ABCMdb

PMID: 18088596

Yang R, Scavetta R, Chang XB
The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function.
Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29., [PubMed]

Sentences

No. Mutations Sentence Comment

21 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala However, substitution of the acidic amino acid D792 in Walker B motif with a hydrophobic residue, such as D792L- or D792A-mutated MRP1 [20,23], caused misfolding of the protein and prevented further analysis of the mutated protein. Login to comment 24 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala However, this mechanism of protein folding may not be applied to the misfolding caused by substitution of the acidic amino acid with a hydrophobic residue, such as D792L- or D792A-mutated MRP1 [20,23]. Login to comment 27 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala We suspected that D792A or D792L mutation abolished the hydrogen-bond formation between D792 and S685 and resulted in misfolding of the mutated MRP1 protein. Login to comment 29 ABCC1 p.Ser685Thr In this report we have mutated S685 to T, A, C, H, D or N and found that only S685T mutation formed complex-glycosylated mature protein, implying that the interaction between the hydroxyl group at 685 in Walker A motif and the carboxyl group at 792 in Walker B motif of NBD1 plays a crucial role for MRP1 protein folding. Login to comment 30 ABCC1 p.Ser685Thr In addition, all these mutants, except S685T, are temperature-sensitive. Login to comment 31 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn ABCC1 p.Ser685Asp However, even if they form mature protein at 27 °C, these mature MRP1 proteins bearing S685A, S685C, S685D, S685H or S685N mutations still do not have full ability to transport LTC4, indicating that the hydroxyl group at 685 (in serine or threonine) plays an important role for interacting with Mg·ATP [30]. Login to comment 45 ABCC1 p.Ser685Thr ABCC1 p.Ser685Thr ABCC1 p.Ser685Cys ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685His ABCC1 p.Ser685Asn ABCC1 p.Ser685Asn ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp The forward and reverse primers used to introduce these mutations are: S685T/forward, 5'-GTG GGC TGC GGA AAG ACG TCC CTG CTC TCA GCC-3'; S685T/reverse, 5'-GGC TGA GAG CAG GGA CGT CTT TCC GCA GCC CAC-3'; S685A/forward, 5'-GTG GGC TGC GGA AAG GCG TCC CTG CTC TCA GCC-3'; S685A/reverse, 5'-GGC TGA GAG CAG GGA CGC CTT TCC GCA GCC CAC-3'; S685C/forward, 5'-GTG GGC TGC GGA AAG TGC TCC CTG CTC TCA GCC-3'; S685C/reverse, 5'- GGC TGA GAG CAG GGA GCA CTT TCC GCA GCC CAC-3'; S685H/ forward, 5'-GTG GGC TGC GGA AAG CAC TCC CTG CTC TCA GCC-3'; S685H/reverse, 5'-GGC TGA GAG CAG GGA GTG CTT TCC GCA GCC CAC-3'; S685N/forward, 5'- GTG GGC TGC GGA AAG AAC TCC CTG CTC TCA GCC-3'; S685N/reverse, 5'-GGC TGA GAG CAG GGA GTT CTT TCC GCA GCC CAC-3'; S685D/forward, 5'-GTG GGC TGC GGA AAG GAT TCC CTG CTC TCA GCC-3'; S685D/reverse, 5'-GGC TGA GAG CAG GGA ATC CTT TCC GCA GCC CAC-3'. Login to comment 46 ABCC1 p.Asp792Ser The mutation of D792S in Walker B motif (Fig. 4A) was also introduced into the full length MRP1 cDNA by using the same strategy shown above. Login to comment 47 ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser The forward and reverse primers used to introduce this mutation are: D792S/forward, 5'-GAC ATT TAC CTC TTC AGT GAT CCC CTC TCA GC-3' and D792S/reverse, 5'-GC TGA GAG GGG ATC ACT GAA GAG GTA AAT GTC-3'. Login to comment 94 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala We suspected that D792A or D792L mutation abolished the hydrogen-bond formation between D792 and S685 and resulted in misfolding of the mutated MRP1 protein. Login to comment 98 ABCC1 p.Ser685Thr ABCC1 p.Ser685Thr ABCC1 p.Ser685Ala ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser Substitution of the S685 with an amino acid that prevents formation of the hydrogen-bond with D792, such as S685A, Table 1 Km (Mg·ATP) and Vmax (LTC4) Values of wild-type and mutant MRP1s Vmax (pmol/mg/min)* Km (μM)* MRP1 164.0±7.0 59.0±2.2 S685T 330.7±8.8 143.0±8.2 S685D 65.3±1.2 249.3±6.3 D792S 79.3±2.1 245.3±8.2 S685D/D792S 99.0±2.9 151.3±6.8 *Km (Mg·ATP) and Vmax (LTC4) values for wild-type, S685T, S685D, D792S and S685D/D792S (n=3) were derived from the corresponding Michaelis-Menten curves shown in Fig. 6. Login to comment 99 ABCC1 p.Ser685Thr The P value for comparison of Km (Mg·ATP) of wild-type versus S685T is 0.0001 and, therefore, the P values for other mutants must be at most 0.0001. Login to comment 108 ABCC1 p.Ser685Ala In addition, majority of S685A is core-glycosylated immature protein at 37 °C, with an apparent molecular weight of ~165 kDa (Fig. 2B). Login to comment 109 ABCC1 p.Ser685Ala Of note, a degradation product (~37 kDa) was detected by monoclonal antibody 42.4 [20] against NBD1 in S685A, but not in wild-type MRP1, implying that this mutant MRP1 protein synthesized in BHK cell is not as stable as wild-type. Login to comment 110 ABCC1 p.Ser685Thr In order to test whether the hydroxyl group in S685 plays such an important role for the protein folding, this serine residue was mutated to threonine (S685T in Fig. 2A). Login to comment 111 ABCC1 p.Ser685Thr Interestingly, S685T mutant produced similar amount of complex-glycosylated mature protein as wild-type (Fig. 2B), implying that the hydroxyl group in threonine plays a similar role as the one in serine residue. Login to comment 112 ABCC1 p.Ser685Cys ABCC1 p.Ser685His ABCC1 p.Ser685Asn We, then, mutated this serine residue to other amino acids, such as S685C, S685N and S685H (Fig. 2A), that could potentially form hydrogen-bond with the D792 residue in Walker B motif. Login to comment 113 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685Asn Although the ratio of mature versus immature MRP1 protein at 37 °C in S685C or S685N is slightly higher than in S685A (Figs. 2B and 3A), substitution of the S685 with either cysteine or asparagine cannot completely rescue the misfolding (Fig. 2B). Login to comment 114 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685His ABCC1 p.Ser685Asn S685H is an interesting mutant that produces similar amount of immature MRP1 protein and 37 kDa degradation product as S685A, but the amount of complex-glycosylated mature S685H is significantly higher than that of S685A, S685C or S685N (Fig. 2B), implying that the histidine residue at that position might form a weak hydrogen bond or salt bridge with the D792 in Walker B motif. Login to comment 116 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn S685A, S685C, S685H and S685N are temperature sensitive mutants Serine residue at 685 may also interact with the metal cofactor [30] and the β-phosphate of the bound ATP [17] and, thus, participates Mg·ATP binding. Login to comment 119 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn As shown in Fig. 3A, all these mutants, including S685H, S685N, S685C and S685A, mainly form complex-glycosylated mature MRP1 proteins at 27 °C. Login to comment 121 ABCC1 p.Ser685Thr However, the amounts of MRP1 mutant proteins accumulated in BHK cells at 27 °C is still much less than that of wild-type or S685T (Fig. 3A), probably due to the mutants are not as stable as wild-type, even at 27 °C. Login to comment 122 ABCC1 p.Ser685Thr ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn This hypothesis is confirmed by the fact that the two degradation products, ~75 kDa and ~35 kDa, were clearly detected by mAb against NBD2 in the mutants of S685H, S685N, S685C or S685A grown at 27 °C, but not in wild-type or S685T (Fig. 3A). Login to comment 123 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn If these mutants, including S685H, S685N, S685C and S685A, form complex-glycosylated mature MRP1 proteins at 27 °C, they should be distinguished from the core-glycosylated immature protein by digestion with endoglycosidase H [36]. Login to comment 124 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn The results in Fig. 3B clearly indicate that the 165 kDa immature MRP1s, including S685H, S685N, S685C and S685A, are sensitive to endoglycosidase H digestion whereas the 190-kDa mature MRP1 proteins, regardless whether they are wild-type or mutants, are not (Fig. 3B). Login to comment 128 ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser We, then, tested whether switching these two residues, i.e., substituting the Walker A S685 with aspartic acid and replacing the Walker B D792 with serine (Fig. 4A, S685D/D792S), would promote the Fig. 2. Login to comment 132 ABCC1 p.Ser685Ala The definition of S685A means that the S685 in Walker A motif was substituted with an alanine residue while the Walker B motif was unmutated. Login to comment 138 ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser The results in Fig. 4B indicated that substitution of the Walker A serine residue with aspartic acid (S685D) or Walker B mutant D792S resulted in misfolding of the protein. Login to comment 139 ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser In addition, switching these two residues, S685D/D792S, also did not rescue the misfolding (Fig. 4B). Login to comment 140 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser All these three mutants, including S685D, D792S and S685D/D792S, are temperature-sensitive variants (Fig. 4C) that are not as stable as wild-type (Fig. 4C) and mainly degraded by proteasome (data not shown). Login to comment 141 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser The core-glycosylated S685D, D792S and S685D/D792S mutants are sensitive to endoglycosidase H digestion whereas the 190-kDa mature MRP1 proteins are not (Fig. 4D). Login to comment 144 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn S685A, S685C, S685H and S685N are temperature-sensitive mutants. Login to comment 155 ABCC1 p.Ser685Thr Although the amounts of MRP1 mutants in the membrane vesicles are much less than that of wild-type or S685T (Fig. 5A), majorities of these mutants in membrane vesicles prepared from the cells grown at 27 °C are complex-glycosylated mature MRP1s (Fig. 5A). Login to comment 157 ABCC1 p.Ser685Thr For un-known reasons, the ATP-dependent LTC4 transport activity catalyzed by S685T is significantly Fig. 4. Login to comment 160 ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser The highlighted letters indicate that the S685 in Walker A motif was substituted with the D residue (S685D) whereas the D792 in Walker B motif was substituted with the S residue (D792S). Login to comment 163 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser (C) S685D, D792S and S685D/D792S are temperature-sensitive mutants. Login to comment 170 ABCC1 p.Ser685Ala In contrast, substitution of the serine residue with an alanine (S685A) that prevents the interactions with the magnesium co-factor and the β-phosphate of the bound ATP significantly reduced the ATP-dependent LTC4 transport activity (Fig. 5B). Login to comment 171 ABCC1 p.Ser685Ala However, S685A mutation exerts much higher transport activity than that of the membrane vesicles containing CFTR (Fig. 5B), implying that this mutation did not completely abolish the ATP-dependent LTC4 transport activity. Login to comment 172 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn ABCC1 p.Ser685Asp Interestingly, substitution of the Walker A serine residue with a cysteine (S685C), a histidine (S685H), an aspartic acid (S685D) or an asparagine (S685N) that may potentially interact with metal co-factor and the β-phosphate of the bound ATP exerts approximately two fold higher transport activity than that of S685A (Fig. 5B). Login to comment 173 ABCC1 p.Ser685Ala ABCC1 p.Asp792Ser Substitution of the Walker B aspartic acid with a serine residue (D792S) that potentially interacts with the magnesium co-factor also exerts approximately two fold higher transport activity than that of S685A (Fig. 5B). Login to comment 174 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser In addition, switching these two residues, i.e., S685D/D792S, exerts even higher transport activity than that of S685D or D792S (Fig. 5B), implying that the interactions of these residues with metal cofactor and the β-phosphate of the bound ATP participate the ATP-dependent LTC4 transport. Login to comment 176 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser The Km (Mg·ATP) values of S685D, D792S and S685D/D792S are significantly higher than that of wild-type MRP1 Six oxygen atoms, including the γ-oxygen of S685 residue in the Walker A motif and the δ-oxygen of D792 in the Walker Fig. 5. Login to comment 192 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp Since the amounts of MRP1 proteins determined in Fig. 5A were different, the amounts of membrane vesicles used in these experiments were adjusted to a similar amount of MRP1 by adding varying amounts of membrane vesicles prepared from parental BHK cells: 0.81 μg of wild-type MRP1+2.19 μg BHK; 0.82 μg of S685T+2.18 μg BHK; 3.00 μg of S685D; 2.31 μg of S685D/D792S+0.69 μg BHK; 1.88 μg of D792S+1.12 μg BHK. Login to comment 198 ABCC1 p.Ser685Thr As shown in Fig. 6 and Table 1, the Vmax (LTC4) value of S685T is significantly higher than that of wild-type, consistent with the results derived from a solution containing 4 mM ATP and 10 mM MgCl2 (Fig. 5B). Login to comment 199 ABCC1 p.Ser685Thr ABCC1 p.Ser685Thr Although the exact mechanism of why S685T is more active than the wild-type is not clear, the higher Km (Mg·ATP) value of S685T (Table 1) might be a factor. Login to comment 200 ABCC1 p.Ser685Thr ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser However, the Km (Mg·ATP) values of S685D, D792S and S685D/D792S are even higher than that of S685T (Table 1), whereas the Vmax (LTC4) values of these mutants are much lower than that of wild-type. Login to comment 201 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser We simply interpreted these results as that the mutations of S685D, D792S and S685D/D792S not only affected ATP binding but also ATP hydrolysis and ATP-dependent solute transport. Login to comment 202 ABCC1 p.Ser685Ala ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser These results imply that the Km (Mg·ATP) value of S685A should even be higher than that of S685D or D792S. Login to comment 203 ABCC1 p.Ser685Ala Unfortunately, the Km (Mg·ATP) value of S685A cannot be accurately determined due to the low transport activity. Login to comment 212 ABCC1 p.Ser685Ala (B) S685A-mutated MRP1 mainly affects ATP binding at the mutated NBD1, but not the ATP hydrolysis at the un-mutated NBD2. Login to comment 213 ABCC1 p.Ser685Thr ABCC1 p.Ser685Ala S685A or S685T mutation was introduced into the pDual/N-half/C-half and expressed in Sf21 insect cells [24]. Login to comment 216 ABCC1 p.Ser685Thr ABCC1 p.Ser685Ala ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser wild-type, S685A-, S685T-, S685D-, D792S- and S685D/ D792S-mutated MRP1s were used to do photo-affinity labeling at 37 °C with either [α-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP in the presence of vanadate. Login to comment 220 ABCC1 p.Ser685Ala ABCC1 p.Ser685Ala For S685A-mutated MRP1, the amounts of labeling with either [α-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP are much less than the corresponding labeling on wild-type MRP1 (Fig. 7A), implying that S685A mutation significantly affects the Mg·ATP binding. Login to comment 221 ABCC1 p.Ser685Ala However, the amount of labeling on S685A with [α-32 P]-8-N3ATP is higher than that of labeling with [γ-32 P]-8-N3ATP (Fig. 7A), implying that certain amount of the bound ATP is hydrolyzed. Login to comment 222 ABCC1 p.Ser685Ala ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser Similar results (as S685A) were obtained from S685D-, D792S- and S685D/D792S-mutated MRP1s (Fig. 7A), implying that all these mutations affect Mg·ATP binding. Login to comment 223 ABCC1 p.Ser685Thr In contrast, the labeling of S685T with [γ-32 P]-8-N3ATP is significantly higher than the corresponding labeling on wild-type MRP1 (Fig. 7A). Login to comment 224 ABCC1 p.Ser685Thr In addition, the labeling of S685T with [α-32 P]-8-N3ATP is also higher than the corresponding labeling on wild-type MRP1 (Fig. 7A). Login to comment 225 ABCC1 p.Ser685Thr ABCC1 p.Ser685Thr The ratio of [γ-32 P]-8-N3ATP labeling, after subtracting the labeling without UV-irradiation, versus [α-32 P]-8-N3ATP labeling on wild-type MRP1 is approximately 0.3, whereas this ratio for S685T is approximately 0.6, implying that ATP bound to the S685T-mutated MRP1 may not be efficiently hydrolyzed. Login to comment 226 ABCC1 p.Ser685Thr However, the ATP-dependent LTC4 transport activity of S685T-mutated MRP1 is much higher than that of wild-type (Figs. 5 and 6). Login to comment 227 ABCC1 p.Ser685Thr In order to solve this puzzle, S685T was introduced into pDual/N-half/ C-half expression vector [24,25] and expressed in Sf21 cells. Login to comment 230 ABCC1 p.Ser685Ala ABCC1 p.Ser685Ala The [γ-32 P]-8-N3ATP labeling of S685A-mutated-NBD1-containing N-half fragment is significantly lower than the corresponding labeling in wild-type NBD1-containing N-half (Fig. 7B), implying that S685A mutation affects Mg·ATP binding at the mutated NBD1. Login to comment 231 ABCC1 p.Ser685Ala However, the [α-32 P]-8-N3ATP labeling of the un-mutated NBD2-containing C-half of S685A-mutated MRP1 is much higher than the [γ-32 P]-8-N3ATP labeling of the same fragment (Fig. 7B), indicating that ATP bound to this NBD2 is hydrolyzed. Login to comment 232 ABCC1 p.Ser685Thr ABCC1 p.Ser685Thr In contrast, the [γ-32 P]-8-N3ATP or [α-32 P]-8-N3ATP labeling of S685T-mutated-NBD1-containing N-half is significantly higher than the corresponding labeling on the wild-type N-half (Fig. 7B), presumably the S685T mutation increased affinity for 8-N3ATP at the mutated NBD1. Login to comment 233 ABCC1 p.Ser685Thr ABCC1 p.Ser685Thr In addition, the [γ-32 P]-8-N3ATP labeling of S685T-mutated- NBD1-containing N-half is much higher than that of the labeling on the un-mutated NBD2-containing C-half (Fig. 7B) and the [α-32 P]-8-N3ATP labeling of the un-mutated NBD2-containing C-half of S685T-mutated MRP1 is much higher than the [γ-32 P]-8-N3ATP labeling of the same fragment (Fig. 7B), indicating that ATP bound to this NBD2 is efficiently hydrolyzed. Login to comment 238 ABCC1 p.Ser685Cys ABCC1 p.Ser685Cys Indeed, substitution of S685 with an alanine residue that eliminates the hydroxyl group in this serine residue resulted in misfolding of the protein at 37 °C (Figs. 2B and 3), indicating that the hydrogen-bond formation between these two residues may play a crucial role for the protein folding. If that is the case, replacement of the hydroxyl group in this serine residue with a thiol group, i.e., S685C mutation [Computer simulation (Fig. 1) of S685C-mutated NBD1 indicates that the distance between the thiol group of C685 and the carboxyl group of D792 is ~2.85 Å], should result in a complex-glycosylated mature MRP1 protein. Login to comment 239 ABCC1 p.Ser685Cys ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala In fact, although the ratio of mature S685C versus immature protein at 37 °C is slightly higher than that of S685A (Fig. 3A), majority of S685C are high-mannose core-glycosylated (endoglycosidase H-sensitive) immature protein at 37 °C (Figs. 2B, 3A and B), indicating that the hydrogen-bond formation between these two residues is not the only factor that affects the protein folding. Login to comment 240 ABCC1 p.Ser685His ABCC1 p.Ser685Asn ABCC1 p.Asp792Ser The results derived from S685H, S685N and D792S also support the above conclusion. Login to comment 241 ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685His ABCC1 p.Ser685His For example, although majority of the S685H [The five-member imidazole ring in histidine might be partial positively charged under physiological pH and computer simulation (Fig. 1) of S685H-mutated NBD1 indicates that the distance between the nitrogen atom of H685 and the carboxyl group of D792 is ~1.93 Å] are core-glycosylated immature protein at 37 °C (Figs. 2B and 3A), the ratio of mature S685H versus immature protein at 37 °C is much higher than that of S685A (Figs. 2B and 3A), implying that the hydrogen-bond formation between these two residues does play a role for the protein folding. Login to comment 242 ABCC1 p.Ser685Asp Substitution of the hydroxyl group in S685 with a carboxyl group (S685D) will introduce a negatively charged group at that position and may repel each other with the negatively charged D792. Login to comment 243 ABCC1 p.Ser685Ala ABCC1 p.Ser685Asp As S685A mutant, elimination of the hydrogen-bond formation between D685 (the S685D mutation) and D792 resulted in misfolding of the protein (Fig. 4B and C). Login to comment 244 ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser Interestingly, switching these two residues, i.e., S685D/D792S, might resume the hydrogen-bond formation between them but did not rescue the misfolding of the protein (Fig. 4B and C). Login to comment 250 ABCC1 p.Ser685Thr In contrast to other mutations, substitution of the Walker A serine residue with a threonine (S685T) that does not change the position of the hydroxyl group, except introducing an extra methyl group, results in complex-glycosylated mature MRP1 protein (Figs. 2B and 3). Login to comment 253 ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu In addition, substitutions of the Walker B motif D1454 and E1455 in NBD2 of MRP1 with a leucine residue (D1454L/E1455L) also did not cause misfolding of the protein [20]. Login to comment 254 ABCC1 p.Asp1454Leu ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu ABCC1 p.Asp1454Asn ABCC1 p.Ser1334Asn ABCC1 p.Ser1334Asp ABCC1 p.Ser1334Ala ABCC1 p.Ser1334Cys ABCC1 p.Ser1334Thr ABCC1 p.Ser1334His In order to rule out the possibility that the double mutant D1454L/E1455L might rescue the misfolding caused by D14 54L mutation, we have made single mutants including D1454L, D1454N, S1334A, S1334T, S1334C, S1334H, S1334D and S1334N. Login to comment 261 ABCC1 p.Asp1454Leu ABCC1 p.Asp1454Asn It is also true for the corresponding mutations in Walker B motif of NBD2 in MRP1, such as D1454L (manuscript in preparation) and D1454N [46], have a significant effect on the ATP-dependent LTC4 transport. Login to comment 262 ABCC1 p.Ser685Cys ABCC1 p.Ser685Ala ABCC1 p.Ser685His ABCC1 p.Ser685Asn ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser Functional analyses of the Walker A serine mutants in NBD1, including membrane vesicles containing the complex-glycosylated mature and endoglycosidase H- resistant (Figs. 3B and 4D) S685H, S685C, S685A, S685D, S685N, D792S and S685D/D792S prepared from these temperature sensitive variants (Figs. 3 and 4) grown at 27 °C, indicate that these mutations affect ATP binding and ATP-dependent LTC4 transport (Table 1 and Figs. 5-7). Login to comment 263 ABCC1 p.Ser685Asp ABCC1 p.Ser685Asp ABCC1 p.Asp792Ser ABCC1 p.Asp792Ser Interestingly, although the double mutant S685D/D792S did not rescue the misfolding of the protein, the ATP-dependent LTC4 transport activity of this double mutant was significantly higher than the single mutant S685D or D792S (Fig. 5 and Table 1), implying that the hydrogen-bond formation between D685 and S792 may play a role in stabilizing the protein in a proper conformation [30]. Login to comment 264 ABCC1 p.Ser685Thr In addition, substitution of the Walker A serine residue with a threonine (S685T) without changing the position of the hydroxyl group exerted higher affinity for 8-azido-ATP (Fig. 7) at the mutated NBD1 and significantly increased ATP-dependent LTC4 transport activity (Table 1 and Figs. 5 and 6), emphasizing the importance of the hydroxyl group located at this position. Login to comment