Structural Biology of Neuronal Nitric Oxide Synthase

描述（由申请人提供）：一氧化氮（NO）是由l -精氨酸和分子氧通过三种结构不同的一氧化氮合成酶（NOS）合成的气态信使：神经元（nNOS），内皮（eNOS）和诱导（iNOS）。在正常情况下，NO在体内平衡中起关键作用。在中风中，nNOS产生的NO过量会导致显著的神经毒性，而eNOS产生的NO有利于通过血管舒张和抑制血小板聚集来恢复血液流动，nNOS是中风治疗的一个极好的分子靶点，但抑制剂必须对nNOS亚型具有明显的选择性，这样它们才不会阻断eNOS产生NO的积极作用。本研究项目的假设是：1)通过比较nNOS和eNOS亚型的结构研究，可以发现nNOS亚型特异性抑制的差异；2)了解天然NOS抑制剂抑制的结构基础，可能提供新的NOS抑制策略。高分辨率x射线晶体学研究将探讨nNOS抑制的结构基础，主要研究三个特定目的：1)确定nNOS催化血红素结构域的三维结构；2)表征和比较nNOS与eNOS主结构域结合的nNOS特异性异构体衍生物；3)确定天然蛋白质抑制剂caveolin-1的NOS识别位点。这些研究将为nNOS异构体特异性药物设计提供基础科学框架。

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a gaseous messenger synthesized from L-arginine and molecular oxygen by three structurally distinct nitric oxide synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Under normal conditions, NO plays key roles in homeostasis. In stroke, NO overproduction by nNOS results in significant neurotoxicity, while NO generated by eNOS is beneficial to restoring blood flow via vasodilation and inhibition of platelet aggregation, nNOS is an excellent molecular target for stroke therapy, but inhibitors must be clearly selective for the nNOS isoform such that they do not block the positive effects resulting from NO production by eNOS. The hypotheses underlying this research project are that 1) comparative structural study of the nNOS and eNOS isoforms will identify differences, which can be targeted for isoform specific inhibition of nNOS and 2) an understanding of the structural basis of inhibition of a natural NOS inhibitor may provide novel NOS inhibition strategies. The structural basis of nNOS inhibition will be probed by high resolution x-ray crystallographic studies addressing three Specific Aims: 1) the determination of the three dimensional structure of the catalytic heme domain of nNOS, 2) characterization and comparison of nNOS specific isoform derivatives bound to the nNOS and eNOS home domain, and 3) determination of the NOS recognition site of a natural protein inhibitor, caveolin-1. These studies will provide the basic science framework for nNOS isoform specific drug design.