Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain
Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain
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DOI：
10.1074/jbc.m114.564005
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发表时间：
2014-06-13
影响因子：
4.8
通讯作者：
Southworth, Daniel R.
中科院分区：
文献类型：
--
作者：
Yokom, Adam L.;Morishima, Yoshihiro;Southworth, Daniel R.
一氧化氮合酶（NOS）是哺乳动物产生NO调节血压、突触反应和免疫防御所必需的。NOS是一个大的同源二聚体，具有充分表征的还原酶和加氧酶结构域，其协调多步骤的结构域间电子转移机制以氧化L-精氨酸并产生NO。Ca 2 +-钙调蛋白（CaM）结合在还原酶和加氧酶结构域之间以激活NO合成。虽然NOS长期以来一直被提议采用不同的构象，交替之间的interflavin和FMN-血红素电子转移步骤，全酶的结构仍然难以捉摸和钙调素结合的安排是未知的。在这里，我们应用单粒子电子显微镜（EM）方法来表征全长的神经元亚型（nNOS）复合物，并确定钙调素激活的结构机制。我们已经确定，nNOS采用开放和封闭的构象状态的合奏和钙调素结合诱导的还原酶结构域的戏剧性重排。我们对完整的nNOS-CaM复合物的三维重建揭示了封闭的构象和交叉单体排列，其中FMN结构域从NADPH-FAD中心旋转远离，朝向加氧酶二聚体。这项工作捕获，为第一次，还原酶-加氧酶的结构安排和钙调素依赖的释放FMN域，协调驱动电子转移的域催化过程中。
Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize L-arginine and generate NO. Ca2+-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis.