G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’)1. Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2, 3, 4, 5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80)6, 7 and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Characterization of Nb60 interaction with β2AR. (221 KB)
a–c, Competition equilibrium binding studies using [125I]cyanopindolol (CYP), the cold competitor agonist isoprenaline (ISO), β2AR in HDL particles, and the indicated concentration of Nb80 (a), Gs (b), or Nb60 (c). The dotted vertical line represents log IC50 in absence of modulator, and the change in ligand affinity is depicted with coloured arrows. d, 19F NMR CPMG relaxation dispersion experiment with β2AR–Nb60–carazolol (Cz). Kex, exchange rate. e, Competition equilibrium binding studies using [125I]cyanopindolol, the non-labelled competitor agonist isoprenaline, β2AR in HDL particles, and 1 μM wild-type Nb60 or Nb60(T102A/F103A). f, ELISA depicting capture of β2AR by wild-type Nb60 or the T102A/F103A variant. Inset: Coomassie stain of nanobody input. Radioligand binding and ELISA experiments were performed at least three times with deviation shown as standard error.
- Extended Data Figure 2: Characterization of β2AR–Nb60–carazolol crystals. (598 KB)
a, Monodispersity of T4L–β2AR–Nb60–carazolol (β2AR–Nb60–Cz) complex as assessed by size exclusion chromatography. Inset, Coomassie stain illustrating presence of β2AR and Nb60 in fractions combined for crystallography. b, Representative picture of β2AR–Nb60–Cz lipidic cubic phase (LCP) crystals. c, Insertion of F103 (green) from Nb60 CDR3 (purple) into hydrophobic β2AR pocket, nitrogen and oxygen shown as blue and red shaded surfaces, respectively. d, Example of β2AR–Nb60–Cz crystal lattice. e, Electron density 2Fo–Fc map (Sigma: 1) of carazolol binding pocket (top panels) Nb60 CDR3 binding pocket (bottom panels) within β2AR.
- Extended Data Figure 3: Differential effects of Nb60 and Nb80 on the affinity of 12 different β2AR ligands. (315 KB)
Competition equilibrium binding studies using [125I]cyanopindolol, the indicated non-labelled competitor, β2AR in HDL particles, and 1 μM of Nb60 or Nb80. Data represent at least three independent experiments with deviation depicted as standard error.
- Extended Data Figure 4: Agonist-induced G-protein activation in cellulo correlates with the magnitude of affinity change mediated by Nb80 in vitro. (256 KB)
a, Table representing cell signalling and ligand affinity data. Ligand-dependent G-protein activation was quantified by measuring cAMP levels (GloSensor, Promega) from HEK293 cells overexpressing β2AR. Ligand affinity was measured in membranes prepared from the same cells as above using competition binding assays with [125I]cyanopindolol. Ligand efficacy (log τ) was calculated as previously described36. See methods and Supplementary Information for cooperativity (α) determination. b, c, Correlation plot of log τ and αNb80 (b), or αNb60 (c). All data represent at least three independent experiments with deviation shown as standard error.
- Extended Data Figure 5: Positive correlation between allosteric properties of Nb80 and Gs. (403 KB)
a, Equilibrium binding studies using HDL β2AR, [125I]cyanopindolol, the indicated unlabelled competitor, and 100 nM purified heterotrimeric Gs protein. b, Correlation plot of cooperativity values (α) for Nb80 and Gs. c, Sequence alignment of Nb60 and NbA11. Radioligand competition binding studies with Nb80, Nb60 or NbA11, [125I]cyanopindolol, the unlabelled competitor isoprenaline or clenbuterol, and HDL β2AR. All data represent at least three independent experiments with deviation shown as standard error.
- Extended Data Figure 6: Affinity determination for Nb60 and Nb80 for unliganded β2AR. (58 KB)
ELISA assay detecting capture of increasing concentrations of Nb60 or Nb80 by immobilized HDL β2AR in the absence of ligand. All data represent at least three independent experiments with deviation shown as standard error.
- Extended Data Figure 7: Theoretical framework illustrating the two views of allostery. (138 KB)
a, Nested reaction schemes at equilibrium indicating the correspondence (arrowed light-blue shades) between binding site cooperativity (ternary complex model in outer box) and changes of allosteric conformations (inner cubes). Arrows stand for reversible equilibrium interactions. b, Change of the macroscopic dissociation constant (1/K) of a ligand L (shifting the equilibrium towards r1) induced by increasing the concentrations of nine different N-ligands with diverse allosteric effects (γ1, γ2) on receptor states. Simulations were made using a three-state model based on the parameter values listed on the right side of the plot (curves on the left side are colour coded in red/blue tones corresponding to the boxes on the right). The change in K (that is, log difference between presence and absence of N) is calculated from equation 1 in the Supplementary Information (analysis of nanobody allostery).
- Extended Data Figure 8: Comparison of experimental and theoretical cooperativities predicted according to a two-state or three-state allosteric model. (433 KB)
See also the Supplementary Information section on analysis of nanobody allostery. a–d, Theoretical log α values were computed according to a two-state model for a series of hypothetical ligands (L) (log β1 range: 4/8) and a positive (PAN, log γ1 >> 0) or negative (NAN, log γ1 << 0) nanobody. a–d, Observed data overlaid on values simulated at J1 = 8.9 × 10−4 in histogram form (with experimental bars drawn on the closest theoretical log β1 bin value) (a), or superimposed (b), on the log αNAN versus log αPAN relationships predicted for different J1 values. The same data are replotted as separate graphs for lower J1 (c) and larger J1 (d) values, to show the sigmoidal relationships existing between macroscopic log αs and log β1. e, f, Simulations made according to the three-state allosteric model. e, Predicted (lines) and observed (circles) log α values plotted as functions of log (β1/β2). Three groups of ligands (I to III, defined by the table of a0 and m parameters) produce increasingly stronger reductions of r2 equilibrium. f, Same data plotted as log αNb60 versus log αNb80 relationships (see Fig. 4). All α values derived from at least three independent radioligand binding experiments with deviation depicted as standard error.
Extended Data Tables
- Supplementary Information (411 KB)
This file contains Supplementary Text and Data and Supplementary References.