Structural Metallobiochemistry of Nitric Oxide Synthases

描述(申请人提供)：一氧化氮合酶(NOS)调节一氧化氮(NO)的合成，从而具有双重生物学活性，既是血小板聚集、血压调节、神经传递、长期增强的可扩散信使，也是对抗肿瘤细胞和寄生虫的细胞毒剂。诱导型一氧化氮合酶(INOS)、内皮型一氧化氮合酶(ENOS)和神经型一氧化氮合酶(NNOS)三种亚型通过对其电子传递机制和六种辅因子的组装来实现其关键的生物学功能。NOS二聚体的每个亚基都有两个模块，由钙调蛋白结合连接体连接：一个加氧酶模块(NOSox)，其血红素、四氢生物蝶呤(H4B)、锌离子和精氨酸结合位点形成了NO产生的催化中心；以及一个还原酶模块(NOSred)，其NADPH、FAD和FMN位点为该血红素提供电子。我们的总体目标是描述一氧化氮合酶活性部位相互作用、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的结构生物化学提供了一个强大的框架。到目前为止，我们的进展提出了四个拟议的目标，这些目标是由具体的假设驱动的。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，并解决特定的关键和具有挑战性的未回答的问题。NOSox、NOSred和Cam是如何组合成函数的？从NOSred FMN到NOSox血红素的限速电子转移的机制是什么？同工酶的特性如何调节和调节一氧化氮合酶活性？一氧化氮合酶的活性是如何通过与其蛋白质伙伴的相互作用来调节的？我们对一氧化氮合酶结构域和全长蛋白质的跨学科实验将表征活性部位相互作用、关键组装、构象转换机制和蛋白质间相互作用。我们希望表征一组原型的结构和突变的酶，与抑制剂和蛋白质伙伴的功能复合体，并确定精细调控NO(合成)的结构化学。氢氢交换质谱仪(DXMS)和先进的小角X射线散射(SAXS)结合计算机辅助设计突变体以锁定、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。这项拟议研究的预期结果是详细的分子理解一氧化氮合酶同工酶的活性、抑制和调节，这些同工酶与其生物学和医学上的重要方面有关，对于血压调节、中风、感染性休克、癌症和炎症损害具有重要意义。

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.