ABCB4 p.Leu1082Cys
| Predicted by SNAP2: | A: D (63%), C: N (53%), D: D (85%), E: D (80%), F: D (66%), G: D (80%), H: D (80%), I: N (78%), K: D (80%), M: N (66%), N: D (80%), P: D (85%), Q: D (75%), R: D (80%), S: D (75%), T: D (66%), V: N (66%), W: D (80%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Drug-stimulated ATPase activity of human P-glycopr... J Biol Chem. 2000 Jun 30;275(26):19435-8. Loo TW, Clarke DM
Drug-stimulated ATPase activity of human P-glycoprotein is blocked by disulfide cross-linking between the nucleotide-binding sites.
J Biol Chem. 2000 Jun 30;275(26):19435-8., [PMID:10806188]
Abstract [show]
P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs). Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the cross-linked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cys-less P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol. The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.
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No. Sentence Comment
84 Residue in NBD1 Cys-1074 Residue in NBD2 Cys-431 L425C -a V426C - V1069C - G427C ϩb G1070C - N428C - S1071C - S429C - S1072C - G430C - G1073C - Cys-431 - Cys-1074 - G432C - G1075C - K433C - K1076C - S434C - S1077C - T435C - T1078C - T436C - V1079C - V437C - V1080C - Q438C - Q1081C - L439C ϩ L1082C NDc a ϩ, cross-linked product not detected on SDS-PAGE.
X
ABCB4 p.Leu1082Cys 10806188:84:304
status: NEW