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 Go 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.
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The atomic coordinates of the M2R–βarr1 structure have been deposited in the Protein Data Bank under accession number 6U1N. The electron microscopy maps of M2R–βarr1–Fab30(scFv) and interface M2R–βarr1–Fab30(scFv) have been deposited in the Electron Microscopy Data Bank with accession codes EMD-20612 and EMD-20948, respectively.
Rajagopal, S. & Shenoy, S. K. GPCR desensitization: Acute and prolonged phases. Cell. Signal. 41, 9–16 (2018).
Reiter, E., Ahn, S., Shukla, A. K. & Lefkowitz, R. J. Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52, 179–197 (2012).
Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G. & Kobilka, B. K. Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes. Science 364, 552–557 (2019).
Gurevich, V. V. & Gurevich, E. V. GPCR signaling regulation: the role of GRKs and arrestins. Front. Pharmacol. 10, 125 (2019).
Chen, Q., Iverson, T. M. & Gurevich, V. V. Structural basis of arrestin-dependent signal transduction. Trends Biochem. Sci. 43, 412–423 (2018).
Scheerer, P. & Sommer, M. E. Structural mechanism of arrestin activation. Curr. Opin. Struct. Biol. 45, 160–169 (2017).
Shukla, A. K. et al. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497, 137–141 (2013).
Zhou, X. E. et al. Identification of phosphorylation codes for arrestin recruitment by G protein-coupled receptors. Cell 170, 457–469.e413 (2017).
Miller, W. E. & Lefkowitz, R. J. Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking. Curr. Opin. Cell Biol. 13, 139–145 (2001).
Smith, J. S., Lefkowitz, R. J. & Rajagopal, S. Biased signalling: from simple switches to allosteric microprocessors. Nat. Rev. Drug Discov. 17, 243–260 (2018).
García-Nafría, J. & Tate, C. G. Cryo-EM structures of GPCRs coupled to Gs, Gi and Go. Mol. Cell. Endocrinol. 488, 1–13 (2019).
Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015).
Kruse, A. C. et al. Muscarinic acetylcholine receptors: novel opportunities for drug development. Nat. Rev. Drug Discov. 13, 549–560 (2014).
Staus, D. P. et al. Sortase ligation enables homogeneous GPCR phosphorylation to reveal diversity in β-arrestin coupling. Proc. Natl Acad. Sci. USA 115, 3834–3839 (2018).
Gurevich, V. V., Pals-Rylaarsdam, R., Benovic, J. L., Hosey, M. M. & Onorato, J. J. Agonist-receptor-arrestin, an alternative ternary complex with high agonist affinity. J. Biol. Chem. 272, 28849–28852 (1997).
Peisley, A. & Skiniotis, G. 2D projection analysis of GPCR complexes by negative stain electron microscopy. Methods Mol. Biol. 1335, 29–38 (2015).
Grinkova, Y. V., Denisov, I. G. & Sligar, S. G. Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng. Des. Sel. 23, 843–848 (2010).
Shukla, A. K. et al. Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512, 218–222 (2014).
Lally, C. C., Bauer, B., Selent, J. & Sommer, M. E. C-edge loops of arrestin function as a membrane anchor. Nat. Commun. 8, 14258 (2017).
Noble, A. J. et al. Routine single particle cryoEM sample and grid characterization by tomography. eLife 7, e34257 (2018).
Latorraca, N. R. et al. Molecular mechanism of GPCR-mediated arrestin activation. Nature 557, 452–456 (2018).
Ballesteros, J. A. & Weinstein, H. in Methods in Neurosciences Vol. 25 (ed. Sealfon, S. C.) 366–428 (Academic, 1995).
Koehl, A. et al. Structure of the µ-opioid receptor–Gi protein complex. Nature 558, 547–552 (2018).
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011).
Krishna Kumar, K. et al. Structure of a signaling cannabinoid receptor 1-G protein complex. Cell 176, 448–458.e12 (2019).
Huang, W. et al. Structure of the neurotensin receptor 1 in complex with β-arrestin 1. Nature (in the press).
Gaidarov, I., Krupnick, J. G., Falck, J. R., Benovic, J. L. & Keen, J. H. Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding. EMBO J. 18, 871–881 (1999).
Parruti, G. et al. Molecular analysis of human beta-arrestin-1: cloning, tissue distribution, and regulation of expression. Identification of two isoforms generated by alternative splicing. J. Biol. Chem. 268, 9753–9761 (1993).
Eichel, K., Jullié, D. & von Zastrow, M. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat. Cell Biol. 18, 303–310 (2016).
Eichel, K. et al. Catalytic activation of β-arrestin by GPCRs. Nature 557, 381–386 (2018).
Nuber, S. et al. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature 531, 661–664 (2016).
Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).
Nobles, K. N., Guan, Z., Xiao, K., Oas, T. G. & Lefkowitz, R. J. The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and -2. J. Biol. Chem. 282, 21370–21381 (2007).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user friendly software for single-particle image processing. eLife 7, e35383 (2018).
Heymann, J. B. & Belnap, D. M. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).
Adams, P. D. et al. The PHENIX software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).
Dolinsky, T. J. et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522–W525 (2007).
Tang, C. L., Alexov, E., Pyle, A. M. & Honig, B. Calculation of pK as in RNA: on the structural origins and functional roles of protonated nucleotides. J. Mol. Biol. 366, 1475–1496 (2007).
Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).
Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).
Robertson, M. J., Tirado-Rives, J. & Jorgensen, W. L. Improved peptide and protein torsional energetics with the OPLSAA force field. J. Chem. Theory Comput. 11, 3499–3509 (2015).
Dodda, L. S., Cabeza de Vaca, I., Tirado-Rives, J. & Jorgensen, W. L. LigParGen web server: an automatic OPLS-AA parameter generator for organic ligands. Nucleic Acids Res. 45, W331–W336 (2017).
Kulig, W., Pasenkiewicz-Gierula, M. & Róg, T. Topologies, structures and parameter files for lipid simulations in GROMACS with the OPLS-aa force field: DPPC, POPC, DOPC, PEPC, and cholesterol. Data Brief 5, 333–336 (2015).
Jorgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc. (1996).
Hirsch, J. A., Schubert, C., Gurevich, V. V. & Sigler, P. B. The 2.8 Å crystal structure of visual arrestin: a model for arrestin’s regulation. Cell 97, 257–269 (1999).
Luttrell, L. M. et al. Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9. Sci. Signal. 11, eaat7650 (2018).
O’Hayre, M. et al. Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK. Sci. Signal. 10, eaal3395 (2017).
We thank G. Hodgson, J. Taylor, Q. Lennon and V. Brennand for administrative assistance. Financial support was provided by the National Institutes of Health (grants R01 HL16037 to R.J.L. and R01 NS092695 to G.S.) and the Mathers Foundation (G.S.). R.J.L is a Howard Hughes Medical Institute investigator. A.L.W.K. is a Howard Hughes Medical Institute medical research fellow. We thank A. Masoudi for assistance in structural analysis of the M2R–βarr1 complex, S. Zheng for assistance in initially screening M2R–βarr1 complexes by negative-stain electron microscopy, and A. Inoue for βarr1/2-null HEK293 cells.
The authors declare no competing interests.
Peer review information Nature thanks Oliver Clarke, Lei Shi, John Tesmer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
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]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 the mutant βarr1(V70C) (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 are representative of three independent experiments. d, Statistical analysis of bimane fluorescence data from Fig. 1c. Data are the mean of three independent experiments with error bars representing s.e.m. * indicates significance for the indicated comparison, determined by 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 the final MSP1D1E3-M2Rpp–βarr1–Fab30 complex and SDS–PAGE analysis of peak fractions. g, h, Low resolution cryo-EM analysis of M2Rpp–βarr1–Nb24–scFv30 complex in MSP1D1H5 nanodiscs showing βarr in a ‘hanging’ conformation (g) or ‘core’ conformations with ‘rocking’ relative to the nanodisc density (h). i, Low-resolution cryo-EM map of the 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 s.e.m. * indicates significance compared to control (P < 0.0001, one-way ANOVA).
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 cryo-EM 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 3.6 Å structure providing insights into the binding interface and orientation of βarr1 relative to M2Rpp.
a, ‘Gold standard’ 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 correlation between model and map for the global map and the focused map, respectively. Refined model validation was also performed by calculating the correlation between model and focused half map 1 (green), and the 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.
a, The tilt of β-arrestin relative to the M2Rpp was measured by the angle (red arrow) between V37 (βarr1), I317 (βarr1) and R1213.50 (M2R). b, Superposition of βarr1 in the M2Rpp–βarr1 complex and visual arrestin in the crystal structure of Rho–Arr1 (PDB: 5W0P). c, Superposition of βarr1 in the inactive state (grey, PDB: 1G4M), in the phosphopeptide-activated state (green, PDB:4JQI) and in the receptor-bound active state (gold, M2Rpp–βarr1). The arrows indicate rearrangements of central crest loops.
Extended Data Fig. 5 Computational and experimental examination of interactions between the finger loop and M2Rpp.
a, Depiction of E61 of the finger loop and probable interaction partners R57 and R71 (top) and the R60 of the finger loop and D130 of the middle loop (bottom) from PHENIX/OPLS3 refinement in the cryo-EM map. The mesh depicts the 3.6 Å cryo-EM map contoured at σ = 4.0 with a masked 3.0 Å zone around the atoms depicted (top) or contoured at σ = 3.0 with a masked 3.0 Å zone around the atoms depicted (bottom). b, An overlay of the last frame from 200 ns of simulation for five of the molecular dynamics simulation trajectories. D69 of arrestin is depicted as bonds in addition to R3.50 and N2.39 of M2Rpp. Lines connect D69 and R3.50 from the same snapshot. c, Plots of the R3.50 zeta carbon–D69 gamma carbon distance (top) and N2.39 gamma carbon–D69 gamma carbon distance (bottom) over the course of the molecular dynamics simulations performed in this work. Grey lines correspond to raw data, whereas coloured lines correspond to a 1-ns sliding average. Teal traces correspond to simulations with membrane, and blue traces correspond to simulations with a small nanodisc. d, Competition radioligand binding experiments using [3H]NMS to measure the affinity of the agonist iperoxo for HDL-M2Rpp in the absence and presence of wild-type βarr1, βarr1(D69A) and the βarr1(Δ62–77) mutant that lacks the finger loop. Data are the mean of four independent experiments with error bars representing s.e.m. Inset, difference in logIC50 between βarr1 variants and control (no βarr1). *denotes significance compared to wild-type βarr1 (P < 0.0001, one-way ANOVA).
a, Detail of crystal structure of activated βarr1with phosphopeptide (left, PDB: 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 cryo-EM structure of M2Rpp–βarr1 (right). The arrow shows the direction of finger-loop untwisting. b, Expanded view of L129 (orange) in the cleft of βarr1 overlaid with a modelled 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 one-letter code is correlated to the frequency with which that residue occurs at that position.
a, Ribbon model of the M2Rpp–βarr1 complex depicting the location of C-edge loops and PtdIns(4,5)P2 (PIP2)-interacting residues. b, Sequence alignment of the arrestin family, showing differential conservation of the C-edge loops and the PtdIns(4,5)P2-binding motif.
a, Atomistic simulations of the M2Rpp–βarr1 complex in a membrane bilayer or a small nanodisc. Time courses are provided for the calculated interdomain twist angle for each replicate in membrane bilayer (left) or a small nanodisc (right). Raw data are provided in grey and 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 the data from Fig. 4c; data represent means of three independent experiments with error bars representing s.e.m. *P < 0.0001, one-way ANOVA; indicating significance from control within the same subset. c, Statistical analysis of data from Fig. 4d; data represent the mean and s.e.m. of three independent experiments. *P < 0.0001, one-way ANOVA; denotes significance compared to control.
a, Flow cytometry analysis of βarr1/2-null cells transiently transfected with Flag–M2R and GFP-βarr(WT) or GFP-βarr1(3×D). Cells were treated with vehicle or iperoxo for 30 min and subsequently stained with Alexa Fluor-650-labelled anti-Flag M1 antibody. GFP+ singlet cells were gated for analysis. Results are representative of data from three independent experiments. b, Quantitation of Flag–M2R surface staining by flow cytometry, as described above. Alexa Fluor-650-labelled anti-Flag M1 staining was normalized to the mean fluorescence of unstimulated cells expressing wild-type 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., not significant. c, Expression of GFP–βarr1(WT) or GFP–βarr1(3×D) and Flag–M2R in βarr1/2-null HEK293 cells as assessed by SDS–PAGE and western blot analysis. Tubulin was used as a loading control. Data are representative of three independent experiments. d, Quantification of GFP–βarr1(WT) or GFP–βarr1(3×D) expression by flow cytometry using βarr1/2-null HEK293 cells. Data represent the mean and standard error from three independent experiments; *P = 0.0034 (two-sided unpaired t-test). e, Localization of GFP–βarr1(WT) or βarr1(3×D) in Flag–vasopressin-2-receptor overexpressing HEK293 cells treated with arginine vasopressin peptide (AVP) for the indicated time. Data are representative of three independent experiments. f, Three-site interaction network of GPCR–β-arrestin 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 the C domain of β-arrestin with the lipid bilayer (3) because 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.
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Staus, D.P., Hu, H., Robertson, M.J. et al. Structure of the M2 muscarinic receptor–β-arrestin complex in a lipid nanodisc. Nature 579, 297–302 (2020). https://doi.org/10.1038/s41586-020-1954-0
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