ABCMdb

ABCC1 p.Ser685Asp

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Publications

PMID: 18088596 [PubMed] Yang R et al: "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."

No. Sentence Comment

31 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 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
98 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
128 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
138 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 In addition, switching these two residues, S685D/D792S, also did not rescue the misfolding (Fig. 4B). Login to comment
140 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 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
160 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 (C) S685D, D792S and S685D/D792S are temperature-sensitive mutants. Login to comment
172 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
174 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 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 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
200 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 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 These results imply that the Km (Mg·ATP) value of S685A should even be higher than that of S685D or D792S. Login to comment
216 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
222 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
242 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 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 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
262 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 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