ABCMdb

ABCC1 p.Ser685Cys

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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
112 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 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
144 S685A, S685C, S685H and S685N 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
238 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 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
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