Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

Journal name:
Nature
Volume:
535,
Pages:
448–452
Date published:
DOI:
doi:10.1038/nature18636
Received
Accepted
Published online

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.

At a glance

Figures

  1. Allosteric nanobodies have opposing effects on agonist affinity for the β2AR.
    Figure 1: Allosteric nanobodies have opposing effects on agonist affinity for the β2AR.

    a, Schematic of the ternary complex model. Ligand (L) affinity to receptor (R) increases in the presence of transducer (T), this allosteric linkage is denoted by dashed line with arrows. b, Compared to the absence of modulator, Nb60 decreases isoprenaline affinity (negative cooperativity) and Nb80 and Gs increases affinity (positive cooperativity) as assessed by radioligand competition assays using β2AR HDL particles. c, The effects of Nb60 and Nb80 or Gs on isoprenaline affinity are saturable functions of their concentration. d, e, The affinity of Nb60 for unliganded β2AR (d), represented by a tight isotherm sigmoidal binding curve23, is reduced in the presence of isoprenaline (iso) (e), as determined by isothermal titration calorimetry. f, Nb60 dose-dependently increases and Nb80 decreases the binding of the radiolabelled antagonist [3H]ICI-118,551 to the β2AR. All radioligand binding studies represent a minimum of three independent experiments with deviation shown as the standard error.

  2. Nb60 stabilizes the S2 inactive state by coordinating the β2AR ionic lock.
    Figure 2: Nb60 stabilizes the S2 inactive state by coordinating the β2AR ionic lock.

    a, b, Cartoon depicting a side (a) or cytoplasmic (b) view of the β2AR transmembranes (TM). Conversion from the two inactive states (S1 and S2) to the active S4 state requires both agonist and transducer (G protein) binding and is represented by a 14 Å outward movement of TM6. c, 19F NMR spectroscopy of the β2AR with the antagonist carazolol (Cz) ± Nb60. d, The 3.2 Å structure of the β2AR bound to carazolol (Cz) and Nb60 (β2AR–Cz–Nb60). e, Coordination of β2AR ionic lock (R131 and E268) by Nb60 CDR3 residues T102 and Y106. For comparison, a disengaged and fully formed ionic lock are shown by the β2AR–Cz (PDB accession code 2RH1) and β1AR–Cz (PDB accession code 2YCZ), respectively. Hydrogen bonds are shown as black dotted lines. f, Overlay of β2AR–Cz and β2AR–Cz–Nb60 structures.

  3. Nb60 and Nb80 have varying effects on the affinity of different β2AR ligands.
    Figure 3: Nb60 and Nb80 have varying effects on the affinity of different β2AR ligands.

    a, Schematic depicting the use of equilibrium radioligand binding studies to quantify the cooperativity (α) between Nb60 or Nb80 binding and ligand affinity (see Methods and Supplementary Information). c.p.m., counts per minute. b, Cooperativity values for Nb60 (αNb60) and Nb80 (αNb80) for β2AR ligands with varying efficacies. Ligands are ordered by magnitude of αNb80. c, Correlation plot of αNb60 and αNb80; regression shown as solid red line with 95% confidence interval (dotted red line). All α values derived from at least three independent radioligand binding experiments with the deviation depicted as standard error. Adr, adrenaline; alp, alprenolol; carv, carvedilol; caraz, carazolol; clen, clenbuterol; fen, fenoterol; form, formoterol; hbi, hydroxybenzyl isoproterenol; ICI, ICI-118,551; iso, isoprenaline; isoe, isoetharine; pin, pindolol; proc, procaterol; salb, salbutamol; salm, salmeterol; zint, zinterol.

  4. β2AR agonists differentially stabilize receptor states to regulate receptor activation.
    Figure 4: β2AR agonists differentially stabilize receptor states to regulate receptor activation.

    a, b, Illustration of a two-state (a), or three-state (b), model of receptor activation describing the effect of β2AR ligands on receptor conformations stabilized by Nb60 (R60) or Nb80 (R80). The equilibrium (J) between receptor states can be influenced by ligand binding through the allosteric factor β. The theoretical cooperativity (α) between nanobody and ligand binding derived from the two-state model (dashed black line) fails to predict the observed α values for a subset of ligands (dashed red oval). However, the observed cooperativity values can be accurately predicted using an allosteric model in which ligands can differentially modulate three independent receptor states (three-state). Certain ligands (orange) primarily stabilize the active R80 state, whereas others (purple or green) can stabilize R80 but simultaneously destabilize the inactive R60 state. All α values are derived from at least three independent radioligand binding experiments with the deviation depicted as standard error.

  5. Characterization of Nb60 interaction with β2AR.
    Extended Data Fig. 1: Characterization of Nb60 interaction with β2AR.

    ac, 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.

  6. Characterization of β2AR–Nb60–carazolol crystals.
    Extended Data Fig. 2: Characterization of β2AR–Nb60–carazolol crystals.

    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 2FoFc map (Sigma: 1) of carazolol binding pocket (top panels) Nb60 CDR3 binding pocket (bottom panels) within β2AR.

  7. Differential effects of Nb60 and Nb80 on the affinity of 12 different β2AR ligands.
    Extended Data Fig. 3: Differential effects of Nb60 and Nb80 on the affinity of 12 different β2AR ligands.

    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.

  8. Agonist-induced G-protein activation in cellulo correlates with the magnitude of affinity change mediated by Nb80 in vitro.
    Extended Data Fig. 4: Agonist-induced G-protein activation in cellulo correlates with the magnitude of affinity change mediated by Nb80 in vitro.

    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.

  9. Positive correlation between allosteric properties of Nb80 and Gs.
    Extended Data Fig. 5: Positive correlation between allosteric properties of Nb80 and Gs.

    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.

  10. Affinity determination for Nb60 and Nb80 for unliganded β2AR.
    Extended Data Fig. 6: Affinity determination for Nb60 and Nb80 for unliganded β2AR.

    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.

  11. Theoretical framework illustrating the two views of allostery.
    Extended Data Fig. 7: Theoretical framework illustrating the two views of allostery.

    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).

  12. Comparison of experimental and theoretical cooperativities predicted according to a two-state or three-state allosteric model.
    Extended Data Fig. 8: Comparison of experimental and theoretical cooperativities predicted according to a two-state or three-state allosteric model.

    See also the Supplementary Information section on analysis of nanobody allostery. ad, 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. ad, 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.

Tables

  1. Effect of Nb60 and Nb80 on [125I]cyanopindolol affinity
    Extended Data Table 1: Effect of Nb60 and Nb80 on [125I]cyanopindolol affinity
  2. Data collection and refinement statistics (molecular replacement)
    Extended Data Table 2: Data collection and refinement statistics (molecular replacement)

Accession codes

Primary accessions

Protein Data Bank

References

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Author information

  1. These authors contributed equally to this work.

    • Dean P. Staus,
    • Ryan T. Strachan &
    • Aashish Manglik

Affiliations

  1. Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA

    • Dean P. Staus,
    • Biswaranjan Pani,
    • Alem W. Kahsai,
    • Laura M. Wingler,
    • Seungkirl Ahn,
    • Arnab Chatterjee,
    • Ali Masoudi &
    • Robert J. Lefkowitz
  2. Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA

    • Ryan T. Strachan
  3. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Aashish Manglik,
    • William I. Weis &
    • Brian K. Kobilka
  4. Department of Chemistry, University of Toronto, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada

    • Tae Hun Kim &
    • R. Scott Prosser
  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Andrew C. Kruse
  6. Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

    • Els Pardon &
    • Jan Steyaert
  7. Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium

    • Els Pardon &
    • Jan Steyaert
  8. Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • William I. Weis
  9. Department of Pharmacology, Istituto Superiore di Sanità, Rome 00161, Italy

    • Tommaso Costa
  10. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA

    • Robert J. Lefkowitz
  11. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815-6789, USA

    • Robert J. Lefkowitz

Contributions

D.P.S. and R.T.S. conceived the project. Pharmacological assessment of the interactions between Nb60 and Nb80 with the β2AR were designed, performed and analysed by D.P.S., R.T.S., B.P., S.A., and A.C. Formation, purification, and crystallization of the β2AR–Nb60–carazolol complex was conducted by D.P.S. and A. Manglik. Data collection, refinement, and structural analysis was done by A. Manglik, A.C.K., and A. Masoudi, and W.I.W. NMR spectroscopy was executed by A. Manglik, T.H.K, and supervised by R.S.P. Isothermal titration calorimetry was conducted by A.W.K. Nanobody reagents were provided by E.P. and J.S. Detailed allosteric analysis of radioligand binding data was implemented by T.C. Figures were created by D.P.S., A. Manglik, T.C., L.M.W., R.T.S., and A. Masoudi. The manuscript was written by D.P.S., T.C., R.T.S., L.M.W., A. Manglik, A.K.C., and R.J.L. Overall research was supervised by B.K.K. and R.J.L.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Coordinates and structure factors for the β2AR–Nb60–carazolol complex are deposited in the Protein Data Bank (accession code 5JQH).

Reviewer Information Nature thanks H. Hamm, P. Scheerer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Characterization of Nb60 interaction with β2AR. (221 KB)

    ac, 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.

  2. 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 2FoFc map (Sigma: 1) of carazolol binding pocket (top panels) Nb60 CDR3 binding pocket (bottom panels) within β2AR.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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).

  8. 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. ad, 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. ad, 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

  1. Extended Data Table 1: Effect of Nb60 and Nb80 on [125I]cyanopindolol affinity (58 KB)
  2. Extended Data Table 2: Data collection and refinement statistics (molecular replacement) (315 KB)

Supplementary information

PDF files

  1. Supplementary Information (411 KB)

    This file contains Supplementary Text and Data and Supplementary References.

Additional data