Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase
Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase
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DOI：
10.1074/jbc.m406204200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Getzoff, ED
中科院分区：
文献类型：
--
作者：
Garcin, ED;Bruns, CM;Getzoff, ED
三种一氧化氮合酶 (NOS) 同工酶通过催化 Ca2+/钙调蛋白触发的 NO 合成，在神经传递、血管稳态和宿主防御中发挥着至关重要但又不同的作用。在这里，我们通过将诱变和生物化学与完全组装的、电子供应的神经元 NOS 还原酶二聚体的晶体结构测定相结合来解决有关 NOS 活性和调节的当前问题。通过整合这些结果，我们从结构上阐明了 NOS 中同工酶特异性调节电子转移的独特机制。我们对自抑制螺旋的发现、其在结构域之间的位置以及与经典钙调蛋白结合基序的惊人相似性，支持了 NOS 抑制的新机制。 NADPH、同工酶特异性残基 Arg(1400) 和 C 末端尾部通过将 FMN 结合结构域锁定在电子接受位置来协同抑制 NOS 活性。我们的分析表明，钙调蛋白结合或 C 端尾部磷酸化释放了整个 FMN 结构域的大规模摆动运动，以将电子传递到全酶中的催化模块。
Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.