ABCB1 p.Lys1061Arg
| Predicted by SNAP2: | A: N (61%), C: D (59%), D: N (53%), E: N (82%), F: D (66%), G: N (61%), H: N (72%), I: D (63%), L: N (72%), M: N (53%), N: N (87%), P: D (53%), Q: N (87%), R: N (93%), S: N (82%), T: N (78%), V: D (66%), W: D (75%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Both ATP sites of human P-glycoprotein are essenti... Biochemistry. 1999 Oct 19;38(42):13887-99. Hrycyna CA, Ramachandra M, Germann UA, Cheng PW, Pastan I, Gottesman MM
Both ATP sites of human P-glycoprotein are essential but not symmetric.
Biochemistry. 1999 Oct 19;38(42):13887-99., 1999-10-19 [PMID:10529234]
Abstract [show]
Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.
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No. Sentence Comment
84 To introduce the FspI site, nucleotides 3181-3183 were changed from AAG to CGC, which also resulted in a single amino acid substitution (K1061R).
X
ABCB1 p.Lys1061Arg 10529234:84:137
status: NEW330 In constructing this protein, three amino acid changes were introduced: K1061R, V1214I, and Q1215D.
X
ABCB1 p.Lys1061Arg 10529234:330:72
status: NEW