Qin Lei, Zheng Jimin, Grant Caroline E, Jia Zongchao, Cole Susan P C, Deeley Roger G
Division of Cancer Biology and Genetics, Cancer Research Institute, Departments of Biochemistry, Pathology and Molecular Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Biochemistry. 2008 Dec 30;47(52):13952-65. doi: 10.1021/bi801532g.
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 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 Mg(2+) 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.
ATP结合盒（ABC）转运蛋白的两个核苷酸结合结构域（NBD）二聚化形成复合核苷酸结合位点（NBS），每个位点包含来自一个结构域的沃克A和B基序以及来自另一个结构域的ABC“C”特征序列。在许多ABC蛋白中，NBS在功能上被认为是等效的。然而，ABCC蛋白如MRP1并非如此，其中包含来自N端NBD1的沃克A和B基序的NBS1通常以高亲和力结合ATP，但水解活性较低，而NBS2则相反。ABCC蛋白NBD1的一个显著特征是在核心沃克B基序之后缺乏催化性谷氨酸残基。在多药耐药蛋白（MRP）1中，该残基为天冬氨酸（D793）。此前，我们证明将D793突变为谷氨酸足以增加NBS1处的ATP水解，但矛盾的是，由于突变的NBS1处ADP紧密结合，转运活性降低了50 - 70%。在此，我们在NBD1中鉴定出两个非典型氨基酸，它们有助于ADP的保留。我们发现将色氨酸653转换为酪氨酸和/或将脯氨酸794转换为丙氨酸可增强D793E突变体的转运活性以及ADP从NBS1的释放。此外，将P794A突变引入野生型MRP1可使白三烯C4的转运增加约2倍。分子动力学模拟表明，虽然D793E突变增加了ATP水解，但相邻脯氨酸794而非更典型的丙氨酸的存在降低了连接沃克B和D环区域的灵活性，显著降低了镁离子和ADP的释放速率。总体而言，这些结果表明在D793E背景下NBD1释放ADP的速率可能是MRP1转运循环中的限速步骤。
