Structural Metallobiochemistry of Nitric Oxide Synthases

描述：（由申请人提供）项目的总体目标是了解 一氧化氮合酶（NOS）同工酶如何调节一氧化氮的合成 (NO)因此它的双重生物活性是（i）一种可扩散的信使 用于神经传递、长时程增强、血小板聚集和血液 压力调节，和（ii）用于防御肿瘤细胞的细胞毒性剂 和寄生虫诱导型（iNOS）、内皮型（eNOS）和神经型（nNOS） 同种型通过一种有趣的钙调节的 电子转移机制和至少五个辅因子的独特组装。 NOS二聚体的每个亚基都有两个模块， (CaM)铰链区：1）具有血红素加氧酶结构域（NOSox）， 四氢生物蝶呤（H4 B）和形成催化中心的L-Arg结合位点 对于NO的产生，加上二聚体界面处的单个结构Zn位点，以及 2)具有NADPH、FAD和FMN位点的还原酶模块（NOSred）， 电子到血红素。所有三种同工酶的系统表征， 单独的NOSox、CaM结合和NOSred组分将处理复合物 结构生化基础NOS活性，同工酶特异性，和 调控结合Stuehr、Tamner和Getzoff小组的努力， 统一结构-功能研究的有效应用。生化和 Stuehr小组的突变表征将与 耦合实验晶体学、溶液散射和电子 微观结果加上Getzoff的计算结构分析， Tamner集团。作为一个整体，该项目将为以下方面提供基础： 发展和测试假设，从而弥合巨大的差距， 增加详细的NOS结构和生化数据， 了解NOS活性。这项工作的重点是定义保守和 负责1）催化活性和调节的可变同工酶特征 NOSox的结构和活性，2）NOSox同工酶的配体结合，3）NOSox的结构和活性， NOSred和4）组装的NOS中的结构域相互作用。设计的NOS突变体将 用于实验测试NOS结构的新兴原理， 功能这种协调的结构生物化学循环旨在提供一种 对NOS活性、抑制和调节的分子理解 与其生物学的重要方面相关的同工酶。这些结果将 进一步构建统一认识NOS的基本框架 与设计基于结构的抑制剂作为理想的化学品有关， 研究NOS功能的工具，并作为中风、败血症 休克和炎症损伤。

DESCRIPTION: (provided by applicant) The overall Project goal is to understand how nitric oxide synthase (NOS) isozymes regulate the synthesis of nitric oxide (NO) and thereby its dual biological activities as (i) a diffusible messenger for neurotransmission, long-term potentiation, platelet aggregation and blood pressure regulation, and (ii) a cytotoxic agent for defense against tumor cells and parasites. The inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) isoforms achieve their key functions via an intriguing calcium-regulated electron-transfer mechanism and a unique assembly of at least five cofactors. Each subunit of the NOS dimer has two modules joined by a calmodulin-binding (CaM) hinge region: 1) an oxygenase domain (NOSox) with heme, tetrahydrobiopterin (H4B), and L-Arg binding sites forming the catalytic center for NO production, plus a single structural Zn site at the dimer interface, and 2) a reductase module (NOSred) with NADPH, FAD, and FMN sites supplying electrons to the heme. Systematic characterizations of all three isozymes and individual NOSox, CaM-binding, and NOSred components will address the complex structural biochemistry underlying NOS activity, isozyme specificity, and regulation. Coupled Stuehr, Tamner, and Getzoff group efforts will insure efficient application of unified structure-function studies. Biochemical and mutational characterizations by the Stuehr group will proceed in concert with coupled experimental crystallographic, solution scattering and electron microscopic results plus computational structural analyses by the Getzoff and Tamner groups. As an integrated whole, this project will provide the basis to develop and test hypotheses, and to thereby bridge the growing gap between huge increases in detailed NOS structural and biochemical data and in-depth comprehension of NOS activities. This work focuses on defining conserved and variable isozyme features responsible for 1) catalytic activity and regulation of NOSox, 2) ligand binding to NOSox isozymes, 3) structure and activity of NOSred, and 4) domain interactions in assembled NOS. Designed NOS mutants will be used to experimentally test emerging principles for NOS structure and function. This coordinated structural biochemistry cycle aims to provide a molecular understanding of the activity, inhibition, and regulation of NOS isozymes relevant to important aspects of their biology. These results will furthermore build the essential framework for a unified understanding of NOS relevant to the design of structure-based inhibitors as desirable chemical tools for studying NOS function and as therapeutic agents for stroke, septic shock, and inflammatory damage.