Ligand binding to cytochrome P450 3A4 in phospholipid bilayer nanodiscs - The effect of model membranes
Ligand binding to cytochrome P450 3A4 in phospholipid bilayer nanodiscs - The effect of model membranes
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DOI：
10.1074/jbc.m703568200
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发表时间：
2007-09-28
影响因子：
4.8
通讯作者：
Atkins, William M.
中科院分区：
文献类型：
--
作者：
Nath, Abhinav;Grinkova, Yelena V.;Atkins, William M.
细胞色素P450 3A 4（CYP 3A 4）是一种主要的药物代谢酶。大多数CYP 3A 4配体结合的研究目前在溶液中进行，没有模型膜。因此，关于细胞膜对CYP 3A 4配体结合行为影响的信息很少。磷脂双层纳米盘是一种新型的高密度脂蛋白膜系统，其稳定性，单分散性和一致性是由它们的自组装保证的。我们探讨了四个配体（6-（对甲苯胺基）-2-萘磺酸（TNS），α-萘甲酮（ANF），咪康唑，溴隐亭）结合到纳米盘的CYP 3A 4的能量学。通过结合环境敏感性配体荧光和配体诱导的纳米盘支架蛋白中存在的色氨酸残基的荧光位移监测配体与纳米盘的结合;通过配体诱导的血红素Soret带吸光度位移监测与CYP 3A 4活性位点的结合。与CYP 3A 4-Nanodisks中活性位点结合的解离常数为：TNS为4.0 μ M，ANF为5.8 μ M，咪康唑为0.45 μ M，溴隐亭为0.45 μ M。这些值是结合到脂质双层中的CYP 3A 4的值，因此可能比使用溶液中的CYP 3A 4测量的值更具生物学相关性。在某些情况下，使用Nanodisces中的CYP 3A 4进行的亲和力测量与溶液值显著不同。我们还研究了配体与CYP 3A 4和膜结合之间的平衡。TNS没有表现出显着的偏好，无论是环境; ANF优先绑定到膜，咪康唑和溴隐亭优先绑定到CYP 3A 4活性位点。
The membrane-bound protein cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning the membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high density lipoprotein particles, whose stability, monodispersity, and consistency are ensured by their self-assembly. We explore the energetics of four ligands (6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), alpha-naphthoflavone (ANF), miconazole, and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the scaffold proteins of Nanodiscs; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 mu M for TNS, 5.8 mu M for ANF, 0.45 mu M for miconazole, and 0.45 mu M for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment; ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site.