Structure of the µ-opioid receptor-Gi protein complex

Abstract

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein G_i. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free G_i. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-G_i complex to previously determined structures of other GPCRs bound to the stimulatory G protein G_s reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the G_i protein-coupling specificity of the µOR.

Extended Data Figure 1. scFv binding characteristics

scFv 16 does not perturb the interfaces between Gα and Gβ at a) its binding epitope or b) the Switch II region located ~40A away. Our structure is colored by chain, while the structure of GDP-bound G_i1 heterotrimer (PDB 1GP2) is colored grey. c) In the nucleotide-free state, there is a ~7˚ rotation of G βγ relative to the Gα_s Switch II domain when compared to the GDP-bound form. This rotated conformation is similar to that observed in nucleotide-free G_s coupled to the β₂AR (PDB ID 3SN6) as shown in panel d).

Extended Data Figure 2. Cryo-EM data processing

a, Representative cryo-EM micrograph of the μOR-G_i complex. Scale bar, 20nm. b, Representative two-dimensional averages showing distinct secondary structure features from different views of the complex. c, Flow chart of cryo-EM data processing. The unmasked map in the middle of the chart has been colored by subunit. The inset shows the fit of the crystal structure of the α-helical domain in the corresponding density of the unmasked reconstruction. Three-dimensional density maps colored according to local resolution. d, “Gold standard” Fourier shell correlation (FSC) curves from Phenix indicates overall nominal resolutions of 3.5 Å and 3.6 Å using the FSC=0.143 criterion for the scFv-subtracted map (green curve) and scFv-retained maps (purple curve), respectively.

Extended Data Figure 3. Cryo-EM map vs. refined structure

a) EM density map (scFv subtracted) and model are shown for all seven transmembrane α-helices of the μOR, DAMGO, and Gα helices α5 and αN. b,c) Cross-validation of model to EM density map. The model was refined against one half map after displacement of atoms by 0.2A, and FSC curves were calculated between this model and the final cryo-EM map (full dataset, black), of the outcome of model refinement with a half map versus the same map (red), and of the outcome of model refinement with a half map versus the other half map (green). The results of the scFv-retained model vs. map and of scFv subtracted model vs. map are shown in b) and c), respectively.

Extended Data Figure 4. Selected cryo-EM densities of μOR-G_i Complex

Cryo-EM density (displayed as mesh) surrounding residues involved in a) DAMGO binding, b) μOR-Gα_i interaction around ICL2, c) ICL3, and d) cytoplasmic ends of the μOR transmembrane helices. These figures accompany the models shown in figures 1e, 4b, 5a, and 5b respectively.

Extended Data Figure 5. Stability of DAMGO in MD Simulations

a. Over the course of MD simulations, the positions of the first 4 residues of DAMGO do not significantly change, while the 5^th residue (Gly-ol) shows significant variability in position. Frames from the first and last 100 ns are shown with an intermediate to highlight both the relative stability of the first 4 amino acids, as well as the flexibility of the fifth. Arrows show the extent of motion in the N- and C-terminal residues over the course of simulation. Cryo-EM density for DAMGO is shown as mesh. b. Root mean standard deviations (RMSDs) from the modeled pose of DAMGO to the pose during MD simulations. The RMSD calculations include heavy atoms on the peptide backbone. Data from three independent simulations are plotted. The RMSDs for residues 1 to 4 (black) and the C-terminal Gly-ol (blue) are plotted separately to highlight their stability and mobility, respectfully.

Extended Data Figure 6. Water occupancy in orthosteric binding site

Left panel, water occupancy in MD simulations of DAMGO-bound μOR overlaid with a representative conformation from MD simulations. ‘Occupancy relative to bulk solvent’ is the ratio of the rate at which water is observed in a given volume to the rate at which water is expected to be observed in an equivalent volume in the bulk solvent. For example, blue regions (occupancy ratio = 2) are occupied by water twice as often as an equivalent region in the bulk solvent. Right panel, crystallographic waters in the BU72-bound μOR binding pocket (PDB ID: 5C1M). Waters are shown as black spheres, BU72 is shown as yellow sticks, and hydrogen bonds are shown as dashed lines.

Extended Data Figure 7. Comparison of the C-termini of Gα_s and Gα_i

The C-terminus of Gα_s is bulkier than that of Gα_i due to substitution of small amino acids C (−4 position) and G (−3 position) in Gα_i to Y and E respectively in Gα_s. This leads to steric clashes with TMs 3 and 7 of the μOR. Top - ribbon view of μOR (green) with WT Gα_i (gold, left) and a Gα_is model (right) created by substituting C and G for Y and E based on the β₂AR-G_s crystal structure. Substituted positions are colored in light purple. The −4 to −2 positions have their side chains shown as spheres, and the rest as a ribbon. Bottom - space filling view of the μOR showing the steric clashes that result from these substitutions.

Extended Data Figure 8. Comparison of Gai C terminal peptide binding modes

Side (top half), and cytoplasmic (bottom half) views of a) the μOR (green) with the last 11 residues of Gα_i (gold) alone, b) compared to the β₂AR(orange) with the last 11 residues of Gα_s (light purple) (PDB ID 3SN6), or c) compared to MetaII Rhodopsin (pink) in complex with an 11 residue G_transducin (G_t) C-terminal peptide (dark purple) (PDB ID 3PQR). The mOR-Gi complex aligns best with the MetaII-G_t complex both in terms of TM6 displacement as well as position of the α5 peptide.

Figure 1. Cryo-EM structure of the μOR-G_i complex

a, Orthogonal views of the cryo-EM density map of the μOR-G_i heterotrimer complex colored by subunit (μOR in green, DAMGO in orange, G⍺_s Ras-like domain in gold, Gβ in cyan, Gγ in purple). b, Model of the μOR-G_i complex in the same views and color scheme as shown in a. c, Residues that line the μOR orthosteric binding pocket are shown as sticks for the μOR-G_i complex (green) and the μOR-Nb39 complex (PDB 5C1M; blue). The binding pocket residues of DAMGO and BU-72 occupied μOR show nearly identical conformations, despite differences in ligand structure. d, Comparison of BU-72 (yellow carbons) in the orthosteric pocket of the μOR-Nb39 complex (blue) with DAMGO (orange carbons) in the orthosteric pocket of the μOR-G_i complex (green). e, view of DAMGO in the orthosteric binding pocket with critical residues shown. f, A frame from every 100 ns of a 1 μs MD simulation (yellow for t = 0 fading to red for t = 1 μs) shows that the first 4 residues of DAMGO (bottom) are stable, whereas the C-terminal Gly-ol (top) is dynamic but frequently returns to the modeled pose.

Figure 2. Structural changes in the μOR stabilized by nucleotide-free G_i

a, Comparison of inactive μOR (brown) and the G_i stabilized active state of μOR (green). b, Comparison of Nb39 and G_i stabilized active states of the μOR (blue and green, respectively). The structures are nearly identical except for a slight shift of TM6 towards TM7 in the G_i -bound state. c, Residues important for activation of the μOR show nearly identical conformations despite the difference in ligands. d, Comparison of G_s-stabilized β₂AR (orange) and G_i-stabilized μOR (green). While most transmembrane helices align well between the two receptors, TM6 is kinked further outward by 9Å in the β₂AR. Distance calculated between Cα of residue 6.29 (Ballesteros-Weinstein numbering) in TM6.

Figure 3. Changes in Gi upon coupling to the μOR

a, b, Comparison of GDP-bound Gα_i (PDB 1GP2, grey) and nucleotide-free Gα_i from the μOR-G_i complex (gold). GDP is shown as blue spheres in panel a and sticks in panel b. The primary differences between these two structures are the opening and outward movement of the alpha helical domain (AHD), and an upward shift of the α5 helix by 6Å to engage the receptor core. The α-carbons of the TCAT motif are represented as spheres in panel b. The TCAT motif coordinates the guanosine base of GDP. The upward shift of the α5 helix and repositioning of the TCAT motif leads to nucleotide release. c, d, e, The interface between the α1 helix and the N-terminal end of the α5 helix and TCAT motif for GDP-bound Gα_i (c), nucleotide free Gα_i (d), and nucleotide free G_s from the β₂AR -G_s complex (e). The upward movement of the α5 helix disrupts the interaction between the α1 and α5 helices leading to changes in the P-loop that coordinates the phosphates of GDP.

Figure 4. Comparison of the receptor-G protein binding interfaces of the μOR-Gi and β₂AR-Gs complexes

a, Comparison of the conformation of the α5 helix of Gα and receptor TM6 in β₂AR-G_s and μOR-G_i complexes after alignment on the receptor. b, Interactions between ICL2 of the μOR (green) and Gα_i (gold). Asp 350 of Gα_i is depicted with narrow lines to indicate uncertainty in its conformation due to poor cryo-EM density for its side chain. c, Interactions between ICL2 of the β₂AR (orange) and Gα_s (blue). d, Surface view of the hydrophobic pockets in Gα_i (top panel) and Gα_s (bottom panel) that interact with a non-polar amino acid in ICL2 of the μOR and β₂AR, respectively.

Figure 5. Comparison of the receptor-G protein binding interfaces of the μOR-Gi and β₂AR-Gs complexes

Top panels show interactions between ICL3 of μOR and Gα_i (a) and between the cytosolic ends of TMs 3,5,6, of the μOR and the α5 helix of G_i (b). Asp 350 of Gα_i is depicted with narrow lines to indicate uncertainty in its position due to poor cryo-EM density for its side chain. Bottom panels show these same interfaces between β₂AR and G_s (c,d).