Determination of the membrane contact residues and solution structure of the helix F/G loop of prostaglandin I2 synthase.
Determination of the membrane contact residues and solution structure of the helix F/G loop of prostaglandin I2 synthase.
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前列腺素 I2 合酶螺旋 F/G 环的膜接触残基和溶液结构的测定。
DOI：
10.1016/s0003-9861(02)00728-2
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发表时间：
2003
影响因子：
3.9
通讯作者：
Ruan,Ke-He
中科院分区：
文献类型：
--
作者：
Wu,Jiaxin;So,Shui-Ping;Ruan,Ke-He
根据我们通过同源建模和拓扑研究创建的前列腺素 I2 合酶 (PGIS) 的拓扑排列模型，我们假设 PGIS 的螺旋 F/G 环包含与 N 端膜锚定域不同的膜接触区域。为了提供直接的实验数据，我们使用约束合成肽来模拟通过添加了半胱氨酸残基的二硫键在两端环化的 PGIS 残基 208-230，探索了内质网 (ER) 膜和 PGIS F/G 环之间的关系。通过高分辨率 1H 二维核磁共振 (2D NMR) 实验和自旋标记掺入技术研究了溶液结构和对受限 PGIS F/G 环肽的膜接触重要的残基。通过在模拟膜环境的十二烷基磷酸胆碱 (DPC) 胶束存在下结合 2D NMR 实验，获得了 F/G 环段的完整 1 H NMR 归属，并确定了肽的溶液结构。 PGIS F/G环段显示出明确的螺旋转螺旋构象，其与相应区域中P450BM3的三维晶体结构相似。通过将自旋标记的12-硬脂酸酯掺入具有肽的DPC胶束中的效果来评估与PGIS F/G环的膜接触的方向和残基。肽中对应于 PGIS 残基 L217 (L11)、L222 (L16) 和 V224 (V18) 的三个残基已被证明与 DPC 胶束接触，这意味着这些残基参与与天然膜结合 PGIS 中的 ER 膜接触。这些结果为定位微粒体 P450 F/G 环区域中的膜接触残基提供了第一个实验证据，对于进一步定义和理解天然膜环境中 PGIS 和其他微粒体 P450 的膜拓扑结构具有重要意义。
From our topological arrangement model of prostaglandin I2synthase (PGIS) created by homology modeling and topology studies, we hypothesized that the helix F/G loop of PGIS contains a membrane contact region distinct from the N-terminal membrane anchor domain. To provide direct experimental data we have explored the relationship between the endoplasmic reticulum (ER) membrane and the PGIS F/G loop using a constrained synthetic peptide to mimic PGIS residues 208–230 cyclized on both ends through a disulfide bond with added Cys residues. The solution structure and the residues important for membrane contact of the constrained PGIS F/G loop peptide were investigated by high-resolution1H two-dimensional nuclear magnetic resonance (2D NMR) experiments and a spin label incorporation technique. Through the combination of 2D NMR experiments in the presence of dodecylphosphocholine (DPC) micelles used to mimic the membrane environment, complete1H NMR assignments of the F/G loop segment have been obtained and the solution structure of the peptide has been determined. The PGIS F/G loop segment shows a defined helix turn helix conformation, which is similar to the three-dimensional crystallography structure of P450BM3 in the corresponding region. The orientation and the residues contacted with the membrane of the PGIS F/G loop were evaluated from the effect of incorporation of a spin-labeled 12-doxylstearate into the DPC micelles with the peptide. Three residues in the peptide corresponding to the PGIS residues L217 (L11), L222 (L16), and V224 (V18) have been demonstrated to contact the DPC micelles, which implies that the residues are involved in contact with the ER membrane in the native membrane-bound PGIS. These results provided the first experimental evidence to localize the membrane contact residues in the F/G loop region of microsomal P450 and are valuable to further define and understand the membrane topology of PGIS and those of other microsomal P450s in the native membrane environment.