Dataset Information

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

ABSTRACT: The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs.

SUBMITTER: Livingston KE

PROVIDER: S-EPMC6042960 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

Livingston Kathryn E KE Mahoney Jacob P JP Manglik Aashish A Sunahara Roger K RK Traynor John R JR

eLife 20180622

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 ...[more]

PMID: 29932421

Similar Datasets

Project description:Chronic pain is a prevalent problem affecting approximately one out of every five adults in the U.S. The most effective way to treat chronic pain is with opioids, but they cause dangerous side effects such as tolerance, addiction, and respiratory depression, which makes them quite deadly. Opioids, such as fentanyl, target the μ-opioid receptor (MOR), which can then bind to the intracellular Gi protein or the β-arrestin protein. The Gi pathway is primarily responsible for pain relief and potential side effects, but the β-arrestin pathway is chiefly responsible for the unwanted side effects. Ideally, an effective pain medication without side effects would bind to MOR, which would bias signaling solely through the Gi pathway. We used the Bio3D library to conduct principal component analysis to compare the cryo-electron microscopy MOR structure in complex with the Gi versus an X-ray crystallography MOR structure with a nanobody acting as a Gi mimic. Our results agree with a previous study by Munro, which concluded that nanobody-bound MOR is structurally different than Gi-bound MOR. Furthermore, we investigated the structural diversity of opioids that can bind to MOR. Quantum mechanical calculations show that the low energy solution structures of fentanyl differ from the one bound to MOR in the experimental structure, and pKa calculations reveal that fentanyl is protonated in aqueous solution. Glide docking studies show that higher energy structures of fentanyl in solution form favorable docking complexes with MOR. Our calculations show the relative abundance of each fentanyl conformation in solution as well as the energetic barriers that need to be overcome to bind to MOR. Docking studies confirm that multiple fentanyl conformations can bind to the receptor. Perhaps a variety of conformations of fentanyl can stabilize multiple conformations of the MOR, which can explain why fentanyl can induce different intracellular signaling and multiple physiological effects.

Dataset Information

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

Publications

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets