Single-molecule analysis of ligand efficacy in β2AR-G-protein activation

Abstract

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β₂ adrenergic receptor (β₂AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G_s heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β₂AR-G_s complexes are formed and the efficiency of nucleotide exchange leading to G_s activation. These data also reveal transient nucleotide-bound β₂AR-G_s species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

Extended Data Figure 1

Ligand binding properties of β₂Δ6-148C/266C. a, [³H]-dihydroalprenolol (DHA) saturation binding on purified β₂AR, comparing wild-type (WT; blue squares) versus unlabeled (orange circles) and labeled (green triangles) β₂Δ6-148C/266C. Affinities (K_d) are shown in the table. Non-specific binding controls are shown as corresponding open symbols. Error bars represent the standard error of the mean (s.e.m.) from triplicate measurements. b, [³H]-DHA–iso competition binding on purified β₂AR, comparing wild-type (WT; blue circles) versus unlabeled (orange squares) and labeled (green triangles) β₂Δ6-148C/266C. Affinities (K_i) are reported in the table. Error bars represent the s.e.m. from triplicate measurements. c, Dose-response curves of the BRET-based cAMP biosensor CAMYEL with wild-type (WT) β₂AR (black triangles) and the mutants β₂Δ6 (blue squares) and β₂Δ6-148C/266C (red circles). Data from untransfected cells are shown in green triangles. Data from five independent experiments were normalized to the maximal isoproterenol response by WT β₂AR in each experiment and globally fit to the entire data set, with the error bars representing the s.e.m., as reported in the table. d, Skeletal structures of ligands used in the current study. e, [³H]-DHA competition binding on unlabeled β₂Δ6-148C/266C for ligands used in the current study, except for cz and BI, for which the ultra-high affinities reported (32 pM and 84 pM, respectively) would not allow us to determine them accurately in our assay. Instead, we used concentrations of 1 μM for both in all our measurements. The calculated K_is are shown on the table.

Extended Data Figure 2

Fluorophore structures and properties. a–b, Skeletal structures of the modified Cy3B* donor (a) and Cy7* acceptor (b) fluorophores. c–d, Normalized donor fluorophore emission (Cy3: green; Cy3B*: dark green) and acceptor fluorophore absorbance (Cy5: red; Cy7*: dark magenta) spectra for Cy3/Cy5 (c) and Cy3B*/Cy7* (d) FRET pairs. The spectral overlap integral (shaded region) was calculated and used to determine the Förster distance (R₀) values for each pair. e, Inter-dye FRET efficiencies of the Cy3/Cy5 (black) and Cy3B*/Cy7* (blue) donor and acceptor fluorophore pairs as a function of inter-dye distances calculated based on R₀ values. f, Bulk anisotropy measurements on Cy3B*-labeled β₂Δ6-148C/266C.

Extended Data Figure 3

smFRET experimental controls. a, Site-specific labeling. SDS-PAGE gels under green (540 nm) or near IR (740 nm) epi-illumination for fluorescence visualization of Cy3B* or Cy7* labeling of β₂Δ6 and β₂Δ6-148C/266C. Coomassie-stained gel image is shown as a gel-loading control. Digestion with Factor Xa protease and deglycosylation with PNGase F leads to separation of the 148C and 266C labeling sites on generated N-terminal and C-terminal fragments, respectively. For gel source data, see Supplementary Figure 1. b, Quantification of Cy3B*/Cy7*-labeling specificity of full-length β₂Δ6-148C/266C. Data is normalized to β₂Δ6-148C/266C labeling. c, Specificity of streptavidin-mediated receptor immobilization. Frame capture from immobilization movies showing labeled β₂Δ6-148C/266C on streptavidin-free (-SA) or streptavidin-coated (+SA) surfaces. Bar graph shows the number of immobilized, labeled β₂AR in these conditions. Error bars represent the standard deviation from two replicates. d, Schematic of labeled β₂AR immobilization via biotinylated alp (alp-biotin). e, FRET population contour plot and histogram for alp-biotin-immobilized receptor shows correspondence with the FRET population distribution of biotin-M1-Fab-immobilized, alp-bound, labeled β₂AR (Fig. 2b). Histogram error bars represent the standard deviation from four replicates with N total molecules analyzed. f, FRET population contour plots (top row) and histograms (bottom row) for epi titration on biotin-M1-Fab-immobilized, labeled β₂Δ6-148C/266C (Fig. 2c). Dashed lines (blue) highlight the mean FRET values for the lowest (2 nM; top dashed line) and highest (200 μM; bottom dashed line) epi concentrations tested. Histogram error bars represent the standard deviation from three replicates with N total molecules analyzed. The scale bar on the right indicates relative populations for the contour plots.

Extended Data Figure 4

a, Sample fluorescence (green for Cy3B*; magenta for Cy7*) and FRET (blue) time traces for biotin-M1-Fab-immobilized, labeled β₂Δ6-148C/266C imaged in the absence and presence of saturating ligands. b, Same as Fig. 2b but with the full FRET efficiency range (0–1) shown for the population histograms (bottom row). c, Plots showing the mean cross correlation values of donor and acceptor fluorescence as a function of lag time for the ensemble of individual fluorescence traces obtained from experiments shown in Figure 2b. d, Plots showing the mean cross correlation values of donor and acceptor fluorescence as a function of lag time for an ensemble of simulated fluorescence trajectories rapidly fluctuating between high (0.75) and intermediate (0.55) FRET values with varying low- to high-FRET transition rate constants (colored lines), where the high- to low-FRET transition rate is held constant at 100 s⁻¹ (Methods). e, FRET distributions of the simulated data, as described in panel d.

Extended Data Figure 5

All-atom molecular dynamics simulations of the Cy3B*/Cy7*-labeled β₂AR in a detergent micelle. a, Time evolution of the distance between the dyes. Time dependence of the distances between the midpoints of the dyes along the simulation trajectories is shown for the β₂AR-cz (gray) and β₂AR-BI/G_s (black) systems. The distributions are displayed as histograms on the right; gray bars: β₂AR-cz; clear bars: β₂AR-BI/G_s. The estimated inter-dye distances derived from the experimental mean FRET values (Figs. 2b,3b; Extended Data Fig. 2e) are indicated by solid lines topped with circles (β₂AR-cz: red; β₂AR-BI/G_s: blue). b, Time evolution of the distance between C_alpha carbons at the labeling site. C_alpha-C_alpha distances for β₂AR-cz: red and β₂AR-BI/G_s: blue. c, The simulated dye-tethered β₂AR-BI/G_s system embedded in a DDM micelle (gray sticks). β₂AR is rendered in gray, with TM6 and the agonist (BI) highlighted in blue. The G_s protein is rendered in wheat color surrounded by its molecular surface to indicate the excluded volume for dye movements. The Cy3B* and Cy7* dyes are colored green and magenta, respectively. Water molecules, ions, and detergent molecules distant from the β₂AR structure are omitted. d, Positions explored by the midpoints of the dyes during the simulations are shown as clusters of dots in the context of the β₂AR-cz (transparent red dots) and β₂AR-BI/G_s (transparent blue dots) complexes; the center of mass of each collection of dots is indicated by a solid sphere. C_alpha carbons for labeled positions 148^4.40 and 266^6.28, are shown as magenta and green spheres, respectively.

Extended Data Figure 6

smFRET imaging of biotin-G_s-immobilized, labeled β₂AR. a, Schematic of labeled β₂AR immobilization via biotinylated G_s heterotrimer. b–c, Representative fluorescence (green for Cy3B*; magenta for Cy7*) and FRET (blue) time traces for biotin-G_s-immobilized, labeled β₂Δ6-148C/266C imaged in the presence of clen (b) or epi (c) in nucleotide-free conditions (apyrase-treated). d, FRET population contour plots (top row) and histograms (bottom row) for biotin-G_s-immobilized β₂Δ6-148C/266C imaged in the presence of partial and full agonists in nucleotide-free conditions. The dashed line indicates the invariant mean FRET value (~0.38) for all agonists tested. Histogram error bars represent the standard deviation from three replicates with N total molecules analyzed. e–f, FRET population contour plots of biotin-G_s-immobilized β₂Δ6-148C/266C in the presence of agonists exhibiting FRET transitions upon rapid addition (arrow) of 30 μM GDP (e) or 100 μM GTP (f). Scale bar on the right indicates the relative population for the contour plots.

Extended Data Figure 7

a, Sample fluorescence and FRET time traces for biotin-M1-Fab-immobilized, labeled β₂Δ6-148C/266C imaged in the absence and presence of saturating ligands plus 8 μM G_s in nucleotide-free (after apyrase treatment) conditions. b–c, FRET population contour plots for biotin-M1-Fab-immobilized, labeled β₂Δ6-148C/266C imaged in the presence of the agonists clenbuterol (b) or epinephrine (c), 8 μM G_s and increasing concentrations of GDP (Fig. 5a) or GTP in the presence of saturating GDP (30 μM) (Fig. 5e) with N indicating the total number of molecules analyzed from two replicates. Nucleotide-free (0 μM GDP; from Fig. 3b) and ligand only (from Fig. 2b) conditions are included as references. Scale bar on the right indicates the relative population for the contour plots.

Extended Data Figure 8

a–b, Transition rates from high- to low-FRET states (k_high→low) of labeled β₂AR with different agonists, saturating G_s (8 μM) and increasing GDP concentrations (2–100 μM)(a) or increasing GTP concentrations (0–100 μM) in 30 μM GDP (b). c, Low-FRET state distributions for labeled β₂AR with different agonists in 100 μM GDP and saturating G_s showing their overlap with the distribution in the presence of epi and saturating G_s in nucleotide-free conditions (apyr) (Fig. 3b). The dashed line shows the mean FRET value for apyr. d, Mean low FRET values from c. e, The percent change in the area of the low-FRET state population distributions for clen (black squares) and epi (dark yellow triangles)(as shown in Fig. 5b and c, respectively) was plotted with increasing GDP concentration (0–100 μM) and fitted to a single exponential decay function to derive the GDP EC₅₀. f–g, Sample FRET traces (blue line) of labeled β₂AR in the presence of clen (f) or epi (g) plus 100 μM GDP and saturating G_s. Dashed lines indicate each ligand’s corresponding mean high FRET value (black), mean low FRET value (light green) and mean FRET value in nucleotide-free conditions (dark green). h, The ratio of the low-FRET state lifetime of β₂AR in the presence of saturating G_s and GDP (τ_GDP) over the low-FRET state lifetime in saturating GTP plus 30 μM GDP (τ_GDP+GTP)(Fig. 5f) is shown for different agonists. All error bars in the figure represent the standard deviation from two replicates.

Figure 1

High-resolution perspective of β₂AR-G_s activation. a, Labeling sites N148C (magenta) in TM4 and L266C (green) in TM6 (blue) are shown on the inactive β₂AR structure (PDB:2RH1). Agonist activation leads to outward TM6 displacement (~14Å) and G_s coupling (shown GDP-bound G_i; PDB:1GP2). Within the GDP-free complex the α5 helix (red) of Gα_s (wheat) engages the cytoplasmic face (red mesh) of β₂AR (PDB:3SN6). GTP binding causes Gα_s (PDB:1AZT) and Gβγ subunit (not shown) to separate. b, Ligand efficacy profiles determined using a GTP turnover assay (Methods; error bars=s.e.m., 3 replicates).

Figure 2

The extent of TM6 motions correlate with efficacy. a, Schematic of ligand binding experiments (Methods). b, Population FRET contour plots (top; scale bar=relative population) and cumulative population histograms (bottom; error bars=s.d., 3 replicates with N total molecules). Dashed lines indicate the distinct mean FRET values observed. c, Apparent EC₅₀ for epinephrine (Methods; error bars=s.d., 3 replicates). d, Pearson’s correlation histograms (Methods) describing anticorrelated donor and acceptor fluorescence fluctuations in epinephrine with increasing donor lag time. e, Comparison of zero-lag Pearson’s correlation histograms for representative ligands. f, Scatter plot of mean FRET efficiencies vs. mean correlation coefficients observed for each ligand.

Figure 3

Ligand efficacy affects β₂AR-G_s coupling efficiency. a, Schematic of G_s-coupling experiments (Methods). b, Population FRET contour plots (top; scale bar=relative population) and cumulative population histograms (bottom; error bars=s.d., 2 replicates with N total molecules) observed for apyrase-treated (nucleotide free) β₂AR-G_s complexes. Dashed lines show correspondence between high- and low-FRET values. c, Schematic of G_s titration experiments (Methods). d, Example fluorescence (donor [light green]; acceptor [magenta]), and FRET (blue) time traces overlaid with predicted state sequence (red) in the presence of iso, GDP and G_s. e, Transition rates from high- to low-FRET states (k_high→low; error bars=s.d., 2 replicates) with increasing G_s(GDP) concentrations.

Figure 4

Pre-steady state measurements of nucleotide binding to clen- and epi-bound β₂AR-G_s complexes. a–d (panels from left to right), Sample FRET traces (blue) with state idealizations (red line) following nucleotide addition (arrow). Population FRET contour plots (scale bar=relative population; N=total molecules) before and after nucleotide addition (arrow). Population histograms showing the relative occupancies of low- (green) and high- (blue) FRET states post-nucleotide addition. Transition density plots displaying the mean FRET values before (x axis) and after (y axis) each transition (scale bar=transitions per second t/s; N_t=total transitions). Time-dependent changes in the relative occupancies of low- (green) and high- (blue) FRET states.

Figure 5

Nucleotide exchange efficiency is ligand dependent. a, Transition rates from low- to high-FRET states (k_low→high) with agonists, G_s and increasing GDP concentrations. b–c, Low-FRET state distributions with increasing GDP in G_s and clen (b) or epi (c). Vertical dashed lines indicate the mean low-FRET value observed without GDP (black; L) and the mean high-FRET value at 100 μM GDP (purple; H). d, Apparent EC₅₀ for GDP binding to the β₂AR-G_s complex with different agonists. e, Transition rates from low- to high-FRET states (k_low→high) with agonists, G_s, GDP, and increasing GTP concentrations. f, Histograms of low-FRET state lifetimes (τ) for each agonist with G_s and saturating GDP (light gray) or saturating GTP and GDP (dark gray). Error bars represents s.d. (two replicates) except (d), which represents s.e.m.

Figure 6

Proposed kinetic framework underlying β₂AR-G_s coupling and nucleotide exchange. a, Schematic of β₂AR TM6 conformational states within the G protein activation cycle (Note: the low-FRET β₂AR-G_s(GTP) complex was not experimentally observed but is inferred). b, Gα_s α5 helix engaging the intracellular face of β₂AR disrupts helix-proximal β6α5 loop interactions with GDP. c, Ranking of agonist molecular efficacies (ε) relative to epi in terms of the effective rate of generating G_s(GTP) from G_s(GDP) (Methods). d, Ligand efficacy-based cAMP measurements in living cells (Methods; error bars=s.e.m, 3 replicates).