Characterization of the secondary structure and membrane interaction of the putative membrane anchor domains of prostaglandin I2 synthase and cytochrome P450 2C1.
Characterization of the secondary structure and membrane interaction of the putative membrane anchor domains of prostaglandin I2 synthase and cytochrome P450 2C1.
复制标题
前列腺素 I2 合酶和细胞色素 P450 2C1 的假定膜锚定域的二级结构和膜相互作用的表征。
DOI：
10.1006/abbi.1998.0599
复制
发表时间：
1998
影响因子：
3.9
通讯作者：
Ruan,KH
中科院分区：
文献类型：
--
作者：
Lin,Y;Wu,KK;Ruan,KH
前列腺素I2合酶（PGIS）产生的前列腺素I2（PGI 2）与另一种P450酶血栓烷A2合酶（TXAS）从相同底物产生的血栓烷A2（TXA 2）相比，对血小板聚集和血管收缩特性具有相反的作用。PGIS和TXAS只有16%的氨基酸序列相同。亲水性分析表明，推定的NH 2-端膜锚结构域的PGIS是类似于许多其他膜结合微粒体P450，这被认为是锚定由一个单一的跨膜段，因此不同的TXAS锚，它似乎有两个跨膜段。为了表征PGIS NH 2-末端区域的膜锚功能，我们使用肽脂质体重建测定来鉴定PGIS NH 2-末端结构域中的膜锚片段，并将其与P450 2C 1的锚片段进行比较。合成了四种肽，模拟PGIS和P450 2C 1的假定NH 2-末端膜锚片段，含有残基1-28（PGIS-LP 1和P450 2C 1-LP 1）或残基25-54（PGIS-LP 2和P450 2C 1-LP 2），并评价了它们插入脂质双层的能力。结果表明，PGIS和P450 2C 1的LP 1肽都与脂质双层结合，而LP 2肽都不与脂质结合。使用CD光谱进一步表征两种LP 1肽的构象。通过加入三氟乙醇、十二烷基磷酸胆碱或掺入脂质体在这些肽中诱导的螺旋结构表明，这些片段在疏水环境中倾向于采用螺旋结构，因此可以起到膜锚片段的作用。这些结果支持PGIS和TXAS以不同方式与内质网膜相互作用的假设，其中PGIS的NH 2-末端锚结构域与P450 2C 1一样，似乎具有单一的跨膜区段。
Prostaglandin I2synthase (PGIS) produces prostaglandin I2(PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2(TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1–28 (PGIS-LP1 and P450 2C1-LP1) or residues 25–54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.