Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)
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DOI：
10.1074/jbc.m805834200
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发表时间：
2008-10-17
影响因子：
4.8
通讯作者：
Clarke, David M.
中科院分区：
文献类型：
--
作者：
Loo, Tip W.;Bartlett, M. Claire;Clarke, David M.
p -糖蛋白（P-gp, ABCB1）是一种atp依赖性药物泵。它的两个同源半部分都包含一个跨膜结构域（TMD），它有六个跨膜（TM）片段和一个核苷酸结合结构域（NBD）。确定这两半是如何相互作用的，可以提供对P-gp折叠的洞察，因为药物结合袋和核苷酸结合位点被预测在两半之间的界面上。在这里，我们提出NBD1-TMD2和NBD2-TMD1相互作用的证据。我们还表明，TMD-NBD在未成熟和成熟P-gp中的相互作用可以受到加工突变的影响。我们发现NBD-TMD突变体L443C(NBD1)/S909C（TMD2）和A266C(TMD1)/F1086C（NBD2）可以在0℃时与氧化剂（菲罗啉铜）交联。钒酸盐捕获核苷酸抑制交联。存在加工突变(G268V/L443C(NBD1)/S909C(TMD2))；L1260A/A266C（TMD1)/F1086C(NBD2)）以未成熟蛋白（150 kDa）为主要产物，突变体不能与邻菲罗啉铜交联。然而，在药理学伴侣（环孢素a）存在的情况下，加工突变体的表达导致在细胞表面可以交联的成熟蛋白（170 kDa）的表达。同样，CFTR突变体A274C(TMD1)/L1260C（NBD2）和V510C(NBD1)/A1067C（TMD2）可以在0℃时与邻菲罗啉铜交联。然而，在这些突变体中引入Delta F508突变，导致未成熟的CFTR合成无法交联。这些结果表明NBD与相反的TMD相互作用的建立是ABC转运蛋白折叠的关键步骤。
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be crosslinked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of Delta F508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.