Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 2010 Aug 20;285(34):25941-9. doi: 10.1074/jbc.M110.126797. Epub 2010 Jun 7.
Calmodulin (CaM) activates the nitric-oxide synthases (NOS) by a mechanism that is not completely understood. A recent crystal structure showed that bound CaM engages in a bridging interaction with the NOS FMN subdomain. We investigated its importance in neuronal NOS (nNOS) by mutating the two residues that primarily create the bridging interaction (Arg(752) in the FMN subdomain and Glu(47) in CaM). Mutations designed to completely destroy the bridging interaction prevented bound CaM from increasing electron flux through the FMN subdomain and diminished the FMN-to-heme electron transfer by 90%, whereas mutations that partly preserve the interaction had intermediate effects. The bridging interaction appeared to control FMN subdomain interactions with both its electron donor (NADPH-FAD subdomain) and electron acceptor (heme domain) partner subdomains in nNOS. We conclude that the Arg(752)-Glu(47) bridging interaction is the main feature that enables CaM to activate nNOS. The mechanism is bi-modal and links a single structural aspect of CaM binding to specific changes in nNOS protein conformational and electron transfer properties that are essential for catalysis.
钙调蛋白（CaM）通过一种尚未完全阐明的机制激活一氧化氮合酶（NOS）。最近的晶体结构表明，结合的 CaM 与 NOS FMN 亚结构域进行桥接相互作用。我们通过突变主要形成桥接相互作用的两个残基（FMN 亚结构域中的精氨酸（Arg752）和 CaM 中的谷氨酸（Glu47））来研究其在神经元型 NOS（nNOS）中的重要性。旨在完全破坏桥接相互作用的突变阻止结合的 CaM 增加 FMN 亚结构域中的电子流，并使 FMN 到血红素的电子转移减少 90%，而部分保留相互作用的突变则具有中间效应。桥接相互作用似乎控制 FMN 亚结构域与 nNOS 中其电子供体（NADPH-FAD 亚结构域）和电子受体（血红素结构域）伴侣亚结构域的相互作用。我们得出结论，Arg752-Glu47 桥接相互作用是 CaM 激活 nNOS 的主要特征。该机制是双模态的，将 CaM 结合的单个结构方面与 nNOS 蛋白构象和电子转移特性的特定变化联系起来，这些变化对于催化至关重要。
