ABCMdb

PMID: 18636743

Yang R, Scavetta R, Chang XB
Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.
Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18., 2008-08-12 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. Login to comment 4 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Login to comment 7 ABCC1 p.Ser1334Thr In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport. Login to comment 12 ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu In contrast, however, substitution of the corresponding Walker B D1454 in NBD2 with the hydrophobic residue leucine (D1454L/E1455L) did not cause misfolding of the mutated MRP1 protein (8), suggesting distinct structures in NBD1 and NBD2 of MRP1. Login to comment 15 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His In order to test this hypothesis, we have substituted the Walker A S1334 with an A (S1334A), a C (S1334C), a D (S1334D), an H (S1334H), an N (S1334N), or a T (S1334T). Login to comment 16 ABCC1 p.Ser1334Thr Indeed, all of these mutants, except S1334T, almost completely abolished the ATP-dependent LTC4 transport, indicating that the interaction of the hydroxyl group at S1334 with Mg·ATP † This work was supported by a grant from the National Cancer Institute, National Institutes of Health (CA89078 to X.C.). Login to comment 36 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His ABCC1 p.Ser1334His The forward and reverse primers used to introduce these mutations are as follows: S1334A/forward, 5'-CG GGA GCT GGG AAG GCG TCC CTG ACC CTG GGC-3'; S1334A/ reverse, 5'-GCC CAG GGT CAG GGA CGC CTT CCC AGC TCC CG-3'; S1334C/forward, 5'-CG GGA GCT GGG AAG TGC TCC CTG ACC CTG GGC-3'; S1334C/reverse, 5'-GCC CAG GGT CAG GGA GCA CTT CCC AGC TCC CG-3'; S1334D/forward, 5'-CG GGA GCT GGG AAG GAC TCC CTG ACC CTG GGC-3'; S1334D/reverse, 5'-GCC CAG GGT CAG GGA GTC CTT CCC AGC TCC CG-3'; S1334H/forward, 5'-CG GGA GCT GGG AAG CAC TCC CTG ACC CTG GGC-3'; S1334H/reverse, 5'-GCC CAG GGT CAG GGA GTG CTT CCC AGC TCC CG-3'; S1334N/forward, 5'-CG GGA GCT GGG AAG AAC TCC CTG ACC CTG GGC-3'; S1334N/reverse, 5'-GCC CAG GGT CAG GGA GTT CTT CCC AGC TCC CG-3'; S1334T/forward, 5'-CG GGA GCT GGG AAG ACG TCC CTG ACC CTG GGC-3'; S1334T/reverse, 5'-GCC CAG GGT CAG GGA CGT CTT CCC AGC TCC CG-3'. Login to comment 82 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His The results in Figure 1A showed that the 190 kDa wild-type MRP1 protein is resistant to endoglycosidase H digestion whereas the minor band is not, indicating that the majority of wild-type MRP1 protein is complex-glycosylated in vivo. All of the mutants, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, have a major band resistant to the endoglycosidase H digestion but sensitive to the PNGase F digestion (Figure 1B), indicating that all of these mutants mainly form complex-glycosylated mature proteins at 37 °C in vivo. Login to comment 88 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His As shown in Figure 2B, S1334A-, S1334C-, S1334D-, S1334H-, or S1334N-mutated MRP1 almost completely abolished the ATP-dependent LTC4 transport, whereas S1334T mutation, which retained the hydroxyl group at this position, exerted ~175% of wild-type MRP1 transport activity, suggesting that the hydroxyl group at this position plays a crucial role for the ATP binding/hydrolysis and ATP-dependent solute transport by MRP1. Login to comment 89 ABCC1 p.Ser1334Thr The Km (Mg·ATP) Value of S1334T Is Significantly Higher than That of Wild-Type MRP1. Login to comment 93 ABCC1 p.Ser1334Thr As shown in Figure 3 and Table 1, the Km(Mg ·ATP) of S1334T (104 µM Mg·ATP) is significantly higher (with a P value of 0.0024) than that of wild-type MRP1 (61 µM Mg·ATP). Login to comment 94 ABCC1 p.Ser1334Thr In addition, the Vmax (LTC4) value of S1334T (618 pmol mg-1 min-1 ) is also significantly higher (with a P value of 0.0016) than that of wild-type MRP1 (342 pmol mg-1 min-1 ), consistent with the results derived from a solution containing 4 mM ATP and 10 mM MgCl2 (Figure 2B). Login to comment 97 ABCC1 p.Ser1334Thr Indeed, all of the mutants, except S1334T, almost completely abolished the ATP-dependent LTC4 transport (Figure 2B), perhaps owing to affecting the ATP binding/hydrolysis. Login to comment 98 ABCC1 p.Ser1334Thr ABCC1 p.Ser1334Thr In order to prove this hypothesis, a similar amount of MRP1 protein in 10 µg of membrane protein (see Figure 4 legend) was used to label them with either [R-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP (with the same specific activity as [R-32 P]- Table 1: Km (Mg·ATP) and Vmax (LTC4) Values of Wild-Type and Mutant MRP1s Vmax (pmol mg-1 min-1)a Km (µM)a MRP1 342.0 ( 50.3 61.0 ( 3.3 S1334T 618.3 ( 11.9 104.0 ( 8.3 a Km (Mg·ATP) and Vmax (LTC4) values (n ) 3) for wild type and S1334T were derived from the corresponding Michaelis-Menten curves shown in Figure 3. Login to comment 99 ABCC1 p.Ser1334Thr The P values for comparison of Vmax (LTC4) and Km (Mg·ATP) of wild type versus S1334T are 0.0016 and 0.0024. Login to comment 101 ABCC1 p.Ser1334Thr However, the labeling intensity of wild-type MRP1 or S1334T with [R-32 P]-8-N3ATP, including the intact [R-32 P]-8-N3ATP bound at NBD1 and the vanadate-trapped ATP hydrolysis product [R-32 P]-8-N3ADP at NBD2 (8), is stronger than that of [γ-32 P]-8-N3ATP labeling including the intact [γ-32 P]-8-N3ATP bound at NBD1 and NBD2 and the 32 P-phospharylated products (8), implying that the majority of the bound ATP at NBD2 is hydrolyzed. Login to comment 112 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His The intensities of the MRP1 bands were determined by a scanning densitometer. The mean ratios (n ) 2, including the results derived from 400 and 800 ng of protein), considering the amount of wild-type MRP1 as 1, of the mutant proteins are as follows: S1334A, 1.46 ( 0.06; S1334C, 0.84 ( 0.14; S1334D, 1.07 ( 0.69; S1334H, 1.17 ( 0.16; S1334N, 0.97 ( 0.08; S1334T, 1.30 ( 0.11. Login to comment 115 ABCC1 p.Ser1334Thr FIGURE 3: S1334T mutation increases the Km (ATP) for ATP-dependent LTC4 transport. Login to comment 118 ABCC1 p.Ser1334Thr Since the amounts of MRP1 proteins determined in Figure 2A were different, the amounts of membrane vesicles used in these experiments were adjusted to a similar amount by adding varying amounts of membrane vesicles prepared from parental BHK cells: 3 µg of wild-type MRP1; 2.308 µg of S1334T plus 0.692 µg of BHK. Login to comment 120 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His labeling intensity of MRP1 mutants, including S1334A, S1334C, S1334D, S1334H, and S1334N, with [R-32 P]-8-N3ATP is weaker than that of wild type or S1334T (Figure 4), indicating that these mutants without the hydroxyl group at this position affect ATP binding, hydrolysis, and vanadate-dependent [R-32 P]-8-N3ADP trapping. Login to comment 121 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His Comparison of labeling intensities with [R-32 P]-8-N3ATP and [γ-32 P]-8-N3ATP further supports this conclusion: (1) The labeling intensity of MRP1 mutants, including S1334A, S1334C, S1334D, S1334H, and S1334N, with [R-32 P]-8-N3ATP is either similar to or weaker than that of [γ-32 P]-8-N3ATP labeling (Figure 4), indicating that the bound ATP in these mutants is not efficiently hydrolyzed. Login to comment 122 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His (2) The intensity of [γ-32 P]-8-N3ATP labeling on the mutants, including S1334A, S1334C, S1334D, S1334H, and S1334N, is either stronger than wild type or similar to wild type (Figure 4), implying that most of those [γ-32 P]-8-N3ATP labeling might occur at the unmutated NBD1 (8). Login to comment 123 ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Thr In order to prove this hypothesis, S1334A and S1334T mutations were introduced into pDual/N-half/C-half expression plasmid (12, 13) and expressed in Sf21 cells. Membrane vesicles prepared from Sf21 cells (Figure 5A) were used to do ATP-dependent LTC4 transport and the photoaffinity labeling with either [R-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP. Login to comment 124 ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Thr As shown in Figure 5B, the ATP-dependent LTC4 transport activity of wild type N-half plus S1334T-mutated C-half is more or less similar to that of wild-type, whereas the transport activity of the wild type N-half plus S1334A-mutated C-half is significantly reduced. Login to comment 125 ABCC1 p.Ser1334Thr ABCC1 p.Ser1334Thr ABCC1 p.Ser1334Thr The [γ-32 P]-8-N3ATP labeling of wild type NBD1-containing N-half fragment is much higher than the corresponding labeling in wild type or S1334T-mutated NBD2-containing C-half fragment (Figure 5C), whereas the [R-32 P]-8-N3ATP labeling of wild type or S1334T-mutated NBD2-containing C-half fragment in the presence of vanadate is much higher than the corresponding labeling in NBD1-containing N-half fragment (Figure 5C), implying that ATP bound to wild type or S1334T-mutated NBD2 is efficiently hydrolyzed. Login to comment 126 ABCC1 p.Ser1334Ala In contrast, the [γ-32 P]-8-N3ATP or [R-32 P]-8-N3ATP labeling of the unmutated NBD1-containing N-half fragment is stronger than the corresponding labeling on the S1334A-mutated NBD2-containing C-half (Figure 5C), suggesting that most of the [γ-32 P]-8-N3ATP or [R-32 P]-8-N3ATP labeling occurred at FIGURE 4: Substitution of S1334 with an amino acid that eliminates the hydroxyl group at this position impaired ATP binding/hydrolysis at the mutated NBD2. Login to comment 134 ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Thr The intensities of the MRP1 bands were determined by a scanning densitometer. The mean ratios (n ) 2, including the results derived from 200 and 300 ng of protein), considering the amount of wild type MRP1 as 1, of the mutant proteins are as follows: S1334A, 0.68 ( 0.06; S1334T, 0.90 ( 0.06. Login to comment 136 ABCC1 p.Ser1334Ala (C) S1334A-mutated MRP1 affects both ATP binding and hydrolysis at the mutated NBD2. Login to comment 137 ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Thr Wild type MRP1 or S1334A- or S1334T-mutated MRP1 was introduced into the pDual/N-half/C-half and expressed in Sf21 insect cells (12, 25). Login to comment 141 ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Ala In addition, the labeling intensity of the S1334A-mutated NBD2-containing C-half with [γ-32 P]- 8-N3ATP is stronger than that of corresponding labeling with [R-32 P]-8-N3ATP (Figure 5C), implying that the trace amount of ATP bound to the S1334A-mutated NBD2 is not efficiently hydrolyzed. Login to comment 145 ABCC1 p.Ser1334Ala However, based on the labeling intensities of the S1334A mutant in Figure 5C, the labeling of the mutated NBD2 fragments, after the partial trypsin digestion, might not be detected. Login to comment 153 ABCC1 p.Lys1333Leu However, if a mutant significantly affected the ATP binding, such a mutant, such as the Walker A mutation of K1333L, would abrogate the ATP-enhanced nucleotide binding at NBD2 (16). Login to comment 155 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His As shown in Figure 6, 2.5 or 10 µM ATP significantly enhanced the vanadate-dependent [R-32 P]-8-N3ADP trapping in wild type MRP1 (Figure 6A) or in S1334T (Figure 6G), whereas there is no enhancement effect in S1334A (Figure 6B), S1334C (Figure 6C), S1334D (Figure 6D), S1334H (Figure 6E), or S1334N (Figure 6F), suggesting that these mutations significantly impaired the nucleotide binding at the mutated NBD2. Login to comment 160 ABCC1 p.Glu1455Gln ABCC1 p.Ser1334Thr In order to test this hypothesis, membrane vesicles containing these mutants were used to label them with 3 H-LTC4 in the presence or absence of 1 mM ATP and vanadate. As shown in Figure 7, the 3 H-LTC4 labeling on wild type MRP1 and S1334T as well as E1455Q is almost completely inhibited by the presence of ATP and vanadate. Login to comment 161 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His In contrast, the labeling on S1334A, S1334C, S1334D, S1334H, or S1334N is only partially diminished, implying that ATP binding or vanadate-dependent ATP hydrolysis product ADP trapping at the mutated NBD2 is impaired. Login to comment 163 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His In contrast, mutation of the corresponding residue in NBD2 of MRP1, such as S1334A, S1334C, S1334D, S1334H, or S1334N, has no effect on the protein folding and processing (Figure 1), implying that the stereo structure surrounding the hydroxyl group of S1334 in NBD2 might be different from that of NBD1. Login to comment 166 ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu Indeed, elimination of the carboxyl group at D1454, such as D1454L/E1455L (8), had no effect on the protein folding and processing. Login to comment 172 ABCC1 p.Ser1334Ala Indeed, substitution of S1334 with an alanine residue (S1334A) reduced the ATP-dependent LTC4 transport activity to ~10% of wild type (Figure 2B). Login to comment 175 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His group (S1334C) exerted slightly higher transport activity than that of S1334A, S1334D, S1334H, or S1334N (Figure 2B), implying that the sulfhydryl group in S1334C might weakly bind to the magnesium cofactor and the -phosphate of the bound ATP. Login to comment 176 ABCC1 p.Ser1334Thr In contrast to other mutants, substitution of the S1334 with a threonine (S1334T) increased ATP-dependent LTC4 transport to ~175% of the wild-type MRP1 (Figures 2B and 3 and Table 1), indicating that the interactions of the hydroxyl group at the position of 1334 with the Mg2+ cofactorandthe -phosphateoftheboundMg·ATP(1,2,4-7) play a crucial role for ATP binding/hydrolysis at NBD2 and ATP-dependent LTC4 transport. Login to comment 178 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His The labeling intensities of [R-32 P]-8-N3ATP in wild type or S1334T are stronger than that of [γ-32 P]-8-N3ATP (Figure 4), whereas the labeling intensities of [R-32 P]-8-N3ATP in S1334A, S1334C, S1334D, S1334H, or S1334N are slightly weaker than that of [γ-32 P]-8-N3ATP (Figure 4), suggesting that even though there might be a trace amount of ATP binding at the mutated NBD2, the bound nucleotide might not be efficiently hydrolyzed. Login to comment 179 ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Thr Indeed, a certain amount of [R-32 P]-8-N3ATP hydrolysis product [R-32 P]-8-N3ADP was trapped in wild-type or S1334T-mutated NBD2, whereas there was no detectable amount of [R-32 P]-8-N3ADP trapped in S1334A-mutated NBD2 (Figure 5C). Login to comment 180 ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His All of the S1334 mutants, including S1334A (Figure 6B), S1334C (Figure 6C), S1334D (Figure 6D), S1334H (Figure 6E), and S1334N (Figure 6F), lost the ATP-enhanced vanadate-dependent ADP trapping (16, 17) at the mutated NBD2, suggesting that these mutations significantly decreased the affinity for nucleotide at the mutated NBD2. Login to comment 181 ABCC1 p.Ser1334Thr ABCC1 p.Ser1334Thr In contrast, although substitution of the S1334 with a threonine (S1334T) introduced an extra methyl group, but kept the hydroxyl group at the original position, the S1334T mutation retained the ATP-enhanced vanadate-dependent ADP trapping (Figure 6G), indicating that the interactions of the hydroxyl group at position 1334 with the Mg2+ cofactor and the -phosphate of the bound Mg· ATP (1, 2, 4-7) play a very important role for ATP binding at NBD2. Login to comment 182 ABCC1 p.Lys1333Glu ABCC1 p.Lys684Glu ABCC1 p.Gly771Ala ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334His The reduced nucleotide binding at the mutated NBD2, such as S1334A, S1334C, S1334D, S1334H, and S1334N, significantly decreased the ability to inhibit the LTC4 binding (Figure 7), similar to the mutations of K684E, G771A, or K1333E (19). Login to comment 183 ABCC1 p.Glu1455Gln This inhibitory effect is directly related to nucleotide binding, but not hydrolysis, at NBD1 and NBD2 since the E1455Q mutation, which abolished ATP hydrolysis but not binding (12), exerted similar ability as wild type MRP1 to inhibit the LTC4 binding (Figure 7). Login to comment