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
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.
|Manglik, Aashish; Kruse, Andrew C; Kobilka, Tong Sun et al. (2012) Crystal structure of the µ-opioid receptor bound to a morphinan antagonist. Nature 485:321-6|