Extended Data Fig. 1: Characterization of the M2R–βarr1 complex.
From: Structure of the M2 muscarinic receptor–β-arrestin complex in a lipid nanodisc
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).
