CYP2C8 Exists as a Dimer in Natural Membranes
CYP2C8 Exists as a Dimer in Natural Membranes
复制标题
DOI：
10.1124/dmd.110.034942
复制
发表时间：
2010-11-01
影响因子：
3.9
通讯作者：
Kemper, Byron
中科院分区：
文献类型：
--
作者：
Hu, Gang;Johnson, Eric F.;Kemper, Byron
带有修饰的N-末端序列(2C8H)的CYP2C8以二聚体的形式结晶，但尚不清楚天然的CYP2C8是否以二聚体的形式存在于自然膜中。我们通过半胱氨酸扫描和巯基的交联或氧化，分别检测了细菌膜和哺乳动物内质网膜中表达的2C8H和CYP2C8的组织结构。在这两种形式的CYP2C8中，都观察到了通过连接区的Cys-24突变而消除的交联二聚体。在N端21个氨基酸的跨膜信号锚中引入单独的半胱氨酸导致了与信号锚的α-螺旋结构一致的交联型式。在连接区，观察到在22、23或24位残基上取代的半胱氨酸发生了交联，恰好在三个Arg残基之前，这表明尽管相邻的正电荷存在，但两个连接体序列接近对齐。将半胱氨酸对的F-G环区引入Cys-24突变体中，根据晶体结构使半胱氨酸对处于最佳交联区，从而产生了交联型二聚体。信号锚序列的缺失消除了Cys-24或F-G环区引入的半胱氨酸介导的交联，表明信号锚相互作用是稳定形成二聚体所必需的。这些结果表明，信号锚序列和F-G环区形成了天然膜中CYP2C8分子间相互作用的界面。
CYP2C8 with a modified N-terminal sequence (2C8H) crystallizes as a dimer, but it is not known whether native CYP2C8 exists as a dimer in natural membranes. We have examined the organization of 2C8H and CYP2C8 expressed in bacterial membranes and mammalian endoplasmic reticulum membranes, respectively, by cysteine scanning and cross-linking or oxidation of sulfhydryl groups. In both forms of CYP2C8, cross-linked dimers were observed that were eliminated by mutation of Cys-24 in the linker region. Introduction of individual cysteines in the N-terminal 21-amino acid membrane-spanning signal anchor resulted in a pattern of cross-linking consistent with an alpha-helical structure for the signal anchor. In the linker region, cross-linking was observed for cysteine substituted at residues 22, 23, or 24, just before three Arg residues, indicating close apposition of the two linker sequences despite the neighboring positive charges. Introduction into the F-G loop region of cysteine pairs optimally located for cross-linking based on the crystal structure resulted in cross-linked dimers in the Cys-24 mutant. Deletion of the signal anchor sequence eliminated cross-linking mediated by Cys-24 or by cysteines introduced in the F-G loop regions, indicating that the signal anchor interaction is required for stable dimer formation. These results indicate that the signal anchor sequence and the F-G loop region form interfaces for CYP2C8 intermolecular interactions in natural membranes.