Structure of the M2 muscarinic receptor-β-arrestin complex in a lipid nanodisc

Abstract

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit β-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis¹. Additionally, β-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins². In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of β-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of β-arrestin 1 (βarr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-βarr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of βarr1 to phosphorylated receptor residues and insertion of the finger loop of βarr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric G_o protein complex³. Moreover, the cryo-electron microscopy map reveals that the C-edge of βarr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, βarr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of β-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility.

Extended Data Figure 1:. M2R-βarr1 complex characterization.

(a) Schematic showing sortase-mediated ligation of GGG-V2Rpp onto GPCRs containing a C-terminal sortase consensus sequence (LPETGGH). (b) Competition radioligand binding experiments using [³H]-N-methylscopolamine (NMS) to measure the affinity of iperoxo for HDL-M2Rpp in the absence (control; Ctl) (logIC50 −6.98 ± 0.07) or presence of βarr1 (logIC50 −8.38 ± 0.07), βarr1-minimal cysteine (MC) (logIC50 −8.52 ± 0.07), and βarr1-V70C mutant (logIC50 −8.34 ± 0.06). (c) Co-immunoprecipitation (IP) of βarr1and Fab30 in the presence and absence of DDM-FLAG-M2Rpp and DDM-FLAG-M2R. Data representative of three independent experiments. d) Statistical analysis of bimane data from Fig. 1c. Data are the mean of three independent experiments with error bars representing SE. (*) Indicates significance (one-way ANOVA) (e) Competition radioligand binding experiments using [³H]-NMS to measure the affinity of the agonist carbachol for HDL-M2Rpp in the absence (Ctl; logIC50 −5.38 ± 0.12) and presence of LY2119620 (LY211) (logIC50 −6.7 ± 0.10), βarr1 and Fab30 (logIC50 −6.8 ± 0.07), or in combination (logIC50 −7.95 ± 0.06). (f) Size exclusion chromatography of final MSP1D1E3-M2Rpp-βarr1-Fab30 complex and SDS-PAGE analysis of peak fractions. (g,h) Low resolution cryoEM analysis of M2Rpp-βarr1-Nb24-scFv30 complex in MSP1D1H5 nanodiscs showing βarr in a “hanging” conformation (left map) or “core” conformations with “rocking” relative to the nanodisc density. (i) Low resolution cryoEM map of M2Rpp-βarr1-Fab30 complex in the larger MSP1D1E3 nanodiscs shows βarr1 in the “core” conformation involving an additional interaction of the C-domain with the lipid bilayer. All βarr1 variants are truncated at amino acid 393. Radioligand binding experiments are the means of three independent experiments with error bars representing SE. (*) Indicates significance compared to control (p<0.0001, one-way ANOVA).

Extended Data Figure 2:. M2Rpp-βarr1 complex conformers and cryoEM reconstruction workflow.

(a) Conformational variability of the M2Rpp-βarr1 complex. Overlay of two low resolution reconstructions aligned on the 7TM portion reveal variability in the angle of the βarr1-Fab30 segment relative to the receptor. One map is shown as mesh and the other as solid surface. The constant domains of Fab30 have been masked out in the reconstructions. For clarity, only one receptor-nanodisc density is shown. (b) Flow chart of cryoEM data processing towards high-resolution reconstructions. All eight-particle classes show engagement of βarr1 with the lipid nanodisc but only one class (17.4% of particles) could be further processed to high resolution. A final focused refinement yielded a 3.6Å structure providing insights into the binding interface and orientation of βarr1 relative to M2Rpp.

Extended Data Figure 3:. Map resolution and model validation.

(a) “Gold standard” Fourier Shell Correlation (FSC) plots of the reconstruction of M2Rpp-βarr1-Fab30(scFv) (global map, black) and focused refinement reconstruction for Interface M2Rpp-βarr1-Fab30(scFv) (focused map, blue). The red and brown curves represent the model-map correlation for global map and focused map, respectively. Refined model validation was also performed by calculating the correlation between model and focused half map 1 (green), and correlation between the randomly displaced model and focused half map 2 (purple). (b, c) Local resolution estimation map for M2R-βarr1-Fab30(scFv) and focused map of half-M2Rpp-βarr1-Fab30(scFv). (d) Overall model fit to the cryo-EM map. The 7TM portion (orange) comes from the full M2Rpp-βarr1-Fab30(scFv) map contoured at σ=7.5, whereas the βarr1-scFv portion comes from the focused refinement of Interface M2Rpp-βarr1-Fab30(scFv) contoured at σ=5.2.

Extended Data Figure 4:. Structural comparisons of arrestins.

(a) The tilt of β-arrestin relative to the M2Rpp was measured by the angle (red arrow) between V37 (βarr1), I317 (βarr1), and R121^3.50 (M2R). (b) Superposition of βarr1 in M2Rpp-βarr1 complex and visual arrestin in crystal structure of Rho-visual arrestin (PDBID:5W0P). (c) Superposition of βarr1 in inactive state (grey, PDB: 1G4M), in activated state by phosphopeptide (green, PDBID:4JQI) and in receptor-bound active state (gold, M2Rpp-βarr1). The arrows indicate rearrangements of central crest loops.

Extended Data Figure 5:. Computational and experimental examination of finger loop/M2Rpp interactions.

(a) Depiction of the finger loop E61 and probable interaction partners R57 and R71 (above) and the R60 of the finger loop and D130 of the middle loop (below) from Phenix/OPLS3 refinement in the cryoEM map. The mesh depicts the 3.6Å cryoEM map contoured at σ=4.0 with a masked 3.0 Å zone around the atoms depicted (above) or contoured at σ=3.0 with a masked 3.0 Å zone around the atoms depicted (below). (b) An overlay of the last frame from 200 ns of simulation for 5 of the MD simulation trajectories. D69 of arrestin is depicted as bonds in addition to R^3.50 and N^2.39 of M2Rpp. Lines connect D69 and R^3.50 from the same snapshot. (c) Plots of the R^3.50 zeta carbon-D69 gamma carbon distance (left) and N^2.39 gamma carbon-D69 gamma carbon distance (right) over the course of the MD simulations performed in this work. Grey lines correspond to raw data, while colored lines correspond to a 1-ns sliding average. Teal traces correspond to simulations with membrane, and blue traces correspond to simulations with small nanodisc. (d) Competition radioligand binding experiments using [³H]-N-methylscopolamine to measure the affinity of the agonist iperoxo for HDL-M2Rpp in the absence and presence of βarr1 WT, D69A and Δ62–77 mutant lacking the finger loop. Data are the mean of four independent experiments with error bars representing SE. Inset: difference in logIC50 between βarr1 variants and control (no βarr1), (*) denotes significance compared to WT βarr1 (p<0.0001, one-way ANOVA).

Extended Data Figure 6:. Finger loop rearrangements and ICL2.

(a) Detail of crystal structure of activated βarr1with phosphopeptide (left panel, PDBID: 4JQI) showing the N-terminal portion of V2Rpp bound on β-strands 5 and 6, thereby twisting the finger loop fold. Unbinding of the N-terminal portion of V2Rpp is required for the finger loop to adopt the observed conformation in the M2Rpp-βarr1 cryoEM structure (right panel). The arrow shows the direction of finger loop untwisting. (b) Close-up of L129 (orange) in the cleft of βarr1 overlaid with a modeled phenylalanine (green) and methionine (red) at the same position. (c) Plot of the frequency of specific amino acids occurring in the second position of ICL2 for βarr-binding class A GPCRs. The size of the 1-letter code is correlated to the frequency with which that residue occurs at that position.

Extended Data Figure 7:. Conservation of lipid binding regions in arrestin family.

(a) Ribbon model of the M2Rpp-βarr1 complex depicting the location of C-edge loops and phosphatidylinositol 4,5-bisphosphate (PIP2)-interacting residues (b) Sequence alignment of arrestin family to show differential conservation of C-edge loops and PIP2-binding motif.

Extended Data Figure 8:. Effects of lipid membrane on βarr1 coupling to M2Rpp.

(a) Atomistic simulations of M₂Rpp-βarr1 complex in membrane bilayer or a small nanodisc. Time courses are provided for the calculated interdomain twist angle for each replicate in membrane bilayer (left column) or a small nanodisc (right column). Raw data is provided in grey while a 1-ns rolling average is provided in teal for the membrane simulations and blue for the small nanodisc simulations. Horizontal teal and blue lines correspond to an active-like and inactive-like interdomain twist angle, respectively. (b) Statistical analysis of figure 4c data are the mean of three independent experiments with errors bars representing SE. (*) Indicates statistical significance from control (Ctl) within the same subset (p<0.0001, one-way ANOVA). (c) Statistical analysis of data from figure 4d; data represent the mean and standard error of three independent experiments and (*) denotes significance compared to Ctl (p<0.0001, one-way ANOVA).

Extended Data Figure 9:. βarr1 C-edge-lipid interaction facilitates M2R internalization.

(a) Flow cytometry analysis of βarr1/2 null cells transiently transfected with FLAG-M2R and WT or 3xD GFP-βarr1. Cells were treated with vehicle or iperoxo for 30 minutes and subsequently stained with Alexa Fluor-650-labeled anti-FLAG M1 antibody. GFP+ singlet cells were gated for analysis. Figure is representative of data from three independent experiments. (b) Quantitation of FLAG-M2R surface staining by flow cytometry, as described above. Alexa Fluor-650-labeled anti-FLAG M1 staining was normalized to the mean fluorescence of unstimulated cells expressing WT β-arrestin1-GFP in each experiment. These data were used to calculate the percentage of receptor internalized in Fig. 5c. Data represent the mean and standard error from three independent experiments and asterisks (*) indicate statistical significance (one-way ANOVA). N.S. represents non-significance. (c) Expression of GFP-βarr1 wild-type or 3xD and FLAG-M2R in βarr1/2 null HEK293 cells as assessed by SDS-PAGE and western blot analysis. Tubulin used as loading control. Data representative of three independent experiments. (d) Quantification of GFP-βarr1 WT or 3xD expression by flow cytometry using βarr1/2 null HEK293 cells. Data represent the mean and standard error from three independent experiments with asterisk (*) representing statistical significance (p=0.0034, two-sided unpaired T-test). (e) Localization of GFP-βarr1 WT or 3xD in FLAG-Vasopressin-2-receptor overexpressing HEK293 cells treated with arginine vasopressin peptide (AVP) for the indicated time. Data representative of three independent experiments. (f) Three-site interaction network of GPCR-βarrestin binding. In the classic two-site interaction model, conformational changes in β-arrestin induced by binding to phosphorylated receptor (1) leads to transmembrane receptor core coupling (2) to sterically block G protein binding. Our findings suggest an expanded model including interaction of β-arrestin C-domain with the lipid bilayer (3) since it synergistically enhances the interaction of β-arrestin with the phosphorylated receptor tail/loops and transmembrane core. Vertical arrows in the receptor represent direction and strength of cooperativity between the extracellular orthosteric ligand-binding and intracellular transducer-binding sites.

Fig. 1:. βarr1 recruitment by M2R in a native lipid environment.

(a) Ligand (L)-induced conformational changes in GPCRs lead to heterotrimeric G protein activation (GTP hydrolysis) and subsequent GRK-mediated receptor phosphorylation. Initial binding of βarr to phosphorylated receptors leads to its coupling to the transmembrane (TM) bundle, sterically occluding further G protein binding. (b) βarr1 allosterically enhances iperoxo affinity to HDL-M2Rpp but not DDM-M2Rpp as determined by competition radioligand binding. The positive allosteric modulator LY211960 (LY211) enhanced iperoxo affinity regardless of reconstitution environment. Data are the mean of three independent experiments with error bars representing SE. (*) Indicates significance compared to control (one-way ANOVA). (LogIC50 values: DDM-Ctl, −7.10 ± 0.09; DDM-βarr1, −7.03 ± 0.06; DDM-LY211, −8.36 ± 0.04 (p<0.0001); HDL-Ctl, −7.49 ± 0.08; HDL-βarr1 −8.29 ± 0.08 (p<0.0007); HDL-LY211, −9.17 ± 0.05 (p<0.0001) (c) HDL-M2Rpp but not DDM-M2Rpp enhance βarr1 finger loop bimane (red star, inset) fluorescence. Curves represent difference in spectra obtained with antagonist (atropine) and agonist (iperoxo). Data represent means of three independent experiments. (d) Orthogonal views of cryoEM density map of the HDL-M2Rpp-βarr1 complex colored by subunit (orange, M2Rpp; teal, βarr1; gray/white, HDL particle). The orange-colored density on βarr1 corresponds to the phosphorylated C-terminal peptide (V2Rpp) ligated to the receptor. The nanodisc density, omitted in the middle panel for clarity, has been generated earlier in image processing before high-resolution refinement of the M2Rpp-βarr1 complex.

Fig. 2:. Structure of the M2Rpp-βarr1 complex.

Orthogonal views (a,b,c) of the M2Rpp-βarr1 structure colored by subunit (orange, M2Rpp; teal, βarr1; gray sticks, model lipid bilayer). (d) Superposed structures of M2Rpp-βarr1 and M2R-G_o complexes (M2R, gray; G_αo Ras domain, pink; PDBID: 6OIK) aligned by M2R. G_o βγ subunits are omitted for clarity. Top right panel shows the cytoplasmic view of M2R transmembrane alignment in the absence of transducers. Bottom right inset shows similar insertion depth of βarr1 finger loop and G_o α5 helix into the M2R TM bundle. (e) Superposition of M2Rpp-βarr1 and rhodopsin-arrestin1 structures (rhodopsin, gray; arrestin1, green; PDBID: 4ZWJ). Inset shows enlarged view βarr1 (M2Rpp) and visual arrestin (rhodopsin) finger loops.

Fig. 3:. Interaction regions between M₂R and βarr1.

M2Rpp-βarr1 complex, with dashed boxes indicating main interaction sites between βarr1 (teal) and receptor (orange). (a) Enlarged view of the βarr1 finger loop inserting into the M2R TM bundle. The model includes side chain positions from Phenix refinement with OPLS3e electrostatics. (b) Enlarged view of βarr1 N-domain bound to the phosphorylated M2Rpp C-terminus. (c) Expanded view of interaction between M2R ICL2 (ribbon) and a hydrophobic cleft in βarr1 (rendered as electrostatic surface; red, blue and white graduations indicate negative, positive, and neutral surface potential, respectively). The mesh in all panels depict the 3.6Å cryoEM map contoured at σ=3.0 with a masked 2.0Å zone around the atoms depicted.

Fig 4:. Lipid membrane anchoring of the βarr1 C-domain.

Atomistic simulations of βarr1 in complex with M2Rpp (in absence of Fab30) to investigate the (a) position of βarr1 C-edge and (b) βarr1 interdomain twist angle in the absence (dark blue) or presence (cyan) of a lipid membrane (dashed black line). (c) HDL-M2Rpp but not HDL-M2R enhances βarr1 C-edge bimane (inset, red star) fluorescence independent of antagonist (atropine, atrp) or agonist (iperoxo, ipx). (d) The ability of iperoxo-activated HDL-M2Rpp to increase βarr1 finger loop bimane (inset, red star) fluorescence requires receptor phosphorylation (compare to HDL-M2R) and is significantly reduced by the C-edge 3xD mutations (L335D, L338D, and S340D). Data representative of three independent experiments.

Fig 5:. βarr1 functionality depends on C-domain lipid interaction.

(a) Activation of purified heterotrimeric G_i protein by iperoxo-stimulated HDL-M2Rpp in vitro is reduced by βarr1 WT but not βarr1 3xD. Data are the mean of three independent experiments with error bars representing SE. (*) Denotes statistical significance (p<0.0001, one-way ANOVA) compared to WT βarr1 plus iperoxo. (b) Iperoxo stimulation of FLAG-M2R causes plasma membrane recruitment of GFP-βarr1 WT and subsequent receptor internalization, which is impaired by the 3xD mutations as assessed by confocal microscopy. βarr1, nuclei, and M2R colored green, blue, and red, respectively. Confocal images are representative of three independent experiments. (c) Quantification of FLAG-M2R internalization by flow cytometry in same cells as (b). Data are the mean of three independent experiments with error bars representing SE. (*) Denotes statistical significance (p=0.0008, unpaired two-sided T-test) compared to WT βarr1.