ABCC1 p.Asp793Gln
| 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] Role of carboxylate residues adjacent to the conse... J Biol Chem. 2003 Oct 3;278(40):38537-47. Epub 2003 Jul 27. Payen LF, Gao M, Westlake CJ, Cole SP, Deeley RG
Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1).
J Biol Chem. 2003 Oct 3;278(40):38537-47. Epub 2003 Jul 27., 2003-10-03 [PMID:12882957]
Abstract [show]
MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.
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No. Sentence Comment
78 The forward primers for D793Q, D793N, D793S, E1455Q, E1455N, E1455S, and E1455L were 5Ј-GCTGACATTTACCTCTTCGATCAACCGCTCTC- AGCAGTGGATGCC-3Ј, 5Ј-GCTGACATTTACCTCTTCGATAATCCGC- TCTCAGCAGTGGATGCC-3Ј, 5Ј-GCTGACATTTACCTCTTCGATTCT- CCCCTCTCAGCAGTGGATGCC-3Ј, 5Ј-CGAAGATCCTTGTGTTGGA- TCAGGCCACGGCGGCCGTGGACCTGG-3Ј, 5Ј-CGAAGATCCTTGTG- TTGGATA ACGCCACGGCCGCCGTGGACCTGG-3Ј, 5Ј-CGAAGATCC- TTGTGTTGGATTCGGCCACGGCAGCCGTGGACCTGG-3Ј, 5Ј-CGAA- GATCCTTGTGTTGGATTTGGCCACGGCCGCCGTGGACCTGG-3Ј, respectively.
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ABCC1 p.Asp793Gln 12882957:78:24
status: NEW248 Unlike the D793E mutation, which decreased LTC4 transport activity by 80%, NBD1 mutations (D793S, D793N, and D793Q) had little effect on ATP-dependent LTC4 uptake (Fig. 7B).
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ABCC1 p.Asp793Gln 12882957:248:109
status: NEW251 At 4 °C, mutation of Asp793 to Ser, Gln, or Asn decreased photolabeling of NBD1 by 8-azido-[␣-32 P]ATP to a similar extent.
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ABCC1 p.Asp793Gln 12882957:251:26
status: NEW264 Similar to wild-type, in the absence of vanadate, D793S, D793Q, and D793N mutant proteins did not display any nucleotide binding at either NBD (Fig. 8B).
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ABCC1 p.Asp793Gln 12882957:264:57
status: NEW267 Although labeling at NBD2 was decreased in the D793Q and D793N mutants, this domain remained the predominant site of photolabeling (Fig. 8B, lanes 7 and 9).
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ABCC1 p.Asp793Gln 12882957:267:47
status: NEW270 Effect of D793Q, D793N, D793S, E1455S, E1455Q, E1455N, E1455L mutations on [3 H]LTC4 transport activity.
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ABCC1 p.Asp793Gln 12882957:270:10
status: NEW271 A, membrane proteins (1 g) from Sf21 cells expressing both halves of either MRP1 (MRP1 dh) or mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, E1455L) were separated by SDS-PAGE on gradient gels and transferred to Immobilon-P membranes.
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ABCC1 p.Asp793Gln 12882957:271:119
status: NEW275 B, membrane vesicles (2 g) containing MRP1 dh, D793Q, D793S, D793N, E1455Q, E1455S, E1455N, E1455L, or beta-Gus were assayed for ATP-dependent LTC4 transport activity at 23 °C for up to 3 min in transport buffer containing [3 H]LTC4 (50 nM, 0.13 Ci), as described under "Experimental Procedures."
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ABCC1 p.Asp793Gln 12882957:275:55
status: NEW304 Comparison of nucleotide binding and vanadate trapping by wild-type MRP1 and mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L).
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ABCC1 p.Asp793Gln 12882957:304:94
status: NEW305 A, at 4 °C, 8-azido- [␣-32 P]ATP photolabeling by wild-type MRP1 and mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L) was evaluated. Membrane vesicles (20 g) were incubated with 5 M 8-azido-[␣-32 P]ATP for 5 min on ice in transport buffer containing 5 mM MgCl2.
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ABCC1 p.Asp793Gln 12882957:305:98
status: NEW308 The position of the labeled MRP1 NH2-half and COOH-half polypeptides are indicated, and endogenous proteins labeled are indicated by E followed by arrows. B and C, at 37 °C under trapping conditions, 8-azido-[␣-32 P]ADP trapping by wild-type MRP1 mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L) was studied.
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ABCC1 p.Asp793Gln 12882957:308:276
status: NEW339 Mutation of Asp793 to Asn, Gln, and also Ser, which is the residue found at the comparable position of cystic fibrosis transmembrane conductance regulator, all decreased slightly LTC4 transport activity, whereas the comparable mutations of Glu1455 inactivated the protein (Fig. 7B).
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ABCC1 p.Asp793Gln 12882957:339:12
status: NEW