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: 8095476
• 负责人: Roger K Sunahara
• 金额: $ 28.34万
• 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/8095476
• 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 PUBLIC HEALTH RELEVANCE: Our goal is to obtain diffraction quality crystals and a high-resolution structure of the ¿-opioid receptor (¿OR), the major site of analgesic action of morphine, bound to a high-affinity antagonist. The proposed work is high-risk and high-impact and consolidates several innovative approaches in order to achieve our goal. These include the creation of ¿OR-T4Lysozyme fusion proteins, the development of new expression and purification strategies, the use of a new class of detergents, and applying lipid- based crystallography methods. 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.

描述（由适用提供）：G蛋白偶联的阿片受体已成为数百年来的重要治疗靶标，因为它们在伤害感受和早期发现优化药物中的作用。 - 阿片受体（或）是吗啡和其他阿片受体（OR）激动剂的镇痛作用的主要介体。因此，该R21提案将集中在� -Opioid接收器上，将研究扩展到相关的亚型，以实验成功允许。虽然有效的疼痛衰减剂，但在``疼痛''中起作用，或者在``恶心，呼吸抑郁症，成瘾和便秘）上具有各种不良副作用，有时还具有各种不良副作用。可以在没有副作用的情况下可以产生抗伤害感受的药物的发展的主要障碍是缺乏对阿片受体的同性结合和激活的结构基础的了解。最近阐明了几种G蛋白偶联受体（GPCR）的X射线晶体结构（GPCR）为小分子量拮抗剂与GPCR的结合提供了新的见解。现在，人们认为，药物结合和稳定受体的特定考虑的能力不仅取决于结合位点，而且取决于对结合位点的访问。对扩张器受体作用的配体的光谱更为复杂，包括肽和小分子量拮抗剂和拮抗剂。因此，对肽和小分子配体结合之间的相似性和差异的结构见解将极大地支持更有效和选择性药物的发展。 GPCR结构生物学的主要突破是将稳定的蛋白质T4溶菌酶（T4L）掺入GPCR结构不稳定区域中，第三个细胞质循环（1）。这种方法是在Stanford的Kobilka实验室中开发的，用于Stanford，并用于获得高级结构的高分辨率结构（2）。用T4L代替第三圈不仅稳定了受体，而且还贡献了一个很好的有序溶剂可访问的结晶填料表面。事实证明，该方法适用于其他GPCR，如腺苷A2A受体的最新结构所示。我们建议利用类似的方法稳定``稳定'或结晶。我们已经成功地证明了`或从昆虫细胞的功能形式的表达和纯化。现在，我们被中毒以获得这个重要的药物目标的X射线晶体结构。我们选择通过R21机制开始对优化受体的结构生物学进行详细研究。我们的目标是获得与高亲和力拮抗剂结合的OR-T4L的衍射质量晶体和高分辨率结构。拟议的工作是高风险和高影响力。如果成功的话，该R21提案的结果将使我们能够通过“或其他或其他亚型”的结构表征获得更多的资金。随后的提案的长期目标将是获得与激动剂和拮抗剂配体的肽和非肽形式结合的受体结构。具体目的1）生成A€OR-T4L融合蛋白，并调整这两种蛋白质之间的接头以优化或功能（配体结合亲和力），表达和稳定性。 2）建立用于晶体学试验的OR-T4L表达和纯化的条件。 3）晶体化并确定»OR -T4L受体缩写的X射线晶体结构：€或 - �OpioID接收器LCP -脂质立方相Y Y或-yfp -YFP -€或-YFPOP -€或融合蛋白TM-融合蛋白TM -T4L -T4L -T4 Lysozyme icl -t4 lysozyme icl -insoselbrane iCl -inssozyme icl- inclacell -intacellular loop-22 ar-22 ar-22 ar- 22 ar-2ar-2ar-2ar-2ar-2ar-2ar- 22 ar-十二烷基马尔替剂SEC-尺寸排除色谱法 公共卫生相关性：我们的目标是获得衍射质量晶体和 - 阿片受体（OR）的高分辨率结构，这是吗啡的镇痛作用的主要部位，与高亲和力拮抗剂结合。拟议的工作是高风险和高影响力，并巩固了几种创新的方法，以实现我们的目标。其中包括创建»OR-T4lysozyme融合蛋白，新表达和纯化策略的发展，新的确定类别的使用以及应用基于脂质的晶体学方法。如果成功的话，该R21提案的结果将使我们能够通过“或其他或其他亚型”的结构表征获得更多的资金。随后的提案的长期目标将是获得与激动剂和拮抗剂配体的肽和非肽形式结合的受体结构。