeling at NBD1 or NBD2 Samples Ratioa at NBD1 Ratio at NBD2 N-half C-half Wild-type Wild-type 2.12±0.26b 0.70±0.14 H827L Wild-type 1.92±0.45 0.90±0.02 Wild-type H1486L 1.89±0.34 1.26±0.12 H827L H1486L 2.12±0.32 1.38±0.33 H827F Wild-type 2.17±0.15 0.93±0.02 Wild-type H1486F 2.15±0.10 1.42±0.14 H827F H1486F 1.98±0.20 1.42±0.19 a The ratio (n=4) of [γ-32 P]-8-N3ATP/[α-32 P]-8-N3ATP labeling at NBD1 or NBD2 was derived from Fig. 3. Login to comment
108 In contrast, the ratio values of NBD2 from H1486L, H827L/H1486L, H1486F and H827F/H1486F are significantly different from that of wild-type NBD2 (0.70). Login to comment
113 The definition of H827L means that the H827L-mutated N-half is co-expressed with wild-type C-half, whereas H1486L means that the H1486L-mutated C-half is co-expressed with wild-type N-half. Login to comment
128 Interestingly, this mutation also increased the Kd (8-N3ATP) value of NBD2 (60.0 μM, Fig. 2B and Table 1) even though the amino acid sequences in NBD2 were not changed, presumably the decreased ATP binding at the H827L-mutated NBD1 altered the affinity for 8-N3ATP at the un-mutated NBD2. Accordingly, mutation of H1486L increased the Kd (8-N3ATP) value of NBD2 from 32.4 μM (wild-type NBD2, Fig. 2A and Table 1) to 58.7 μM (Fig. 2C and Table 1) and the Kd (8-N3ATP) value of the unmutated NBD1 from 11.7 μM (wild-type NBD1) to 31 μM (Fig. 2C and Table 1). Login to comment
129 Thus, it is not surprising that the double mutant H827L/H1486L increased both Kd (8-N3ATP) values for NBD1 and NBD2 (Fig. 2D and Table 1). Login to comment
142 In contrast, however, the ratio of [γ-32 P]-8-N3ATP/[α-32 P]-8-N3ATP labeling at the mutated NBD2, regardless of whether this H residue is mutated to an L or an F, including H1486L, H827L/H1486L, H1486F and H827F/H1486F, is significantly increased (Table 2), implying that the 8-N3ATP bound to the mutated NBD2 is not efficiently hydrolyzed, consistent with their low LTC4 transport activities (Fig. 1C). Login to comment
144 The non-efficient ATP hydrolysis at the H1486L- or H1486F-mutated NBD2 is not caused by global conformational alterations Since the N-halves and C-halves were expressed in Sf21 cells at 27 °C, the results derived from western blot in Fig. 1B cannot tell whether these N-halves and C-halves were globally folded properly or not. Login to comment
145 Thus, although it was clear that 8-N3ATP bound to the H1486L- or H1486F-mutated NBD2 was not Fig. 2. Login to comment
146 The effects of H827L, H1486L, H827L/H1486L, H827F, H1486F or H827F/H1486F mutations on 8-N3ATP binding at NBD1 and NBD2. Login to comment
152 Since complex-glycosylated form of membrane-bound protein in mammalian cells was used as a criterion to indicate that the membrane glycoprotein was globally folded properly, the H827L, H1486L, H827L/H1486L, H827F, H1486F and H827F/H1486F mutations were introduced into full length of MRP1 cDNA in pNUT/MRP1/His [31] and expressed in BHK cells at 37 °C. Login to comment
154 However, the amount of H827L/H1486L or H827F/H1486F double-mutated MRP1 is slightly less than that of wild-type MRP1 (Fig. 4A), implying that these double mutants might affect the stability of the protein in BHK cells. Login to comment
161 The ATP-dependent LTC4 transport activities (Fig. 4B) of these full-length mutants, including H827L, H1486L, H827L/H1486L, H827F, H1486F and H827F/H1486F, are consistent with the results derived from N-half+C-half (Fig. 1C), reinforcing the conclusions made in Figs. 1, 2 and 3. Login to comment
210 In contrast, mutations in NBD2, such as H1486L, H1486F, H1486D, H1486N, H1486E, H1486Q, H1486Y and H1486W, have variant effects on the ATP-dependent LTC4 transport (Figs. 1C, 4B and 5C)). Login to comment
211 Substitution of the H1486 in H-loop with residues that would not form hydrogen-bond with the γ- phosphate of the bound ATP in NBD2, including H1486L-, H1486F-, H827L/H1486L- or H827F/H1486F-mutated MRP1, severely impaired the ATP-dependent LTC4 transport, presumably because the bound ATP at the mutated NBD2 (Fig. 2C, D, F and G) were not efficiently hydrolyzed (Fig. 3 and Table 2). Login to comment
227 Interestingly, the ATP-dependent LTC4 transport activities of the double mutants, including H827L/H1486L, H827F/ H1486F, H827L/H1486L, H827F/H1486F, H827L/H1486L, H827F/H1486F, H827L/H1486L and H827F/H1486F, are either similar to or lower than their corresponding mutations in NBD2 (Figs. 4B and 5C), supporting our conclusion that ATP hydrolysis at NBD2 plays a crucial role for ATP-dependent solute transport by MRP1 [21]. Login to comment