Both ATP sites of human P-glycoprotein are essential but not symmetric

被引:127
作者
Hrycyna, CA
Ramachandra, M
Germann, UA
Cheng, PW
Pastan, I
Gottesman, MM
机构
[1] NCI, Cell Biol Lab, NIH, Bethesda, MD 20892 USA
[2] NCI, Mol Biol Lab, Div Basic Sci, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1021/bi991115m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Mg2+ binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [I-125]iodoarylazidoprazosin ([I-125]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-P-32]-8-azido-ATP at a subsaturating concentration of 2.5 mu M, 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-P-32]-8-azido-ATP. [alpha-P-32]-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 mu M 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-P-32]-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-P-32]-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-P-32]-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 [I-125]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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页码:13887 / 13899
页数:13
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