ABCMdb

ABCC1 p.Asp792Ser

Predicted by SNAP2:	A: D (95%), C: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] The hydroxyl group of S685 in Walker A motif and t... Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29. 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., [PMID:18088596]

Abstract [show]
Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the beta-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 degrees C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the beta-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.

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No. Sentence Comment
46 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 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
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
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
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
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
240 The results derived from S685H, S685N and D792S also support the above conclusion. 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