Residues Responsible for the Asymmetric Function of the Nucleotide Binding Domains of Multidrug Resistance Protein 1
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- 作者：Lei Qin ; Jimin Zheng ; Caroline E. Grant ; Zongchao Jia ; Susan P. C. Cole ; Roger G. Deeley
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：December 30, 2008
- 年：2008
- 卷：47
- 期：52
- 页码：13952-13965
- 全文大小：663K
- 年卷期：v.47,no.52(December 30, 2008)
- ISSN：1520-4995
文摘
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 MRP1, 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 C4 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 Mgp>2+ p> 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.