Crystallization and structure determination of the mu-opioid receptor

mu-阿片受体的结晶和结构测定

• 批准号: 8280351
• 负责人: Roger K Sunahara
• 金额: $ 15.11万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8280351
• 关键词: Abbreviations; Adenosine A2A Receptor; Adrenergic Receptor; Adverse effects; Affinity; Agonist; Analgesics; Binding; Binding Sites; Cells; Chimeric Proteins; Complex; Constipation; Coupled; Crystallization; Crystallography; Detergents; Development; Exploratory/Developmental Grant; Funding; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Hormones; Insecta; Investigation; Ligand Binding; Ligands; Lipids; Mediator of activation protein; Methods; Molecular Conformation; Molecular Sieve Chromatography; Molecular Weight; Morphine; Muramidase; Nausea; Nociception; Opiates; Opioid Receptor; Outcome; Pain; Peptides; Pharmaceutical Preparations; Phase; Proteins; Research; Resolution; Roentgen Rays; Role; Site; Solvents; Structure; Surface; Ventilatory Depression; Work; addiction; base; drug development; high risk; innovation; insight; mu opioid receptors; receptor; receptor binding; receptor function; small molecule; structural biology; success; therapeutic target

DESCRIPTION (provided by applicant): The G protein-coupled opioid receptors have been important therapeutic targets for centuries due to their role in nociception and the early discovery of the opiate drugs. The ¿ opioid receptor (¿OR) is the main mediator of the analgesic effect of morphine and other opioid receptor (OR) agonists. For this reason, this R21 proposal will focus on the ¿-opioid receptor, expanding the research to the related subtypes as experimental success allows. While effective attenuators of pain, analgesics that work on ¿OR have various adverse and sometimes fatal side-effects including nausea, respiratory depression, addiction, and constipation. A major impediment to the development of drugs that can achieve anti-nociception without the concomitant side effects has been a lack of understanding of the structural basis of hormone binding and activation of the opioid receptors. The recent elucidation of the x-ray crystal structures of the several G protein-coupled receptors (GPCRs) has provided new insights into the binding of small molecular weight antagonists to GPCRs. It is now appreciated that the capacity of drugs to bind and stabilize specific conformations of the receptor is dependent not only on the binding site but also on the access to the binding site. The spectrum of ligands that act on opiate receptors is more complex including both peptide and small molecular weight agonists and antagonists. Therefore structural insights into the similarities and differences between peptide and small molecule ligand binding would greatly facilitate the development of more effective and selective drugs. A major breakthrough in GPCR structural biology was the incorporation of a stable protein, T4 lysozyme (T4L), into a structurally unstable region of a GPCR, the 3rd cytoplasmic loop(1).This approach was developed in the Kobilka lab at Stanford, and used to obtain a high-resolution structure of the ¿2AR(1, 2). Replacing the 3rd loop with T4L not only stabilized the receptor but it also contributed a nicely ordered and solvent-accessible crystallization packing surface. This method has proven to be applicable to other GPCRs as demonstrated in the more recent structure of the adenosine A2A receptor. We propose to utilize a similar approach to stabilize the ¿OR for crystallogenesis. We have already successfully demonstrated the expression and purification of a functional form of the ¿OR from insect cells. We are now poised to obtain an X-ray crystal structure of this important pharmacological target. We chose to begin a detailed investigation of the structural biology of opiate receptors through an R21 mechanism. Our goal is to obtain diffraction quality crystals and a high-resolution structure of the ¿OR-T4L bound to a high-affinity antagonist. The proposed work is high-risk and high-impact. If successful, the outcome of this R21 proposal will enable us to obtain funding for a more through structural characterization of the ¿OR and other OR subtypes. The long-term goals of subsequent proposals will be to obtain structures of receptors bound to peptide and non-peptide forms of agonist and antagonist ligands. Specific Aims 1) Generate a ¿OR-T4L fusion protein and adjust the linkers between these two proteins to optimize ¿OR function (ligand binding affinity), expression and stability. 2) Establish conditions for expression and purification of ¿OR-T4L for crystallography trials. 3) Crystallize and determine the x-ray crystal structure of the ¿OR-T4L receptor Abbreviations: ¿OR - ¿ opioid receptor LCP - lipidic cubic phase Y¿OR - YFP-¿OR fusion protein TM - transmembrane segment T4L - T4 lysozyme ICL - intracellular loop ¿2AR - 22 adrenergic receptor DDM - dodecylmaltoside SEC - size exclusion chromatography

描述（由申请人提供）：由于 G 蛋白偶联阿片受体在伤害感受中的作用以及阿片类药物的早期发现，几个世纪以来一直是重要的治疗靶点。阿片受体 (OR) 是吗啡和其他阿片受体 (OR) 激动剂镇痛作用的主要介质，因此，本 R21 提案将重点关注 ¿ -阿片受体，在实验成功的情况下将研究扩展到相关亚型，同时有效减轻疼痛，镇痛药可以发挥作用。 OR 具有各种不良甚至有时致命的副作用，包括恶心、呼吸抑制、成瘾和便秘，开发能够实现抗伤害感受而不伴随副作用的药物的一个主要障碍是缺乏对其结构基础的了解。最近对几种 G 蛋白偶联受体 (GPCR) 的 X 射线晶体结构的阐明为小分子量拮抗剂的结合提供了新的见解。现在人们认识到，药物结合和稳定受体特定构象的能力不仅取决于结合位点，还取决于作用于阿片受体的配体谱。包括肽和小分子量激动剂和拮抗剂，因此对肽和小分子配体结合之间的相似性和差异的结构了解将极大地促进更有效和选择性药物的开发。稳定的蛋白质， T4 溶菌酶 (T4L) 进入 GPCR 的结构不稳定区域，即第三个细胞质环 (1)。这种方法是在斯坦福大学的 Kobilka 实验室开发的，用于获得 ¿ 2AR(1, 2)。用 T4L 替换第三个环不仅稳定了受体，而且还提供了良好有序且溶剂可访问的结晶堆积表面，如最新结构所示，该方法适用于其他 GPCR。我们建议使用类似的方法来稳定 ¿ OR 用于晶体发生，我们已经成功证明了 ¿ 功能形式的表达和纯化。我们现在准备获得这一重要药理学靶标的 X 射线晶体结构。我们选择通过 R21 机制开始对阿片受体的结构生物学进行详细研究。以及 ¿ 的高分辨率结构OR-T4L 与高亲和力拮抗剂结合，如果成功，该 R21 提案的结果将使我们能够通过结构表征获得更多资金。 OR 和其他 OR 亚型后续提案的长期目标是获得与肽和非肽形式的激动剂和拮抗剂配体结合的受体的结构。 OR-T4L 融合蛋白并调整这两种蛋白之间的接头以优化 ¿ OR 功能（配体结合亲和力）、表达和稳定性 2) 建立 ¿ 的表达和纯化条件。 OR-T4L 用于晶体学试验 3) 结晶并确定 ¿ 的 X 射线晶体结构。 OR-T4L 受体缩写：¿或者 - 阿片受体 LCP - 脂质立方相 Y¿或-YFP-¿ OR 融合蛋白 TM - 跨膜片段 T4L - T4 溶菌酶 ICL - 细胞内环 ¿ 2AR - 22 肾上腺素受体 DDM - 十二烷基麦芽糖苷 SEC - 尺寸排阻色谱