Structure and Dynamics of Cyclooxygenase Catalysis and Inhibition

环加氧酶催化和抑制的结构和动力学

基本信息

批准号：
9116265
负责人：
MICHAEL G MALKOWSKI
金额：
$ 38.32万
依托单位：
HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9116265
关键词：
Active Sites Acute Affect Amphipathic Alpha Helix Anabolism Arachidonic Acids Aspirin Binding Biochemical Cardiovascular Diseases Cardiovascular system Catalysis Catalytic Domain Cessation of life Chronic Communication Complex Consumption Coupled Cysteine Detergents Development Diet Disease Electron Spin Resonance Spectroscopy Enzymes Event Fatty Acids Goals Health Health Professional Inflammation Investigation Isoenzymes Ligand Binding Ligands Lipid Bilayers Malignant Neoplasms Maps Masks Mediating Membrane Methods Modeling Molecular Molecular Conformation Motion Movement Nature Non-Steroidal Anti-Inflammatory Agents PTGS2 gene Pathology Patients Pharmaceutical Preparations Play Property Prostaglandin-Endoperoxide Synthase Prostaglandins Proteins Regulation Reporter Risk Roentgen Rays Role Signal Transduction Site Source Spin Labels Structure Technology Testing Time Work base biophysical analysis cyclooxygenase 1 cyclooxygenase 2 inhibitor/antagonist innovation insight monomer mutant nanodisk novel strategies novel therapeutics reconstitution tyrosine radical

项目摘要

DESCRIPTION (provided by applicant): Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid to generate prostaglandins and are inhibited by aspirin, nonselective nonsteroidal anti-inflammatory drugs (NSAIDs), and COX-2 selective diarylheterocyclic based drugs (coxibs). COX-1 and COX-2 are membrane-associated homodimers that bind to one leaflet of the lipid bilayer via a membrane-binding domain comprised of amphipathic helices. As a consequence, the enzymes have traditionally required the presence of detergents to maintain the protein in a stable and active form during functional and structural characterization. A new paradigm has emerged with respect to COX catalysis and regulation. In this model, COX functions as a conformational heterodimer with one monomer active at a given time. Common dietary FAs and nonselective NSAIDs bind to one monomer of the homodimer to modulate the oxygenation of substrates in the other monomer through an allosteric/catalytic couple. The molecular mechanism governing crosstalk between monomers is not known and static pictures derived from crystal structures of COX do not provide any insight into the conformational motions responsible for this dynamical interplay. We surmise that detergent binding and the restricted confines of the crystal lattice have masked these conformational motions. Hence, a new approach to study COX catalysis in solution and in the absence of detergent is needed. We propose to couple the use of nanodisc-reconstituted COX-2 with site- directed spin-labeling ESR spectroscopic technologies to characterize the protein conformational dynamics associated with COX catalysis and inhibition. The objectives are to identify the conformational motions responsible for: a) ligand access to the cyclooxygenase channel and b) communication between monomers. Understanding the molecular basis of how the binding of different ligands induces conformational motions responsible for crosstalk between monomers bridges the gap between the static information derived from COX crystal structure analysis and the dynamical character of COX as it relates to the modulation of COX function.

描述（由申请人提供）：环加氧酶（COX-1 和 COX-2）氧化花生四烯酸生成前列腺素，并被阿司匹林、非选择性非甾体抗炎药 (NSAID) 和 COX-2 选择性二芳基杂环类药物 (昔布类) 抑制COX-1 和 COX-2 是膜相关同型二聚体，与细胞的一个小叶结合因此，传统上，酶需要去污剂的存在，以在功能和结构表征过程中保持蛋白质的稳定和活性形式。 COX 催化和调节在该模型中，COX 作为构象异二聚体，在给定时间内具有一个活性单体，并与同二聚体的一个单体结合。通过变构/催化对调节另一个单体中底物的氧化作用控制单体之间串扰的分子机制尚不清楚，并且来自 COX 晶体结构的静态图片无法提供对导致这种动态相互作用的构象运动的任何见解。我们推测去垢剂结合和晶格的限制掩盖了这些构象运动，因此，一种研究溶液中和无去垢剂情况下 COX 催化的新方法。我们建议将纳米圆盘重构的 COX-2 与定点自旋标记 ESR 光谱技术结合起来，以表征与 COX 催化和抑制相关的蛋白质构象动力学。 a) 配体进入环氧合酶通道，b) 理解不同配体的结合如何诱导构象运动，从而在单体之间架起桥梁。来自 COX 晶体结构分析的静态信息和与 COX 功能调制相关的 COX 动态特性。