µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive1 and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor3. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.
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We acknowledge support from INSERM (S.G.) and CNRS (H.D.) and from the National Institutes of Health Grant (NIDA-DA036246 to B.K.K. and S.G.). We also acknowledge the National Institute of Drug Abuse Drug Supply Program for providing [Dmt1]DALDA.
A.M., T.L., J.S. and B.K.K. have filed a patent for active-state stabilizing nanobodies for opioid receptors.
Extended data figures and tables
Extended Data Figure 1 Overall strategy for the preparation of 13C-dimethylated µOR and quality control.
The wild-type µOR contains a total of 12 lysine residues strategically positioned to sense conformational dynamics in both extracellular and G-protein-coupling domains upon receptor activation. To monitor the µOR activation process in solution in an amphiphile membrane-like environment, we exploit the sensitivity of two 13C-methyl groups covalently bound to the ε-NH2 of lysine side chains (ε-N[13CH3]2-lysines) and to the α-NH2 of the receptor N terminus as NMR probes, an approach recently validated in the GPCR field19. We slightly modified the wild-type sequence to facilitate receptor purification and to remove flexible N and C termini for improvement of sample stability in NMR measurements. a, Snake plot presenting the µOR–2x construct and highlighting the 12 endogenous lysines (red circles), the protease cleavable motifs (black circles) and the Flag and 6 × histidine tags (grey circle). Extracellular and intracellular surfaces are coloured yellow and blue, respectively. b, The different biochemical steps were analysed by SDS–PAGE (left panel) and a typical final size-exclusion chromatography highlighting the monodispersity of 13C-dimethylated µOR is shown (right panel). c, Shown is the reductive methylation of the N terminus and of lysine side chains after reaction with 13C-formaldehyde (13C in green) in the presence of sodium cyanoborohydride (NaBH3CN). Amino acids are shown in ball-and-stick representation and, for clarity, side-chains of unmodified amino acids are replaced by large spheres. This reaction is known to minimally affect protein structure and function and, as previously observed with β2-AR (see ref. 19), it did not affect the functionality of µOR as 13C-dimethylated µOR binds both antagonist (Naloxone) and agonist (BU72) with a similar affinity than unlabelled µOR (d). The left panel in d represents the mean ± s.d. of triplicates for one experiment representative of three experiments in total (n = 3). The right panel in d represents normalized values ± s.d. (larger binding value being 100% and smaller 0%) for one experiment representative of three experiments in total (n = 3). Kd and IC50 values are the mean ± s.d. of the three independent experiments for both receptor preparations. In addition, we observed similar agonist-induced interaction with Nb39 between µOR or 13C-dimethylated µOR using pull-down experiments (e), demonstrating that methylation does not prevent the agonist-dependent interaction with Nb39. Saturation binding experiments on soluble µOR or 13C-dimethylated µOR (both at 100 nM) were done in the presence of an increasing amount of radiolabelled naloxone (up to 1 µM) and non-specific binding was determined in the presence of 100 µM of naloxone. Competition assays on soluble µOR or 13C-dimethylated µOR were done with 100 nM of radiolabelled naloxone and increasing amount of BU72. Free and receptor-bound radioligands were separated using gel-filtration columns. Total binding was plotted as a function of [3H]-naloxone or BU72 concentration and data were analysed using Prism with saturation or competitive binding analyses.
Each single mutant was expressed, purified and labelled as described for µOR–2x. For each mutant, we analysed the R and LRNb spectra. a–k, Left panels represent the 2D spectra with the indicated lysine to arginine mutant in red. On the right panels, we highlight the peak disappearance for each mutant (red line) as compared to the wild-type µOR preparation (black line) in the 1H dimension in the receptor alone condition (R). In some instances, peaks are overlapping in the area of interest and to assign these peaks we had to record the NMR spectra in the ternary complex situation (LRNb). This was the case for K269 (h), K271 (i), and K303 (j). In h, i and j, # indicates the position of the lysine peak from the Flag tag that we measured in some samples and that is due to an incomplete cleavage of the N terminus by the 3C protein. For samples used in the analysis of ligand and Nb effects, we almost eliminated this peak by optimizing the 3C cleavage step as described in the Methods (treatment with 100 µM TCEP). The peak denoted with a # in panel i between wild type (no TCEP) and K271R (100 µM TCEP) is an example of the improvement of 3C cleavage in TCEP-treated samples as indicated by the almost complete disappearance of the peak in both R and LRNb conditions (K271R, red line). The K98 peak was assigned by deduction (Fig. 1a). Indeed, we generated and recorded NMR spectra of 11 mutants of the 12 endogenous lysines and the only peaks that were never affected in all the mutant spectra and left unassigned necessarily correspond to the K98 residue. l, The assignments of D1 (full-length N terminus) and G52 (cleaved N terminus) were inferred from the spectra obtained before (red line) and after cleavage with 3C (black line). The G52 loss of signal is better observed in the LRNb situation (G52* almost completely disappears). Asterisks indicate the chemical shifts of methyl probes in the ternary complex condition.
HMQC spectrum of 13C-dimethylated µOR in the ternary complex situation (R, 30 µM; BU72, 150 µM; Nb, 60 µM) for Nb33 acquired with a 700 MHz spectrometer (blue trace) and for Nb39 acquired with a 500 MHz spectrometer (red trace). Both spectra are very similar and are characteristic to the fully agonist-induced state of µOR. 1D slice of HMQC spectra in the 1H dimension (right panels) highlighting the similar effect of Nb33 and Nb39 in the presence of BU72 on the indicated set of dimethyllysines and compared to the untreated sample (black dotted line).
Extended Data Figure 4 Partial reversal of agonist effects by treatment with the antagonist naloxone.
a–c, HMQC spectra of unliganded 13C-dimethylated µOR (30 µM, R, black) (a), the same sample bound to a saturating concentration of ligand (150 µM, LR, [Dmt1]-DALDA (blue) or naloxone (red) (b) and the same sample treated with a saturating concentration of Nb33 (60 µM, LRNb, [Dmt1]DALDA (green) or naloxone (light pink) (c). d, Spectra of the [Dmt1]DALDA–RNb sample treated with naloxone (900 µM) (L2RNb, magenta). Green arrows in 1D traces highlight the peaks movement in the activation process (c) and for the partial reversal effect of naloxone (d) for K209 and K344/K98 peaks.
a, HMQC spectra of unliganded 13C-dimethylated µOR (30 µM, R, black), 13C-dimethylated µOR treated with a saturating concentration of [Dmt1]DALDA (150 µM, LR, blue), 13C-dimethylated µOR treated with [Dmt1]DALDA and a saturating concentration of Nb33 (60 µM, LRNb, green) and of unliganded 13C-dimethylated µOR treated with a saturating concentration of Nb33 (60 µM, RNb, orange). The small crosses indicate the position of peaks visible in the apo-state that are reduced in intensity or no longer visible in the other spectra. Arrows indicate the position of the 1D slice represented in b. b, 1D slice of HMQC spectra in the 1H dimension highlighting the effect of [Dmt1]DALDA (blue) and [Dmt1]DALDA + Nb33 (green) on the N terminus (G52) and on the indicated set of dimethyllysines (ECL2, TM5, TM6, ICL1 and H8 lysines). The yellow and blue boxes indicate the positions in the extracellular and intracellular domains respectively. The zoom level for each peak and the 13C chemical shifts are indicated in the top left of each panel. Asterisks indicate the chemical shifts of methyl probes in the ternary complex condition.
a, HMQC spectra of unliganded 13C-dimethylated µOR (30 µM, R, black), 13C-dimethylated µOR treated with a saturating concentration of DAMGO (150 µM, LR, blue), 13C-dimethylated µOR treated with DAMGO and a saturating concentration of Nb33 (60 µM, LRNb, green) and of unliganded 13C-dimethylated µOR treated with a saturating concentration of Nb33 (60 µM, RNb, orange). The small crosses indicate the position of peaks visible in the apo-state that are reduced in intensity or no longer visible in the other spectra. Arrows indicate the position of the 1D slice represented in b. b, 1D slice of HMQC spectra in the 1H dimension highlighting the effect of DAMGO alone (blue) and DAMGO + Nb33 (green) on the N terminus (G52) and on indicated set of dimethyllysines (ECL2, TM5, TM6, ICL1 and H8 lysines). The yellow and blue boxes indicate the positions in the extracellular and intracellular domains respectively. The zoom level for each peak and the 13C chemical shifts are indicated in the top left of each panel. Asterisks indicate the chemical shifts of methyl probes in the ternary complex condition.
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Sounier, R., Mas, C., Steyaert, J. et al. Propagation of conformational changes during μ-opioid receptor activation. Nature 524, 375–378 (2015). https://doi.org/10.1038/nature14680
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