Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
- University of Michigan, United States
- University of California, San Francisco, United States
- University of California, San Diego, United States
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.
Data availability
All data generated and analyzed during the study are included in the manuscript and supporting files. Source files have been provided for Fig 3.
Article and author information
Author details
Funding
National Institutes of Health (T32 DA007267)
- Kathryn E Livingston
American Heart Association (13PRE17110027)
- Jacob P Mahoney
National Institutes of Health (T32GM007767)
- Jacob P Mahoney
National Institutes of Health (R01 DA03339)
- John R Traynor
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Livingston et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,670
- views
-
- 758
- downloads
-
- 42
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
eLife 7:e32499.
https://doi.org/10.7554/eLife.32499
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
In the bacterium M. smegmatis, an enzyme called MftG allows the cofactor mycofactocin to transfer electrons released during ethanol metabolism to the electron transport chain.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Both immunoglobulin light-chain (LC) amyloidosis (AL) and multiple myeloma (MM) share the overproduction of a clonal LC. However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic-soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered the understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a2 conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.
