Structural Metallobiochemistry of Nitric Oxide Synthases

一氧化氮合成酶的结构金属生物化学

• 批准号: 7211759
• 负责人: ELIZABETH D GETZOFF
• 金额: $ 44.66万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7211759
• 关键词: Active Sites; Address; Affect; Affinity; Arginine; Arthritis; Binding; Binding Sites; Biochemical; Biological; Biological Process; Biology; C-terminal; Calmodulin; Catalysis; Caveolins; Complement; Complex; Crystallography; Cytotoxic agent; Cytotoxin; Deuterium; Diabetes Mellitus; Digestion; Disulfide Linkage; Docking; Electron Transport; Electrons; Enzymes; Equilibrium; FMN reductase; Flavin Mononucleotide; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Helix (Snails); Heme; Hydrogen; Inflammatory; Ions; Isoenzymes; Length; Literature; Long-Term Potentiation; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Medical; Modeling; Molecular; Molecular Biology; Molecular Sieve Chromatography; Motion; Movement; Mutagenesis; NADP; Neurodegenerative Disorders; Neurons; Nitric Oxide; Nitric Oxide Signaling Pathway; Nitric Oxide Synthase; Outcome; Oxidation-Reduction; Oxidoreductase; Oxygenases; Parasites; Peptides; Phosphorylation; Platelet aggregation; Pliability; Production; Property; Protein Isoforms; Proteins; Pterins; Rate; Regulation; Research; Resolution; Roentgen Rays; Sepsis; Septic Shock; Signal Transduction; Site; Specificity; Spectrum Analysis; Stroke; Structural Biochemistry; Structural Chemistry; Structure; Surface Plasmon Resonance; System; Tail; Techniques; Testing; Therapeutic; Trypsin; base; blood pressure regulation; caveolin 1; cofactor; design; dimer; ear helix; improved; inhibitor/antagonist; light scattering; mutant; neoplastic cell; neurotransmission; research study; tetrahydrobiopterin

DESCRIPTION (provided by applicant): Nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a diffusible messenger for platelet aggregation, blood pressure regulation, neurotransmission, long-term potentiation, and also as a cytotoxic agent for defense against tumor cells and parasites. Three NOS enzymes, the inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) isoforms, achieve their key biological functions via intriguing regulations of their electron transfer mechanism and an assembly of six cofactors. Each subunit of the NOS dimer has two modules joined by a calmodulin-binding linker: an oxygenase module (NOSox) with heme, tetrahydrobiopterin (H4B), Zn ion, and Arginine binding sites forming the catalytic center for NO production, and a reductase module (NOSred) with NADPH, FAD, and FMN sites supplying electrons to the heme. Our overall goal is to characterize the detailed structural biochemistry underlying the active site interactions, catalysis, isozyme-specificity, assembly, regulation, and both inter-domain and inter-protein interactions of NOS enzymes. Our characterizations of the independently functional dimeric NOSox and NOSred modules, and of calmodulin (CaM) bound to the CaM-binding peptide, provide a powerful framework for interpreting NOS structural biochemistry. Our progress to date prompts four proposed Aims, which are driven by specific hypotheses. We now propose integrated structural, mutational and biophysical experiments to test these hypotheses and to address specific critical and challenging unanswered questions. How do NOSox, NOSred and CaM assemble for function? What is the mechanism for rate-limiting electron transfer from the NOSred FMN to the NOSox heme? How do isozyme-specific features tune and regulate NOS activity? How is NOS activity regulated through interactions with its protein partners? Our interdisciplinary experiments on NOS domains and full-length proteins will characterize active-site interactions, key assemblies, conformational switching mechanisms, and inter-protein interactions. We expect to characterize prototypical sets of structures and mutant enzymes, functional complexes with inhibitors and with protein partners, and to define the structural chemistry underlying the exquisite regulation of NO( synthesis. Deuterium Hydrogen Exchange Mass Spectrometry (DXMS) and advanced Small-Angle X- ray Scattering (SAXS) combined with computationally-aided design of mutants to lock, strengthen or block interactions (including designed disulfide linkages) will test and complement high resolution crystallographic structures. The expected outcome of the proposed research is a detailed molecular understanding of the activity, inhibition, and regulation of NOS isozymes relevant to important aspects of their biology and medical importance for blood pressure regulation, stroke, septic shock, cancer and inflammatory damage.

描述（由申请人提供）：一氧化氮合酶（NOS）调节一氧化氮（NO）合成，从而调节其作为血小板聚集、血压调节、神经传递、长时程增强的扩散信使以及细胞毒性的双重生物活性防御肿瘤细胞和寄生虫的药剂。三种 NOS 酶，即诱导型 (iNOS)、内皮型 (eNOS) 和神经型 (nNOS) 亚型，通过对其电子转移机制的有趣调节和六个辅助因子的组装来实现其关键生物学功能。 NOS 二聚体的每个亚基都有两个由钙调蛋白结合接头连接的模块：加氧酶模块 (NOSox)，具有血红素、四氢生物蝶呤 (H4B)、锌离子和精氨酸结合位点，形成 NO 生成的催化中心，以及还原酶模块(NOSred) 具有 NADPH、FAD 和 FMN 位点，为血红素提供电子。我们的总体目标是表征 NOS 酶活性位点相互作用、催化、同工酶特异性、组装、调节以及结构域间和蛋白质间相互作用的详细结构生物化学。我们对独立功能的二聚体 NOSox 和 NOSred 模块以及与 CaM 结合肽结合的钙调蛋白 (CaM) 的表征，为解释 NOS 结构生物化学提供了强大的框架。迄今为止，我们的进展提出了四个拟议目标，这些目标是由具体假设驱动的。我们现在提出综合的结构、突变和生物物理实验来测试这些假设并解决特定的关键和具有挑战性的未解答的问题。 NOSox、NOSred 和 CaM 如何组装以发挥功能？从 NOSred FMN 到 NOSox 血红素的限速电子转移机制是什么？同工酶特异性特征如何调节和调节 NOS 活性？ NOS 活性如何通过与其蛋白质伙伴的相互作用进行调节？我们对 NOS 结构域和全长蛋白质的跨学科实验将表征活性位点相互作用、关键组装、构象转换机制和蛋白质间相互作用。我们期望表征结构和突变酶、具有抑制剂和蛋白质伴侣的功能复合物的原型组，并定义 NO( 合成的精细调节背后的结构化学。氘氢交换质谱 (DXMS) 和先进的小角 X - 射线散射（SAXS）与突变体的计算辅助设计相结合以锁定、加强或阻断相互作用（包括设计的二硫键）将测试和补充高分辨率晶体结构的预期结果。拟议的研究是对 NOS 同工酶的活性、抑制和调节的详细分子理解，这些同工酶与其生物学和医学上的重要方面相关，对血压调节、中风、感染性休克、癌症和炎症损伤具有重要意义。