Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity
Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity
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DOI：
10.1073/pnas.050406897
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发表时间：
2000-03-28
影响因子：
11.1
通讯作者：
Hatziris, Z
中科院分区：
文献类型：
--
作者：
Cupp-Vickery, J;Anderson, R;Hatziris, Z
几种哺乳动物细胞色素P450 （P450）同工异构体与许多底物（包括类固醇和多环芳烃）表现出同向协同性。为了确定类固醇和多环芳烃与P450酶结合的结构因素，并确定变构位点的位置，我们研究了糖多孢菌（Saccharopolyspora erythrea）大环内酯羟化酶P450eryF与雄烯二酮和9-氨基菲的相互作用。结合光谱分析表明，P450eryF与雄烯二酮的亲和力为365 μ M， Hill系数为1.31 +/- 0.6；与9-氨基菲的配位性为91 μ M， Hill系数为1.38 +/- 0.2。生长雄烯二酮和9-氨基菲与P450eryF的配合物晶体，并分别衍射到2.1埃和2.35埃。电子密度图表明，对于这两种配合物，两个配体分子同时存在于活性位点。P450eryF/雄烯二酮模型细化到r = 18.9%，两种雄烯二酮分子具有相似的构象。近端雄烯二酮定位于碳-6的α面最靠近血红素铁，第二类固醇分子位于活性位点远端5.5埃处。P450eryF/9-氨基菲南模型的r = 19.7%，近端9-氨基菲南通过9-氨基与血红素铁配合，第二配体位于活性位点远端约6埃处。这些结果表明，配体结合中的同向性协同性可以由活性位点口袋内的两个底物分子结合而产生，而不会引起蛋白质的主要构象变化。
Several mammalian cytochrome P450 (P450) isoforms demonstrate homotropic cooperativity with a number of substrates, including steroids and polycyclic aromatic hydrocarbons. To identify structural factors contributing to steroid and polycyclic aromatic hydrocarbon binding to P450 enzymes and to determine the location of the allosteric site, we investigated interactions of the macrolide hydroxylase P450eryF from Saccharopolyspora erythraea with androstenedione and 9-aminophenanthrene. Spectroscopic binding assays indicate that P450eryF binds androstenedione with an affinity of 365 mu M and a Hill coefficient of 1.31 +/- 0.6 and coordinates with 9-aminophenanthrene with an affinity of 91 mu M and a Hill coefficient of 1.38 +/- 0.2. Crystals of complexes of androstenedione and 9-aminophenanthrene with P450eryF were grown and diffracted to 2.1 Angstrom and 2.35 Angstrom, respectively. Electron density maps indicate that for both complexes two ligand molecules are simultaneously present in the active site. The P450eryF/ androstenedione model was refined to an r = 18.9%, and the two androstenedione molecules have similar conformations. The proximal androstenedione is positioned such that the alpha-face of carbon-6 is closest to the heme iron, and the second steroid molecule is positioned 5.5 Angstrom distal in the active site, The P450eryF/9-aminophenanthrene model was refined to an r = 19.7% with the proximal 9-aminophenanthrene coordinated with the heme iron through the 9-amino group and the second ligand positioned approximate to 6 Angstrom distal in the active site. These results establish that homotropic cooperativity in ligand binding can result from binding of two substrate molecules within the active site pocket without major conformational changes in the protein.