期刊
BIOCHEMISTRY
卷 47, 期 52, 页码 13952-13965出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi801532g
关键词
-
资金
- Canadian Institutes of Health Research [MOP-62824]
- Canada Research Chair in Cancer Biology and Bracken Chair in Genetics and Molecular Medicine
The two nucleotide binding domains (NBDs) of ATP binding cassette (ABC) transporters dimerize to form composite nucleotide binding sites (NBSs) each containing Walker A and B motifs from one domain and the ABC C signature from the other. In many ABC proteins, the NBSs are thought to be functionally equivalent. However, this is not the case for ABCC proteins, such as MRPI, in which NBS1 containing the Walker A and B motifs from the N-proximal NBD1 typically binds ATP with high affinity but has low hydrolytic activity, while the reverse is true of NBS2. A notable feature of NBD1 of the ABCC proteins is the lack of a catalytic Glu residue following the core Walker B motif. In multidrug resistance protein (MRP) 1, this residue is Asp (D793). Previously, we demonstrated that mutation of D793 to Glu was sufficient to increase ATP hydrolysis at NBS1, but paradoxically, transport activity decreased by 50-70% as a result of tight binding of ADP at the mutated NBS1. Here, we identify two atypical amino acids in NBD1 that contribute to the retention of ADP. We found that conversion of Trp653 to Tyr and/or Pro794 to Ala enhanced transport activity of the D793E mutant and the release of ADP from NBS1. Moreover, introduction of the P794A mutation into wild-type MRP1 increased transport of leukotriene C-4 approximately 2-fold. Molecular dynamic simulations revealed that, while the D793E mutation increased hydrolysis of ATP, the presence of the adjacent Pro794, rather than the more typical Ala, decreased flexibility of the region linking Walker B and the D-loop, markedly diminishing the rate of release of Mg2+ and ADP. Overall, these results suggest that the rate of release of ADP by NBD1 in the D793E background may be the rate-limiting step in the transport cycle of MRP1.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据