Structural Metallobiochemistry of Nitric Oxide Synthases

描述：(申请人提供)项目的总体目标是了解 一氧化氮合酶同工酶如何调节一氧化氮的合成 (NO)，从而其作为(I)可扩散信使的双重生物活性 用于神经传递、长时程增强、血小板聚集和血液 压力调节，以及(Ii)抗肿瘤细胞的细胞毒剂 还有寄生虫。诱导型(INOS)、内皮型(ENOS)和神经型(NNOS) 异构体通过耐人寻味的钙调节来实现其关键功能 电子转移机制和至少五个辅因子的独特组装。 一氧化氮合酶二聚体的每个亚基都有两个模块，通过钙调素结合连接在一起 (CaM)铰链区：1)含血红素的加氧酶结构域(NOSox)， 四氢生物蝶呤(H4B)和形成催化中心的L-精氨酸结合部位 对于不产生，加上在二聚体界面的单一结构锌位置，以及 2)由NADPH、FAD和FMN位点提供的还原酶模块(NOSred) 电子到亚铁血红素。三种同工酶的系统学性质及 单独的NOSox、CaM绑定和NOSred组件将解决复杂的问题 一氧化氮合酶活性的结构生物化学，同工酶特异性，以及 监管。联合Stuehr，Tamner和Getzoff团队的努力将确保 高效应用统一的结构-功能研究。生化和 Stuehr小组的突变特征将与 耦合实验结晶学、溶液散射和电子 微观结果加上Getzoff和 塔姆纳集团。作为一个整体，该项目将为 开发和测试假设，从而弥合巨大的 增加详细的一氧化氮合酶结构和生化数据，并深入 对一氧化氮合酶活性的理解。这项工作的重点是定义守恒和 1)催化活性和调控的可变同工酶特征 2)与NOSOx同工酶结合的配体，3)结构和活性 NOSred，以及4)组装的NOS中的结构域相互作用。设计的一氧化氮合酶突变体将 用于实验测试一氧化氮合酶结构的新原理和 功能。这种协调的结构生物化学循环旨在提供一种 一氧化氮合酶活性、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 进一步为统一理解NOS建立必要的框架 与基于结构的缓蚀剂的设计相关，作为理想的化学物质 研究一氧化氮合酶功能的工具和作为中风、脓毒症治疗药物的工具 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.