Structural Metallobiochemistry of Nitric Oxide Synthases

描述（由申请人提供）：一氧化氮合酶（NOS）调节一氧化氮（NO）的合成，从而调节其作为血小板聚集、血压调节、神经传递、长时程增强的可扩散信使以及作为防御肿瘤细胞和寄生虫的细胞毒性剂的双重生物活性。三种NOS酶，诱导型（iNOS）、内皮型（eNOS）和神经型（nNOS）亚型，通过其电子传递机制的有趣调节和六种辅因子的组装来实现其关键生物学功能。NOS二聚体的每个亚基具有通过钙调蛋白结合接头连接的两个模块：具有血红素、四氢生物蝶呤（H4B）、Zn离子和精氨酸结合位点的加氧酶模块（NOSox）形成NO产生的催化中心，以及具有NADPH、FAD和FMN位点的还原酶模块（NOSred）向血红素提供电子。我们的总体目标是表征详细的结构生物化学活性位点的相互作用，催化，同工酶特异性，组装，调节，以及结构域间和蛋白质间的相互作用NOS酶。我们的独立功能的二聚体NOSox和NOSred模块，和钙调素（CaM）结合的钙调素结合肽的表征，提供了一个强大的框架解释NOS结构的生物化学。迄今为止，我们的进展促使提出了四个目标，这些目标是由特定的假设驱动的。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，并解决具体的关键和具有挑战性的悬而未决的问题。NOSox、NOSred和CaM如何组装以实现功能？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NOS活性？NOS活性如何通过与其蛋白质伴侣的相互作用来调节？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的相互作用，关键组件，构象转换机制和蛋白质间的相互作用。我们期望表征原型组的结构和突变的酶，与抑制剂和蛋白质伴侣的功能复合物，并定义结构化学的精细调控NO（合成）。氘氢交换质谱（DXMS）和先进的小角X射线散射（SAXS）结合突变体的计算机辅助设计以锁定，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。拟议研究的预期成果是对NOS同工酶的活性、抑制和调节进行详细的分子理解，这些同工酶与其生物学的重要方面以及对血压调节、中风、感染性休克、癌症和炎症损伤的医学重要性相关。

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.