Expression and purification of β2AR in Sf9 cells
The β2AR construct PN1 was expressed and purified as previously described39,42. In brief, Sf9 cells were infected with a PN1-containing baculovirus produced using the BestBac method. Cells were then collected and resuspended in chilled lysis buffer containing 10 mM HEPES, pH 7.4, 1 mM EDTA, 1 μM alprenolol and protease inhibitors (leupeptin and benzamidine). Lysed cells were then pelleted at 18,600 rpm for 20 min and dounced to homogeneity in chilled solubilization buffer containing 20 mM HEPES pH 7.4, 350 mM NaCl, 1% n-dodecyl β-D-maltoside (DDM), 0.1% cholesteryl hemisuccinate (CHS), 2 mM MgCl2, 1 μM alprenolol, protease inhibitors and benzonase. After stirring for 90 min at 4 °C and centrifugation at 18,600 rpm for 30 min, 2 mM CaCl2 was added to the soluble fraction, which was then applied to anti-Flag (DYKDDDDK) M1 immunoaffinity resin. The receptor was then washed (20 mM HEPES, pH 7.4, 350 mM NaCl, 0.1% DDM, 0.01% CHS, 2 mM CaCl2, leupeptin and benzamidine), eluted (20 mM HEPES, pH 7.4, 350 mM NaCl, 0.1% DDM, 0.01% CHS, 5 mM EDTA and 200 μg ml−1 Flag peptide), and further purified on Superdex 200 10/300 Increase gel filtration column equilibrated in NH buffer (20 mM, HEPES pH 7.4, 100 mM NaCl) plus 0.1% DDM and 0.01% CHS.
To produce homogeneous β2AR dimer, Sf9 cells expressing PN1 were resuspended at room temperature in 20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1 μM alprenolol, protease inhibitors and 10 μM AP. After incubation for 30 min at room temperature, membrane solubilization was initiated by adding 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% CHS. The purification steps followed the same protocol as for the monomeric receptor, except that 0.01% LMNG replaced DDM in all buffers, and 10 μM AP was maintained throughout the purification.
Expression and purification of heteromeric Gαsβ1γ2
As previously described43,44, heterotrimeric Gs was expressed and purified from Trichoplusia ni Hi5 cells. In brief, two baculoviruses were generated using the BestBac method, one encoding the wild-type human Gαs subunit and the other encoding the wild-type human β1γ2 subunits containing a histidine tag on the N terminus of the β subunit. Cells were infected with both viruses for 48 h and collected by centrifugation. The pellet was then resuspended and stirred for 30 min at 4 °C in hypotonic buffer containing 10 mM HEPES pH 7.4, 100 μM MgCl2, 5 mM β-mercaptoethanol, 20 μM GDP and protease inhibitors. Lysed cells were then pelleted at 18,600 rpm for 15 min and dounced to homogeneity in chilled NH buffer plus 1% sodium cholate, 0.05% DDM, 1 mM MgCl2, 5 mM β-mercaptoethanol, 20 μM GDP and protease inhibitors. After solubilization for 1.5 h while stirring at 4 °C and centrifugation at 18,600 rpm for 35 min, 20 mM of imidazole was added to the soluble fraction, which was then allowed to batch-bind to washed nickel-chelated Sepharose for 2 h. Pelleted resin was then loaded into a narrow column, washed with buffers containing gradually declining amounts of cholate and eluted with NH buffer plus 0.05% DDM, 1 mM MgCl2, 20 μM GDP, 100 μM tris(2-carboxyethyl)phosphine (TCEP) and 250 mM imidazole. Human rhinovirus 3C protease was added to cleave the histidine tag and the eluate was dialysed overnight at 4 C in 2 l of dialysis buffer (NH buffer plus 1 mM MgCl2, 0.05% DDM, 20 μM GDP and 100 μM TCEP). The protein solution was run through a second nickel-chelated Sepharose column, washed with dialysis buffer supplemented with 20 mM imidazole and dephosphorylated for 30 min on ice with lambda protein phosphatase, calf intestinal phosphatase and Antarctic phosphatase with 1 mM manganese chloride. The heterotrimer was further purified from excess βγ subunits using ion-exchange chromatography on the MonoQ 10/100 GL column. The sample was loaded and washed with 20 mM HEPES, pH 7.4, 1 mM MgCl2, 0.05% DDM, 100 μM TCEP and 20 μM GDP. Heterotrimeric Gs was then eluted with a linear salt gradient from 50 mM NaCl to 500 mM NaCl.
Expression and purification of Nb60
Nb60 was expressed and purified as previously described24. In brief, Nb60 was expressed in Escherichia coli BL21(DE3) cells. The lysates were then purified on a nickel-chelated Sepharose column and subsequently on the Superdex 200 Increase 10/300 column in 20 mM HEPES, pH 7.4, and 150 mM NaCl.
Sample preparation for cryo-EM
For the sample in detergent, purified PN1 in 0.1% DDM/0.01% CHS was loaded onto anti-Flag M1 immunoaffinity resin equilibrated in the same detergent mixture supplemented with 2 mM CaCl2. The receptor was exchanged into a synthetic triglucoside-based detergent, TTG-T10 (ref. 45), during which the receptor bound to the resin was washed with increasing ratios of TTG-T10 to DDM, ultimately transitioning the receptor into 0.01% TTG-T10/0.001% CHS. Each detergent exchange buffer contained NH buffer with 2 mM CaCl2, 1 μM carazolol and 10 μM AP. The receptor was subsequently eluted in NH buffer plus 0.01% TTG-T10, 0.001% CHS, 1 μM carazolol, 10 μM AP, 5 mM EDTA and 200 μg ml−1 Flag peptide. After the detergent exchange, PN1 was incubated for 1 h with twofold molar excess of Nb60. Excess Nb60 was cleared on anti-Flag M1 immunoaffinity resin after washing with NH buffer plus 0.0006% TTG-T10, 0.00006% CHS, 1 μM carazolol, 10 μM AP and 2 mM CaCl2. The complex was then eluted off the resin with NH buffer plus 0.0006% TTG-T10, 0.00006% CHS, 1 μM carazolol, 10 μM AP, 5 mM EDTA and 200 μg ml−1 Flag peptide. The final sample was concentrated to over 10 mg ml−1 and used immediately for cryo-EM grid preparation.
To prepare the AP-bound β2AR dimer in lipid nanodiscs, the purified dimer in LMNG was reconstituted into nanodiscs following a previous protocol42 with modifications. Lipids were prepared by mixing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, Avanti), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, Avanti) and cholesterol (Sigma-Aldrich) at a molar ratio of 7:2:1, followed by drying under argon and vacuum desiccation for 2 h. Lipids were resuspended in NH buffer containing 14 mM DDM at 20 mg ml−1. The receptor was diluted to 10 μM, incubated with 50 μM BI-167107 (PubChem CID: 45483813, MedChemExpress) for 10 min on ice, and then combined with membrane scaffold protein (MSP) 1E3D1 and lipids at a molar ratio of 1:2.5:100. The mixture was incubated on ice for 1 h to allow nanodisc assembly. Detergent was removed by sequential addition of semi-wet Bio-Beads SM2 (60 mg ml−1) three times over 3 h period, followed by overnight incubation at 4 °C. The next day, the Bio-Beads were removed, and empty nanodiscs were separated by M1 affinity purification. The dimeric β2AR in nanodiscs were eluted in buffer containing 1 μM BI, 10 μM AP, 5 mM EDTA and 200 μg ml−1 Flag peptide. The eluate was further polished by SEC in NH buffer plus 1 μM BI and 10 μM AP. Peak fractions were pooled and concentrated to 5 mg ml−1 for grid freezing.
The cryo-EM grids were prepared using Vitrobot Mark IV (Thermo Fisher Scientific). Quantifoil R1.2/1.3 Au grids were glow-discharged with air for 90 s at 10 mA using Plasma Cleaner (PELCO EasiGlow). Aliquots of 3 μl protein sample were applied to the glow-discharged grids. After blotting with filter paper (Ted Pella) for 3.0 s, the grids were plunged into liquid ethane cooled with liquid nitrogen.
Cryo-EM data collection and processing
For detergent- and nanodisc-reconstituted β2AR samples, a total of 7,139 and 17,997 micrograph stacks, respectively, were collected using a Titan Krios G3i operating at 300 kV on a Falcon 4i direct electron detector or on a K3 camera (Gatan) with a Quantum energy filter. Micrographs were recorded at video stack received a total electron dose of about 50 e− Å−2 over 40 frames.
Motion correction and contrast transfer function (CTF) estimation were performed using the patch motion and patch CTF in cryoSPARC46. Particle picking was carried out using the blob picker, and extracted particles were binned 4× for initial 2D classification. 2D classes with recognizable structural features were manually selected, and monomeric and dimeric particles were grouped separately. Ab initio reconstruction was performed using around 10% of the selected particles to generate four initial 3D references. Iterative rounds of heterogeneous refinement were conducted until poor-quality classes accounted for less than 5% of input particles. For the detergent dataset, 48,805 dimeric and 25,201 monomeric particles were selected for non-uniform (NU) refinement47. For the nanodisc dataset, 237,408 particles of dimeric species were selected. Particles were imported into RELION 4 (refs. 48,49) for Bayesian polishing50 and subsequently returned to cryoSPARC for further NU refinement. C2 symmetry was imposed for the final NU refinement of dimer species. After handedness correction, local and CTF refinement were performed using a soft mask centred on the TM domains. The final resolutions were estimated using the gold-standard Fourier shell correlation 0.143 criterion. Local resolution estimation was performed in cryoSPARC.
Model building and refinement
Initial models of β2AR (PDB: 2RH1) were docked into cryo-EM maps using UCSF Chimera51 and manually adjusted in COOT52 to fit the density, including placement of ligands. Real-space refinement was performed in PHENIX53,54 with secondary structure and geometry restraints. Model validation was conducted using EMRinger55 to assess side-chain density fitting. Structural figures were prepared using PyMOL and ChimeraX56.
Mass photometry
Mass photometry measurements were performed using a Refeyn TwoMP instrument (Refeyn) and the AcquireMP software (v.2.3) according to an established protocol57. Microscope coverslips (24 × 50 mm, #1.5 thickness; Corning) were cleaned with deionized water and isopropanol, then dried before use. Silicone gaskets were applied to the coverslips to form individual wells immediately before sample loading. The instrument was calibrated using NativeMark unstained protein standards (Thermo Fisher Scientific) according to the manufacturer’s instructions. Each measurement was conducted by first pipetting 10 μl of NH buffer into a well, followed by focal alignment and locking. Then, 1 μl of β2AR sample at a final concentration of 20 nM after dilution was added, mixed gently and data were acquired for 60 s. At least 2,000 binding events were recorded per sample. Data processing and molecular mass determination were performed using the DiscoverMP software suite (Refeyn).
Cell unroofing and immunogold cryo-EM
Cell unroofing and immunogold labelling were performed as previously described58,59,60, with modifications. Quantifoil R1.2/1.3 300-mesh gold EM grids were glow-discharged for 15 s, rinsed three times with 70% ethanol and washed four times with Dulbecco’s PBS (DPBS). The grids were incubated with 0.1 mg ml−1 poly-D-lysine (Gibco) for 1 h at room temperature, followed by four washes in DPBS (Gibco). Laminin (Sigma-Aldrich, 15 μg ml−1) was applied to the grids and incubated at 37 °C for 2 h, then washed again with DPBS. HEK293F cells in suspension were seeded onto prepared grids and cultured until reaching 60–70% confluency. The HEK293 cell line was obtained from and authenticated by ATCC, and was not routinely tested for mycoplasma contamination. β2AR was overexpressed through baculovirus transduction according to the BacMam protocol (Thermo Fisher Scientific). Around 16 h after transfection, cells were rinsed with DPBS containing calcium and magnesium. Grids bearing adherent cells were held with tweezers and dipped into a hypotonic swell buffer (6 mM HEPES-KOH pH 7.4, 43.3 mM K-gluconate, 1.6 mM NaCl, 0.6 mM MgCl2) for 30 s. An additional 6 μl of swell buffer was added to each grid, followed by blotting with Whatman Grade 5 filter paper (Sigma-Aldrich) to remove the apical membrane. Unroofed samples were blocked with 3% goat serum (Thermo Fisher Scientific) in DPBS containing protease inhibitors (Thermo Fisher Scientific) for 20 min at room temperature. Primary antibodies against β2AR (Thermo Fisher Scientific) diluted in DPBS with 1% goat serum was applied for 1 h. After three washes in DPBS, grids were incubated with a goat anti-mouse secondary antibody conjugated to colloidal gold (Ted Pella) diluted in DPBS with 1% goat serum for 1 h, then washed again three times.
Grids were plunge-frozen in liquid ethane using a Leica EM GP2 system and stored in liquid nitrogen. Cryo-EM imaging was performed on a Glacios G2 operated at 200 kV, equipped with a Falcon 4i detector, at a nominal magnification of ×11,000.
Cell-based BRET
To evaluate the effects of AP on cAMP production and β-arrestin recruitment, HEK293 cells endogenously expressing β2AR were transiently transfected with either the BRET-based intramolecular cAMP sensor CAMYEL or with plasmids encoding β-arrestin-2–GFP10 and β2AR–RlucII. CAMYEL comprises both donor and acceptor fused to the cAMP-binding domain of EPAC and undergoes a conformational change after cAMP binding that alters the BRET signal61. At 48 h after transfection, cells were pre-incubated with increasing concentrations of AP (0.03–100 μM) for 30 min, followed by stimulation with 1 μM ISO (Sigma-Aldrich) for 30 min in the presence of 5 μM coelenterazine H (for CAMYEL) or deep blue coelenterazine (DBC) (Cayman Chemical) for the β-arrestin assay.
For BRET-based β2AR dimerization studies, HEK293 cells were co-transfected with 10 ng per well of either β2AR–RlucII, β2AR(V129L)–RlucII, β2ARTM3–RlucII or β1AR–RlucII (BRET donors), along with 20 ng per well of their respective GFP-tagged counterparts (BRET acceptors). Then, 48 h after transfection, cells were incubated with increasing concentrations of AP (0.03–100 μM) or ISO (0.1–100 μM) for 1 h (or a different incubation time according to the experiment goal), followed by addition of DBC substrate and incubation for 20 min. To control for potential AP autofluorescence effects on the BRET signal, HEK293 cells transfected with β2AR–GFP were treated with AP (0.03–100 μM) for 30 min, and the induced GFP signal was directly measured and represented as fold change over basal.
For saturation studies of β2AR homodimerization, cells were co-transfected with a fixed amount of β2AR–RlucII (10 ng per well) and increasing amounts of GFP-tagged β2AR plasmid (0–100 ng per well). BRET measurements were taken at 48 h after transfection after incubation with DBC for 30 min.
To evaluate GRK5 recruitment to the β2AR, cells were co-transfected with β2AR–Rluc and GRK5–GFP. Cells were either stimulated with increasing concentrations of ISO for 30 min followed by DBC incubation for 20 min or pretreated with increasing concentrations of AP (0.03–100 μM) for 1 h followed by 1 μM ISO stimulation for 30 min in the presence of the Rluc substrate DBC.
All BRET signals were recorded at 395 nm (donor emission) and 510 or 530 nm (acceptor emission) using an Infinite F500 plate reader (Tecan). Induced BRET changes were calculated by subtracting the basal BRET signal (in the absence of ligand) from the BRET signal measured after stimulation.
For the AP-mediated cAMP and β-arrestin assays, as well as GRK5 recruitment, results are presented as percent of the ISO-alone response. For dimerization studies and ISO-induced GRK5 recruitment, data are presented as the percentage of the maximal BRET signal. All dose–response curves were fitted using the log[agonist/inhibitor] versus response (three parameters) or log[agonist/inhibitor] versus response (four parameters) function in GraphPad Prism. Data are shown as the mean ± s.e.m., n = 3 or 4.
SPR analysis on AP binding
SPR measurements were performed using the Biacore T200 system. Monomeric β2AR was captured onto a high-affinity streptavidin (SA) sensor chip (Cytiva) through a biotinylated anti-Flag M2 antibody (Sigma-Aldrich), yielding a final response of about 1,500 resonance units. The running buffer consisted of 20 mM HEPES (pH 7.5), 100 mM NaCl and 0.01% LMNG. AP was injected at increasing concentrations ranging from 0.6 to 10 μM at a flow rate of 30 μl min−1. Association and dissociation phases were recorded for each injection. All sensorgrams were processed using double-referencing to correct for non-specific binding and instrument artifacts. This was accomplished by subtracting both (1) the response obtained from a blank injection of running buffer over the active surface (to account for bulk refractive index changes and injection artifacts); and (2) the response from the compound injection over a reference surface without immobilized protein (to correct for nonspecific binding to the surface and matrix effects). Sensorgrams were processed using Biacore Evaluation Software, and binding curves were fit using a steady-state affinity model.
BLI on Gs binding
BLI measurements were conducted at 30 °C with continuous shaking at 1,000 rpm using an Octet RED384 system (FortéBio). SA biosensor tips (Sartorius) were coated with 10 nM biotinylated anti-Flag M1 fragment antigen-binding region (Fab) in the NH buffer with 0.01% LMNG and 0.001% CHS for 300 s. Flag-tagged β2AR was then captured by incubating the tips in 100 nM β2AR with 10 μM ISO for 600 s. After receptor immobilization, the biosensors were transferred into wells containing a concentration series of Gs (30 nM to 10 μM) in the binding buffer containing 10 μM GDP and 0.1% BSA (Sigma-Aldrich) for 180 s (association phase), followed by transfer into buffer-only wells for 300 s (dissociation phase). Control channels lacking either Gs or immobilized β2AR were used for double-reference subtraction. Association and dissociation kinetics were fitted with a single-exponential model to derive apparent kon and koff. Equilibrium binding responses were used to determine the dissociation constant KD.
Radioligand-binding assay
For saturation binding studies, 50–100 femtomoles of monomeric or dimeric β2AR reconstituted in nanodiscs, following the protocol described in the sample preparation for cryo-EM, were incubated with increasing concentration of [3H]DHA at room temperature for 1 h in a buffer containing 20 mM HEPES, 100 mM NaCl and 0.5% BSA. Non-specific binding of the radioligand was determined by adding 10 μM alprenolol in the same reaction system. For monomeric β2AR, the assay was performed with or without AP. For competition binding studies, monomeric or dimeric β2AR reconstituted in nanodiscs were incubated with 1 nM [3H]DHA and increasing concentrations of ISO or GDP-bound Gs in the same buffer as saturation binding. Nanodiscs were separated from excess [3H]DHA on Whatman GF/B filters using a Brandel 96-well harvester. The bound radioligand was read on the liquid scintillation counter (MicroBeta Jet, PerkinElmer). Data were analysed by GraphPad Prism 10.
GTP Turnover
The GTPase GLO assay was performed using a modified GTPase-Glo assay from Promega as previously described38,39. In brief, 100 nM of monomeric PN1, reconstituted in nanodiscs with MSP1E3D1 following the protocol described in the sample preparation for cryo-EM, was incubated for 1 h at room temperature with 20 μM ISO and a range of concentrations of AP in NH buffer plus 0.2% DMSO and 20 μM GTP. Simultaneously, a 1 μM stock of heterotrimeric Gs protein was prepared in a buffer consisting of NH buffer plus 0.04% DDM, 200 μM TCEP, 20 mM MgCl2 and 20 μM GDP. Equal volumes of PN1 and Gs were then mixed and incubated for 60 min. The final reaction consisted of 50 nM of ligand-bound PN1 and 500 nM Gs in NH buffer plus 0.1% DMSO, 0.02% DDM, 100 μM TCEP, 10 μM MgCl2, 10 μM GTP and 10 μM GDP. An equal volume of GTPase-Glo reagent in NH buffer plus 0.02% DDM and 5 μM ATP was then added and incubated for 30 min. Detection reagent was subsequently added and incubated for 10 min. Luminescence was detected using the MicroBeta counter. For the time-course assay, the experimental setup remained identical except that 200 nM of AP-mediated dimeric PN1 was included as a condition and the PN1–Gs reactions occurred for 30, 60, 90 and 120 min.
Ligation of V2Rpp to receptor and β-arrestin competition radioligand binding
β2AR constructs modified with a C-terminal sortase recognition sequence (LPETGHH inserted after residue 365) were expressed in Sf9 cells and purified as described above for monomeric and AP-stabilized dimeric receptors. Sortase-mediated ligation of synthetic V2Rpp to receptor was performed as previously described36,37. For ligation reaction, 10 μM purified receptor was incubated in NH buffer supplemented with 0.01% LMNG, 0.001% CHS and 5 mM CaCl2 with 50 μM synthetic GGG–V2Rpp peptide and 2 μM evolved sortase A pentamutant (eSrtA)62. The mixture was incubated overnight at 4 °C. Unreacted receptor and eSrtA (bearing the C-terminal His tag) was removed by binding to nickel-chelated Sepharose resins. Labelled monomeric or dimeric β2AR–V2Rpp samples were reconstituted into nanodiscs following the protocol described in the sample preparation for cryo-EM.
The equilibrium competition radioligand binding assays were performed with β2AR–V2Rpp in nanodiscs in the presence of 2 nM [3H]DHA, increasing concentrations of ISO and 1 μM C-tail-truncated β-arrestin-1(382), prepared as previously described63. Then, 10 μM AP was added where applicable. After incubation at room temperature for 1 h, the samples were collected and the radioactivity was measured as described in the previous section to calculate the inhibitory constant values of ISO.
GRK5 radiometric phosphorylation assays
To evaluate the effect of β2AR dimerization on receptor phosphorylation, β2AR monomers or dimers (1 μM), purified in LMNG micelles or reconstituted in nanodiscs, following the protocol described in the sample preparation for cryo-EM, were incubated for 5 min at 30 °C with purified C-terminally Strep-tagged GRK5 (50 nM) in a reaction buffer containing 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 30 mM NaCl, 0.5 mM EDTA, 100 μM [γ32P]ATP (1,000 to 2,000 cpm pmol−1) and 25 μM BI. The β2AR samples in LMNG micelles were additionally supplemented with 20 μM C8-PIP2 to increase efficiency of β2AR phosphorylation in detergent. To evaluate the effect of AP on β2AR phosphorylation, purified β2AR monomers (1 μM) in LMNG micelles were reconstituted into bicelles with PIP2 (ref. 64) and the AP concentration was varied from 0 μM to 24 μM. The reactions were quenched with SDS sample buffer, and the samples were separated by SDS–PAGE. Gels were stained with Coomassie blue (Sigma-Aldrich), dried, exposed to autoradiography film and 32P-labelled proteins were excised and counted to determine the amount of phosphate transferred. The reaction rates were normalized to phosphorylation of the β2AR monomers (β2AR monomers and dimers) or to phosphorylation in the absence of AP (AP effect).
Sample preparation for fluorescence measurements
Site-specific fluorophore labelling of β2AR was performed using engineered cysteine mutants on a minimal cysteine background (Δ6), as previously described38,39. For smFRET experiments, β2ARΔ6 constructs were cloned into the pcDNA-Zeo-tetO vector and transfected into Expi293 cells stably expressing the tetracycline repressor (Thermo Fisher Scientific, A14635). Transfections were carried out using the Expifectamine kit according to the manufacturer’s protocol. Then, 2 days after transfection, receptor expression was induced with 4 μg ml−1 doxycycline and 5 mM sodium butyrate in the presence of 1 μM alprenolol. Cells were collected 40 h after induction and immediately processed for purification.
For studies on the β2AR dimer in liposomes, single-cysteine mutants were introduced at TM5 (R228C) or H8 (I334C). Homogeneous dimers were expressed in Sf9 cells and purified as described above. Labelling was performed by incubating 10 μM purified receptor with a fivefold molar excess of a premixed maleimide-conjugated dye pair: DY549P1 (Dyomics) and Alexa Fluor 647 (Thermo Fisher Scientific) at a 1:1.5 ratio. The reaction was incubated for 30 min at room temperature and quenched with 5 mM L-cysteine. Excess dye was removed by SEC (Superdex 200 Increase 10/300) in 20 mM HEPES (pH 7.4), 150 mM NaCl and 0.01% LMNG/0.001% CHS. Labelled dimers were reconstituted into liposomes consisting of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (Avanti), and cholesterol at a molar ratio of 6:3:1 using an established protocol65.
For studies on TM6 dynamics, double-cysteine mutants (N148C on TM4 and L266C on TM6) were introduced. Monomeric receptor was expressed in Expi293 cells and purified according to the Sf9 purification procedure. For the dimer, Expi293 cells were co-transfected at a 1:1 plasmid ratio with constructs encoding an 8×His-tagged β2ARΔ6 (no Flag tag) and a Flag-tagged β2ARΔ6 carrying the N148C/L266C mutations. Heterodimers were isolated by tandem affinity purification. The clarified lysates were first incubated with nickel-chelated Sepharose resins and washed with buffer containing 20 mM imidazole. Proteins were eluted with 250 mM imidazole, then supplemented with 2 mM CaCl2 and subjected to anti-Flag M1 immunoaffinity purification. This two-step procedure enriched for heterodimers containing only one protomer with the double-cysteine mutations. Fluorophore labelling was performed as described above for the single-cysteine-mutant dimer sample.
Ensemble fluorescence measurements
Ensemble FRET experiments were conducted on the Fluoromax 4C spectrofluorometer (Horiba Scientific) with excitation and emission slit widths set to 5 nm and 3 nm, respectively. Emission spectra were recorded after excitation at 532 nm. AP-bound β2AR dimers labelled with donor and acceptor fluorophores (I334C H8 sensor) were diluted 1,000-fold in NH buffer plus 0.01% LMNG without AP to a final concentration of 1 nM. Fluorescence spectra were collected at 1, 5, 30, 120 min, and 16 and 24 h after dilution. All spectra were normalized to donor intensity. To assess the effects of transducer binding, samples were incubated with 100 μM ISO and (1) 10 μM Gs (in the presence of apyrase) or (2) 20 μM β-arrestin-1(382), together with V2Rpp and Fab30, which stabilizes the active V2Rpp-bound β-arrestin-1 conformation. β-arrestin-1(382) and Fab30 were prepared as previously described63. The samples were incubated for 1 h in the dark to allow full equilibration before measurement. All of the experiments were performed in triplicate.
smFRET microscopy
Flow chambers for smFRET experiments were assembled using mPEG-passivated glass coverslips (VWR), doped with biotin-PEG16 (Laysan Bio), as described previously38,39. Before use, coverslips were incubated with 1 mg ml−1 NeutrAvidin (Thermo Fisher Scientific), followed by 10 nM biotinylated anti-Flag M1 Fab. Labelled β2AR samples were diluted to 100–500 pM in NH buffer plus 2 mM CaCl2 and added to the chambers. After achieving optimal surface density, unbound receptor was washed out using imaging buffer supplemented with 100 μM cyclooctatetraene (Sigma-Aldrich) and an oxygen scavenging system (1% D-glucose, 1 mg ml−1 glucose oxidase, 0.04 mg ml−1 catalase).
Fluorescence imaging was performed on a custom-built, objective-based TIRF microscope as reported previously66. The setup is built on a Zeiss Axiovert S100 TV platform with a ×100, 1.45 NA oil-immersion objective (Zeiss). Donor and acceptor fluorophores were excited with 532 nm and 637 nm lasers (OBIS LS 150 mW and LX 140 mW, Coherent). Emissions were separated by a 652 nm dichroic beamsplitter (Semrock), filtered through 580/60 nm and 731/137 nm band-pass filters, and split using an OptoSplit II beamsplitter (Cairn Research) onto an EMCCD camera (iXon DU897E, Andor). Data acquisition was controlled by μManager using custom BeanShell scripts, and videos were recorded as stacked TIFFs in frame-transfer mode at 100 ms exposure per frame. The laser power was tuned to balance a high signal-to-noise ratio with photobleaching timescales of tens of seconds. Each slide typically yielded 10–20 videos per channel. All imaging was performed at room temperature.
Fluorescence traces were analysed using custom Python scripts. Donor and acceptor channels were aligned using registration images, and individual molecules were identified as local intensity maxima within a five-pixel neighbourhood. Donor-only spots were excluded. For each fluorophore pair, intensities were background-corrected using a local circular region (35-pixel diameter). Donor leakage into the acceptor channel (~7%) was subtracted.
Donor excitation was used to monitor emission for 80 s, followed by direct acceptor excitation for 1 s to confirm fluorophore identity. Traces were selected for analysis on the basis of the following criteria: (1) signal-to-noise ratio ≥ 5; (2) single-step acceptor photobleaching before donor bleaching; (3) γ factor between 0.5 and 2.5; (4) anticorrelated donor and acceptor intensity fluctuations; and (5) single-step donor bleaching, if present.
FRET efficiency (E) was calculated as E = Ia/(Ia + γId), where Ia and Id are the background-corrected acceptor and donor intensities, respectively. γ-correction was applied as described previously. For each trace, FRET values were binned into 30 intervals across the range [–0.25, 1.25] and normalized to the total number of datapoints. Ensemble FRET histograms were generated by averaging the normalized histograms from individual molecules and fit to a two-Gaussian distribution model.
Sample preparation for DEER
For DEER measurements, β2ARΔ6-N148C/L266C was expressed and purified as described above in Sf9 cells. To exchange detergent from 0.1% DDM/0.01% CHS to 0.01% (w/v) LMNG/0.001% CHS, the receptor was extensively washed with a progressive gradient of DDM:LMNG buffer. In parallel, while the receptor was bound to the resin, alprenolol was removed by washing with saturating concentrations of the low-affinity antagonist atenolol. Owing to the fast dissociation kinetics of atenolol from the β2AR, subsequent washes with ligand-free buffer yielded unliganded β2AR for spin labelling. The Flag eluted receptor was labelled with the spin label reagent 3-(2-iodoacetamido)-proxyl in the presence of 100 μM TCEP in buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl and 0.01% LMNG/0.001% CHS. Twentyfold molar excess of 3-(2-iodoacetamido)-proxyl was added to 10 μM β2ARΔ6 receptor for 3 h at room temperature. After quenching of the reaction with 5 mM final L-cysteine, the receptor was separated from the excess spin label by SEC (Superdex 200 10/300) in SEC buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 0.01% LMNG/0.001% CHS) prepared with D2O. The sample was concentrated using a 50 kDa concentrator to a concentration of >25 μM. D8-glycerol was added as a cryoprotectant to 25% (v/v). Then, 13 μl of sample was added to a borosilicate capillary (1.4 mm (inner diameter) × 1.7 mm (outer diameter); VitroCom) and flash-frozen in liquid nitrogen.
DEER spectroscopy
DEER experiments were conducted as previously described39 at Q-band (~33.68 GHz) using the Bruker Elexsys 580 spectrometer equipped with a SpinJet AWG, EN5107D2 resonator, variable-temperature cryogen-free cooling system (ColdEdge Technologies) and the 300W TWT amplifier (Applied Systems Engineering). All measurements were performed at 50 K. Dipolar evolution data were acquired using a dead-time-free 4-pulse DEER sequence with gaussian pulses67 and with 16-step phase cycling.
The experimental parameters used for DEER data collection were: π/2, πobs and πpump pulse lengths of 40 ns; a frequency offset (Δv) of 90 MHz; d1 = 250 ns; d2 = 5,150 ns; shot repetition time = 2000 μs; shots per point = 4; and integration window = 40 ns. The optimal microwave power (that is, pulse amplitude) for the π/2, πobs and πpump pulses were determined using transient nutation experiments, in which pulse amplitudes were adjusted to maximize the inversion of the Hahn echo68. Pump pulses were applied to the maximum intensity of the field swept echo detected absorption spectrum. Observe pulses were applied at a frequency of 90 MHz lower than the pump pulses.
DEER data were processed using DeerAnalysis 2022 (ref. 69), which uses two fitting routines: neural network analysis (DEERNet70, Spinach revision 5662) and Tikhonov regularization (DeerLab v.0.9.1)71. The consensus fit represents the mean of both methods, with reported 95% confidence intervals also incorporating errors from both methods. Time traces were normalized to signal intensity at t = 0, and distance distributions were area normalized. Custom Python scripts were used for plotting the dipolar evolution time traces and the distance distributions.
Reporting summary
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