Structure and Dynamics of Cyclooxygenase Catalysis and Inhibition

环加氧酶催化和抑制的结构和动力学

• 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
• 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

 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.

 性状（由申请方提供）：环氧化酶（考克斯-1和考克斯-2）可通过花生四烯酸生成异甘草定，并可被阿司匹林、非选择性非甾体抗炎药（NSAID）和考克斯-2选择性二芳基杂环类药物（昔布类）抑制。考克斯-1和考克斯-2是膜相关的同源二聚体，通过由两亲性螺旋组成的膜结合结构域与脂质双层的一个小叶结合。因此，酶传统上需要去污剂的存在以在功能和结构表征期间保持蛋白质处于稳定和活性形式。关于考克斯催化和调节已经出现了新的范例。在这个模型中，考克斯作为一个构象异源二聚体的功能与一个单体活性在给定的时间。常见的膳食脂肪酸和非选择性非甾体抗炎药结合到同二聚体的一个单体，通过变构/催化偶来调节另一个单体中底物的氧合。控制单体之间串扰的分子机制尚不清楚，并且来自考克斯晶体结构的静态图片不能提供对这种动态相互作用的构象运动的任何洞察。我们推测，洗涤剂的结合和晶格的局限性掩盖了这些构象运动。因此，需要一种新的方法来研究考克斯催化在溶液中和在没有洗涤剂的情况下。我们建议将纳米盘重构的考克斯-2与定点自旋标记ESR光谱技术结合使用，以表征与考克斯催化和抑制相关的蛋白质构象动力学。目的是确定负责以下的构象运动：a）配体进入环加氧酶通道和B）单体之间的通信。了解不同配体的结合如何诱导导致单体之间串扰的构象运动的分子基础，弥合了源自考克斯晶体结构分析的静态信息与考克斯的动力学特征之间的差距，因为它涉及考克斯功能的调节。