ABCMdb

ABCC1 p.Ser685Ala

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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
108 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 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
113 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 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 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 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
122 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 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 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
132 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
144 S685A, S685C, S685H and S685N are temperature-sensitive mutants. Login to comment
170 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 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 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 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
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
203 Unfortunately, the Km (Mg·ATP) value of S685A cannot be accurately determined due to the low transport activity. Login to comment
212 (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 S685A or S685T mutation was introduced into the pDual/N-half/C-half and expressed in Sf21 insect cells [24]. 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
220 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 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 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
230 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 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
239 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
241 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
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
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