ABCMdb

ABCC1 p.His827Leu

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (91%), F: D (95%), G: D (91%), I: D (95%), K: D (95%), L: D (95%), M: D (91%), N: D (80%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (91%),
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] 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
141 In addition, H827L or H827F mutation did not significantly affect the ratio of the unmutated NBD2 (Table 2), consistent with their high LTC4 transport activities (Fig. 1C). 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
149 Panels B, C, D, E, F and G show the results derived from H827L-mutated N-half+wild-type C-half, wild-type N-half+H1486L-mutated C-half, H827L-mutated N-half+H1486L-mutated C-half, H827F-mutated N-half+wild-type C-half, wild-type N-half+H1486F-mutated C-half and H827F-mutated N-half+H1486F-mutated C-half. 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
166 Consistent with the H827L or H827F mutation in NBD1, all of the NBD1 mutants, including H827D, H827N, H827E, H827Q, H827Y and H827W, have transport activities similar to that of wild-type MRP1 (Fig. 5C), implying that no matter whether or not these NBD1 mutants form hydrogen-bonds with these components, none of them has a significant effect on the ATP hydrolysis at the un-mutated NBD2 and the ATP-dependent solute transport. Login to comment
190 H827L-, H827F-, H827D-, H827N-, H827E-, H827Q-, H827Y- and H827W-mutated MRP1 form complex glycosylated mature protein in BHK cells at 37 °C (Figs. 4A and 5B), implying that these mutations in NBD1 did not significantly alter the protein conformation. Login to comment
191 In addition, BHK cells expressing H827L-mutated MRP1 exerted similar resistance to daunomycin as wild-type (data not shown), implying that H827L-mutated MRP1 was properly routed to the plasma membrane. Login to comment
205 These results were interpreted as that: (1) proper conformation of the ATP binding pocket may play a major role for ATP binding, whereas hydrogen-bond formation between the residue at 827 and the γ-phosphate of the bound ATP may play a minor role for ATP binding; (2) H827L mutation may induce micro- conformational changes in ATP binding pocket that decrease affinity for ATP at the mutated NBD1 and then subsequently at the un-mutated NBD2; (3) substitution of the five-member imidazole ring in histidine with six-member benzene ring in phenylalanine definitely prevented formation of hydrogen bonds with the γ-phosphate of the bound ATP and the putative catalytic base D793, but retained van der Waals interactions with the residues in D-loop of NBD2 [29]. Login to comment
208 Since mutations of H827L and H827F also prevent the formation of this "catalytic dyad" between D793 and L827 or F827, fully functional H827L- or H827F-mutated MRP1 suggests that formation of the "catalytic dyad" between D793 and H827 is not important for MRP1 function. 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

[hide] Molecular mechanism of ATP-dependent solute transp... Methods Mol Biol. 2010;596:223-49. Chang XB
Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.
Methods Mol Biol. 2010;596:223-49., [PMID:19949927]

Abstract [show]
Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem 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. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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No. Sentence Comment
212 We have found that substitution of the H residue in H-loop of MRP1-NBD1 with a residue, such as H827L or H827F, that avoids the interactions with the putative catalytic base D793 in NBD1 does not have a significant effect on the ATP-dependent LTC4 transport, whereas substitution of the H1486 residue in H-loop of NBD2 with the residues that potentially form hydrogen bond with the putative catalytic base E1455 has yielded functional MRP1 proteins with variant effects on the ATP-dependent LTC4 transport (186). Login to comment