ABCMdb

PMID: 17187755

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., [PubMed]

Sentences

No. Mutations Sentence Comment

27 ABCC1 p.Lys1333Leu In contrast, mutations of the residue that should interact with the γ-phosphate of the bound ATP [28,29], such as K1333L [20], significantly decreased the ATP binding at the mutated NBD2 [23] and almost abolished the ATP-dependent solute transport activity completely. Login to comment 28 ABCC1 p.Lys1333Met ABCC1 p.Gly771Ala Accordingly, mutations of the residues that should interact with the γ-phosphate of the bound ATP [28,29], such as K1333M [19] or G771A [30], also almost abolished the ATP-dependent solute transport activity completely. Login to comment 40 ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 43 ABCC1 p.His1486Gln ABCC1 p.His1486Gln ABCC1 p.His1486Gln ABCC1 p.His1486Gln ABCC1 p.His1486Trp ABCC1 p.His1486Trp ABCC1 p.His1486Trp ABCC1 p.His1486Trp ABCC1 p.His827Tyr ABCC1 p.His827Tyr ABCC1 p.His827Tyr ABCC1 p.His827Tyr ABCC1 p.His1486Tyr ABCC1 p.His1486Tyr ABCC1 p.His1486Tyr ABCC1 p.His1486Tyr ABCC1 p.His827Trp ABCC1 p.His827Trp ABCC1 p.His827Trp ABCC1 p.His827Trp ABCC1 p.His827Gln ABCC1 p.His827Gln ABCC1 p.His827Gln ABCC1 p.His827Gln ABCC1 p.His827Asn ABCC1 p.His827Asn ABCC1 p.His827Asn ABCC1 p.His827Asn ABCC1 p.His1486Glu ABCC1 p.His1486Glu ABCC1 p.His1486Glu ABCC1 p.His1486Glu ABCC1 p.His1486Asp ABCC1 p.His1486Asp ABCC1 p.His1486Asp ABCC1 p.His1486Asp ABCC1 p.His827Asp ABCC1 p.His827Asp ABCC1 p.His827Asp ABCC1 p.His827Asp ABCC1 p.His827Glu ABCC1 p.His827Glu ABCC1 p.His827Glu ABCC1 p.His827Glu ABCC1 p.His1486Asn ABCC1 p.His1486Asn ABCC1 p.His1486Asn ABCC1 p.His1486Asn In addition, the following mutations as shown in Fig. 5A (H827D, H1486D, H827D/ H1486D, H827N, H1486N, H827N/H1486N, H827E, H1486E, H827E/ H1486E, H827Q, H1486Q, H827Q/H1486Q, H827Y, H1486Y, H827Y/ H1486Y, H827W, H1486W and H827W/H1486W) were also introduced into the full length MRP1 cDNA by using the following primers: H827D/forward, 5'-CGG ATC TTG GTC ACG GAC AGC ATG AGC TAC TTG-3'; H827D/ reverse, 5'-CAA GTA GCT CAT GCT GTC CGT GAC CAA GAT CCG-3'; H1486D/forward, 5'-GTC CTC ACC ATC GCC GAC CGG CTC AAC ACC ATC-3'; H1486D/reverse, 5'-GAT GGT GTT GAG CCG GTC GGC GAT GGT GAG GAC-3'; H827N/forward, 5'-CGG ATC TTG GTC ACG AAC AGC ATG AGC TAC TTG-3'; H827N/reverse, 5'-CAA GTA GCT CAT GCT GTT CGT GAC CAA GAT CCG-3'; H1486N/forward, 5'-GTC CTC ACC ATC GCC AAC CGG CTC AAC ACC ATC-3'; H1486N/reverse, 5'-GAT GGT GTT GAG CCG GTT GGC GAT GGT GAG GAC-3'; H827E/forward, 5'-CGG ATC TTG GTC ACG GAG AGC ATG AGC TAC TTG-3'; H827E/reverse, 5'-CAA GTA GCT CAT GCT CTC CGT GAC CAA GAT CCG-3'; H1486E/forward, 5'-GTC CTC ACC ATC GCC GAG CGG CTC AAC ACC ATC-3'; H1486E/reverse, 5'-GAT GGT GTT GAG CCG CTC GGC GAT GGT GAG GAC-3'; H827Q/forward, 5'- CGG ATC TTG GTC ACG CAG AGC ATG AGC TAC TTG-3'; H827Q/ reverse, 5'-CAA GTA GCT CAT GCT CTG CGT GAC CAA GAT CCG-3'; H1486Q/forward, 5'-GTC CTC ACC ATC GCC CAG CGG CTC AAC ACC ATC-3'; H1486Q/reverse, 5'-GAT GGT GTT GAG CCG CTG GGC GAT GGT GAG GAC-3'; H827Y/forward, 5'-CGG ATC TTG GTC ACG TAC AGC ATG AGC TAC TTG-3'; H827Y/reverse, 5'-CAA GTA GCT CAT GCT GTA CGT GAC CAA GAT CCG-3'; H1486Y/forward, 5'-GTC CTC ACC ATC GCC TAC CGG CTC AAC ACC ATC-3'; H1486Y/reverse, 5'-GAT GGT GTT GAG CCG GTA GGC GAT GGT GAG GAC-3'; H827W/forward, 5'-CGG ATC TTG GTC ACG TGG AGC ATG AGC TAC TTG-3'; H827W/reverse, 5'-CAA GTA GCT CAT GCT CCA CGT GAC CAA GAT CCG-3'; H1486W/forward, 5'-GTC CTC ACC ATC GCC TGG CGG CTC AAC ACC ATC-3'; H1486W/reverse, 5'-GAT GGT GTT GAG CCG CCA GGC GAT GGT GAG GAC-3'. Login to comment 90 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu ABCC1 p.His827Leu ABCC1 p.His827Leu 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 94 ABCC1 p.His1486Phe ABCC1 p.His827Phe In contrast, the Kd values of NBD1 or NBD2 from H827F or H1486F are not 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu 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 ABCC1 p.His1486Leu 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 ABCC1 p.His1486Leu 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 130 ABCC1 p.His1486Phe However, substitution of this histidine residue in H-loop with an aromatic F residue in either NBD1 (H827F) or NBD2 (H1486F) did not significantly alter the Kd (8-N3ATP) values at either NBD1 or NBD2 (Fig. 2E, F and Table 1), suggesting that replacement of the five-member imidazole ring with the six-member benzene ring did not significantly alter the local conformation in ATP binding pocket, especially the van der Waals interactions with the residues in D-loop of counterpart NBDs [29], and hydrogen-bond formation between the residue at 827 or 1486 and the γ- phosphate of ATP bound at that site played a minor role for ATP-binding. Login to comment 131 ABCC1 p.His1486Phe ABCC1 p.His1486Phe However, the double mutant H827F/H1486F did significantly increase the Kd (8-N3ATP) values for the H827F-mutated NBD1 and H1486F-mutated NBD2 (Fig. 2G and Table 1), presumably the tertiary structure alterations caused by these two mutations affect ATP binding at the mutated NBD1 and NBD2. Login to comment 134 ABCC1 p.His1486Phe Since histidine residue in H-loop may contribute to ATP hydrolysis by interacting with the putative catalytic base to form the "catalytic dyad" [29], we speculated that even though ATP can efficiently bind to the H1486F-mutated NBD2, but the bound ATP cannot be efficiently hydrolyzed. Login to comment 141 ABCC1 p.His827Phe ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Phe 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 ABCC1 p.His1486Leu ABCC1 p.His1486Phe Thus, although it was clear that 8-N3ATP bound to the H1486L- or H1486F-mutated NBD2 was not Fig. 2. Login to comment 146 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe The effects of H827L, H1486L, H827L/H1486L, H827F, H1486F or H827F/H1486F mutations on 8-N3ATP binding at NBD1 and NBD2. Login to comment 149 ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Leu 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 164 ABCC1 p.His1486Gln ABCC1 p.His1486Gln ABCC1 p.His1486Trp ABCC1 p.His1486Trp ABCC1 p.His827Tyr ABCC1 p.His827Tyr ABCC1 p.His1486Tyr ABCC1 p.His1486Tyr ABCC1 p.His827Trp ABCC1 p.His827Trp ABCC1 p.His827Gln ABCC1 p.His827Gln ABCC1 p.His827Asn ABCC1 p.His827Asn ABCC1 p.His1486Glu ABCC1 p.His1486Glu ABCC1 p.His1486Asp ABCC1 p.His1486Asp ABCC1 p.His827Asp ABCC1 p.His827Asp ABCC1 p.His827Glu ABCC1 p.His827Glu ABCC1 p.His1486Asn ABCC1 p.His1486Asn In order to test this hypothesis, H827D, H1486D, H827D/H1486D, H827N, H1486N, H827N/H1486N, H827E, H1486E, H827E/H1486E, H827Q, H1486Q, H827Q/H1486Q, H827Y, H1486Y, H827Y/ H1486Y, H827W, H1486W and H827W/H1486W mutations (Fig. 5A) were introduced into full length of MRP1 cDNA in pNUT/MRP1/His and expressed in BHK cells at 37 °C. Login to comment 166 ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Tyr ABCC1 p.His827Trp ABCC1 p.His827Gln ABCC1 p.His827Asn ABCC1 p.His827Asp ABCC1 p.His827Glu 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 168 ABCC1 p.His1486Gln ABCC1 p.His1486Glu For example, H1486Q had similar transport activity (112%) as that of wild-type MRP1 (Fig. 5C), whereas H1486E dropped this activity to ~36% of the wild-type (Fig. 5C). Login to comment 169 ABCC1 p.His1486Asp ABCC1 p.His1486Asn H1486N had ~60% of wild-type MRP1 transport activity, whereas H1486D dropped this activity to ~20%. Login to comment 170 ABCC1 p.His1486Phe Interestingly, although H1486F dropped this transport activity to ~15%, substitution of the Fig. 4. Login to comment 178 ABCC1 p.His1486Trp In contrast, substitution of this H residue in H-loop with another aromatic W residue (H1486W), which might also form the hydrogen-bond between the nitrogen atom in the ring structure of tryptophan and the γ-phosphate of the bound ATP and the putative catalytic base E1455, dropped this transport activity to ~21% (Fig. 5C). Login to comment 189 ABCC1 p.Lys684Arg ABCC1 p.Asp1454Asn For example, ATP binding to wild-type MRP1 can transport the bound LTC4 from high to low affinity site whereas ATP binding to the K684R- or the D1454N-mutated MRP1 cannot [30]. Login to comment 190 ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Tyr ABCC1 p.His827Trp ABCC1 p.His827Gln ABCC1 p.His827Asn ABCC1 p.His827Asp ABCC1 p.His827Glu 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 ABCC1 p.His827Leu ABCC1 p.His827Leu 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 204 ABCC1 p.His827Phe However, in contrast, H827F mutation did not significantly affect the Kd (8-N3ATP) values of the mutated NBD1 and the un-mutated NBD2 (Fig. 2 and Table 1). Login to comment 205 ABCC1 p.His827Leu 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 ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu 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 210 ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Gln ABCC1 p.His1486Trp ABCC1 p.His1486Tyr ABCC1 p.His1486Glu ABCC1 p.His1486Asp ABCC1 p.His1486Asn 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 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Leu 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 213 ABCC1 p.His1486Phe However, why H1486F mutation did not significantly affect 8-N3ATP binding (Fig. 2F and Table 1) but severely impaired the ATP hydrolysis at the mutated NBD2 (Fig. 3 and Table 2) was not well understood. Login to comment 215 ABCC1 p.His1486Phe ABCC1 p.His1486Tyr ABCC1 p.His1486Tyr The only difference between H1486F and H1486Y is that H1486Y has an extra hydroxyl group on the benzene ring. Login to comment 216 ABCC1 p.His1486Phe ABCC1 p.His1486Tyr H1486F mutation almost abolished the ATP-dependent LTC4 transport activity completely (Figs. 1C and 4B), whereas H1486Y mutation retained ~41% of wild-type LTC4 transport activity (Fig. 5C), presumably the hydroxyl group on the benzene ring of tyrosine provides a possibility to form hydrogen-bond with the γ-phosphate of the bound ATP. Login to comment 223 ABCC1 p.His1486Gln In contrast, substitution of the histidine residue in H-loop with an amino acid that might potentially form the hydrogen-bonds should support the ATP hydrolysis and ATP-dependent solute transport. Indeed, H1486Q-mutated MRP1 is fully active (Fig. 5C), implying that the hydrogen-bond formations between Q1486 and the γ-phosphate of the bound ATP or the putative catalytic base E1455 are as strong as in wild-type. Login to comment 224 ABCC1 p.His1486Gln ABCC1 p.His1486Gln ABCC1 p.His1486Gln ABCC1 p.His1486Glu ABCC1 p.His1486Asn ABCC1 p.His1486Asn ABCC1 p.His1486Asn However, H1486E exerts ~36% of wild-type MRP1 transport activity (Fig. 5C), presumably the carboxyl group of E1486 cannot form strong hydrogen-bonds with the γ-phosphate of the bound ATP and the putative catalytic base E1455 in NBD2. Accordingly, although H1486N has the same side group as the H1486Q mutation, except that the length of the side chain in H1486N is one carbon (or 1.541 Å) shorter than in H1486Q, the transport activity of H1486N dropped from ~112% (H1486Q) to ~60% (Fig. 5C), implying that the distance between two groups forming hydrogen-bond also plays an important role. Login to comment 225 ABCC1 p.His1486Glu ABCC1 p.His1486Asp The transport activity of H1486D dropped from ~36% (H1486E) to ~20% (Fig. 5C), consistent with the above conclusion. Login to comment 227 ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His1486Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Phe ABCC1 p.His827Leu ABCC1 p.His827Leu ABCC1 p.His827Leu ABCC1 p.His827Leu 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