Motion and Flexibility in Human Cytochrome P450 Aromatase

The crystal structures of human placental aromatase in complex with the substrate androstenedione and exemestane have revealed an androgen-specific active site and the structural basis for higher order organization. However, X-ray structures do not provide accounts of movements due to short-range fluctuations, ligand binding and protein-protein association. In this work, we conduct normal mode analysis (NMA) revealing the intrinsic fluctuations of aromatase, deduce the internal modes in membrane-free and membrane-integrated monomers as well as the intermolecular modes in oligomers, and propose a quaternary organization for the endoplasmic reticulum (ER) membrane integration. Dynamics of the crystallographic oligomers from NMA is found to be in agreement with the isotropic thermal factors from the X-ray analysis. Calculations of the root mean square fluctuations of the C-alpha atoms from their equilibrium positions confirm that the rigid-core structure of aromatase is intrinsic regardless of the changes in steroid binding interactions, and that aromatase self-association does not deteriorate the rigidity of the catalytic cleft. Furthermore, NMA on membrane-integrated aromatase shows that the internal modes in all likelihood contribute to breathing of the active site access channel. The collective intermolecular hinge bending and twisting modes provide the flexibility in the quaternary association necessary for membrane integration of the aromatase oligomers. Taken together, fluctuations of the active site, the access channel, and the heme-proximal cavity, and a dynamic quaternary organization could all be essential components of the functional aromatase in its role as an ER membrane-embedded steroidogenic enzyme.

人胎盘芳香化酶（aromatase）与底物雄烯二酮（androstenedione）及依西美坦（exemestane）结合的晶体结构，已揭示出其雄激素特异性活性位点以及高阶组装的结构基础。然而，X射线晶体结构无法阐释由短程涨落、配体结合以及蛋白质-蛋白质相互作用所引发的动态运动。本研究通过正常模式分析（normal mode analysis, NMA）解析了芳香化酶的固有涨落特性，推导了无膜及膜整合单体的内部振动模式，以及寡聚体的分子间振动模式，并提出了适配内质网（endoplasmic reticulum, ER）膜整合的四级组装结构。经NMA得到的晶体寡聚体动力学特性，与X射线分析得到的各向同性热因子结果一致。通过对Cα原子（C-alpha atoms）相对于平衡位置的均方根涨落进行计算，证实了芳香化酶的刚性核心结构是其固有属性，不受类固醇结合相互作用变化的影响，且芳香化酶的自组装并不会削弱催化裂隙的刚性。此外，针对膜整合型芳香化酶的NMA结果显示，其内部振动模式极有可能参与活性位点入口通道的“呼吸”运动。集体性的分子间铰链弯曲与扭转模式，为芳香化酶寡聚体的膜整合所需的四级组装提供了必要的柔性。综上，活性位点、入口通道及血红素近端腔的涨落，以及动态四级组装结构，均是作为内质网（ER）膜嵌入型类固醇生成酶发挥功能的芳香化酶的核心必要组成部分。