Characterization of the substrate mimic bound to engineered prostacyclin synthase in solution using high-resolution NMR spectroscopy and mutagenesis: implication of the molecular mechanism in biosynthesis of prostacyclin.
Characterization of the substrate mimic bound to engineered prostacyclin synthase in solution using high-resolution NMR spectroscopy and mutagenesis: implication of the molecular mechanism in biosynthesis of prostacyclin.
复制标题
使用高分辨率核磁共振波谱和诱变对溶液中与工程前列环素合酶结合的底物模拟物进行表征：前列环素生物合成中分子机制的含义。
DOI：
10.1021/bi701671q
复制
发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Cervantes,Vanessa
中科院分区：
文献类型：
--
作者：
Ruan,Ke-He;Wu,Jiaxin;Cervantes,Vanessa
使用高分辨率NMR光谱来确定底物（前列腺素H2）模拟物（U46619）与溶液中的工程化前列环素（PGI 2）合酶（PGIS）的对接。通过1D NMR滴定证明了U46619与PGIS蛋白的结合，并且在U46619以浓度依赖性方式与工程化PGIS结合时观察到U46619的C-11、H2 C和H2O处质子的化学位移的显著扰动。通过2D 1H NMR实验，使用转移NOE技术，进一步证实了PGIS结合的U46619的详细构象变化和三维结构。质子H20和H2、H18和H2、H18和H4之间的距离在它们与溶液中的PGIS结合后变短至5 μ m以内。这些较短的距离导致广泛开放的构象，其中未结合的U46619的三角形形状变为具有椭圆形形状的更紧凑的构象。U46619的结合构象比未结合的U46619更适合PGIS底物结合口袋的晶体结构。预测了PGIS活性位点口袋中对底物结合重要的残基。例如，Trp 282可能是最重要的残基之一，并且被怀疑在确定特定催化功能中起作用，这已经通过使用U46619的PGIS结合形式的NMR结构和PGIS晶体结构的对接研究来确定。这些研究为PGIS与其底物模拟物的相互作用提供了结构信息。U46619的C-6位置更接近C-9位置的构象变化为了解PGIS催化PGH 2异构化为前列环素的分子机制提供了第一个实验数据。
High-resolution NMR spectroscopy was used to determine the docking of a substrate (prostaglandin H2) mimic (U46619) to the engineered prostacyclin (PGI2) synthase (PGIS) in solution. The binding of U46619 to the PGIS protein was demonstrated by 1D NMR titration, and the significant perturbation of the chemical shifts of protons at C-11, H2C, and H20 of U46619 were observed upon U46619 binding to the engineered PGIS in a concentration-dependent manner. The detailed conformational change and 3D structure of the PGIS-bound U46619 were further demonstrated by 2D1H NMR experiments using the transferred NOE technique. The distances between the protons H20 and H2, H18 and H2, and H18 and H4 are shorter following their binding to the PGIS in solutiondown to within 5 Å. These shorter distances resulted in a widely open conformation, where the triangle shape of the unbound U46619 changed to a more compact conformation with an oval shape. The bound conformation of U46619 fits the crystal structure of the PGIS substrate binding pocket considerably better than that of the unbound U46619. The residues important to the substrate binding in the active site pocket of PGIS were also predicted. For example, Trp282 could be one of the most important residues and is suspected to play a role in the determination of specific catalytic function, which has been established by the docking studies using the NMR structure of the PGIS-bound form of U46619 and the PGIS crystal structure. These studies have provided the structural information for the interaction of the PGIS with its substrate mimic. The noted conformational changes where the C-6 position is closer to the C-9 position of U46619 provided the first experimental data for understanding the molecular mechanism of the catalytic function of PGIS in the isomerization of PGH2to prostacyclin.