Mechanism of intracellular allosteric β2AR antagonist revealed by X-ray crystal structure


G-protein-coupled receptors (GPCRs) pose challenges for drug discovery efforts because of the high degree of structural homology in the orthosteric pocket, particularly for GPCRs within a single subfamily, such as the nine adrenergic receptors. Allosteric ligands may bind to less-conserved regions of these receptors and therefore are more likely to be selective. Unlike orthosteric ligands, which tonically activate or inhibit signalling, allosteric ligands modulate physiologic responses to hormones and neurotransmitters, and may therefore have fewer adverse effects. The majority of GPCR crystal structures published to date were obtained with receptors bound to orthosteric antagonists, and only a few structures bound to allosteric ligands have been reported. Compound 15 (Cmpd-15) is an allosteric modulator of the β2 adrenergic receptor (β2AR) that was recently isolated from a DNA-encoded small-molecule library1. Orthosteric β-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker. Cmpd-15 exhibits negative cooperativity with agonists and positive cooperativity with inverse agonists. Here we present the structure of the β2AR bound to a polyethylene glycol-carboxylic acid derivative (Cmpd-15PA) of this modulator. Cmpd-15PA binds to a pocket formed primarily by the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7 as well as intracellular loop 1 and helix 8. A comparison of this structure with inactive- and active-state structures of the β2AR reveals the mechanism by which Cmpd-15 modulates agonist binding affinity and signalling.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Crystal structure of β2AR–T4L in complex with carazolol and Cmpd-15PA in inactive conformation.
Figure 2: The Cmpd-15PA binding pocket.
Figure 3: Cmpd-15PA stabilized conformational changes and mechanism of allosteric antagonism.
Figure 4: Mutagenesis of Cmpd-15PA binding site.

Accession codes

Primary accessions

Protein Data Bank


  1. 1

    Ahn, S. et al. Allosteric ‘beta-blocker’ isolated from a DNA-encoded small molecule library. Proc. Natl Acad. Sci. 114, 1708–1713 (2017)

    CAS  Article  Google Scholar 

  2. 2

    Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Zheng, Y. et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540, 458–461 (2016)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Oswald, C. et al. Intracellular allosteric antagonism of the CCR9 receptor. Nature 540, 462–465 (2016)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Jazayeri, A. et al. Extra-helical binding site of a glucagon receptor antagonist. Nature 533, 274–277 (2016)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Staus, D. P. et al. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535, 448–452 (2016)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Ring, A. M. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Huang, W. et al. Structural insights into μ-opioid receptor activation. Nature 524, 315–321 (2015)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Kobilka, B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271 (1995)

    CAS  Article  Google Scholar 

  14. 14

    Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009)

    CAS  Article  Google Scholar 

  15. 15

    Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Google Scholar 

  16. 16

    Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. 103, 8060–8065 (2006)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Adams, P. D . et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    CAS  Article  Google Scholar 

  20. 20

    Ten Eyck, L. F. Fast Fourier transform calculation of electron density maps. Methods Enzymol. 115, 324–337 (1985)

    CAS  Article  Google Scholar 

  21. 21

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    CAS  Article  Google Scholar 

  22. 22

    Krissinel, E. Stock-based detection of protein oligomeric states in jsPISA. Nucleic Acids Res. 43 (W1), W314–W319 (2015)

    CAS  Article  Google Scholar 

  23. 23

    Whorton, M. R. et al. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl Acad. Sci. 104, 7682–7687 (2007)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Strachan, R. T. et al. Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR). J. Biol. Chem. 289, 14211–14224 (2014)

    CAS  Article  Google Scholar 

  25. 25

    Ghanouni, P. et al. The effect of pH on β2 adrenoceptor function. Evidence for protonation-dependent activation. J. Biol. Chem. 275, 3121–3127 (2000)

    CAS  PubMed  Google Scholar 

  26. 26

    Ranganathan, A., Dror, R. O. & Carlsson, J. Insights into the role of Asp792.50 in β2 adrenergic receptor activation from molecular dynamics simulations. Biochemistry 53, 7283–7296 (2014)

    CAS  Article  Google Scholar 

  27. 27

    Lomize, M. A., Lomize, A. L., Pogozheva, I. D. & Mosberg, H. I. OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006)

    CAS  Article  Google Scholar 

  28. 28

    Huang, J. & MacKerell, A. D., Jr. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J. Comput. Chem. 34, 2135–2145 (2013)

    CAS  Article  Google Scholar 

  29. 29

    Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010)

    CAS  Article  Google Scholar 

  30. 30

    MacKerell, A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Best, R. B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ1 and χ2 dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012)

    CAS  Article  Google Scholar 

  32. 32

    Best, R. B., Mittal, J., Feig, M. & MacKerell, A. D., Jr. Inclusion of many-body effects in the additive CHARMM protein CMAP potential results in enhanced cooperativity of α-helix and β-hairpin formation. Biophys. J. 103, 1045–1051 (2012)

    ADS  CAS  Article  Google Scholar 

  33. 33

    Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Vanommeslaeghe, K., Raman, E. P. & MacKerell, A. D., Jr. Automation of the CHARMM General Force Field (CGenFF) II: assignment of bonded parameters and partial atomic charges. J. Chem. Inf. Model. 52, 3155–3168 (2012)

    CAS  Article  Google Scholar 

  35. 35

    Vanommeslaeghe, K. & MacKerell, A. D., Jr. Automation of the CHARMM General Force Field (CGenFF) I: bond perception and atom typing. J. Chem. Inf. Model. 52, 3144–3154 (2012)

    CAS  Article  Google Scholar 

  36. 36

    Salomon-Ferrer, R., Götz, A. W., Poole, D., Le Grand, S. & Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013)

    CAS  Article  Google Scholar 

  37. 37

    Case, D. A. et al. AMBER 2016. (2016)

  38. 38

    Hopkins, C. W., Le Grand, S., Walker, R. C. & Roitberg, A. E. Long-time-step molecular dynamics through hydrogen mass repartitioning. J. Chem. Theory Comput. 11, 1864–1874 (2015)

    CAS  Article  Google Scholar 

  39. 39

    Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

    CAS  Article  Google Scholar 

  40. 40

    Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions. Protein Eng. 8, 127–134 (1995)

    CAS  Article  Google Scholar 

Download references


We thank K. Hirata at Beamline BL32XU of Spring-8 for assistance in data collection. A. Wall and T. Xu provided technical assistance. NuEvolution for constructive discussions in the course of the work. We acknowledge support from the National Institute of Health grants NS028471 and GM106990 (B.K.K.), HL16037 (R.J.L.) and T32HL007101 (A.W.K. and A.M.), Amgen-China Postdoc fellowship (X.L.) and the Mathers Foundation (B.K.K. and W.I.W.). R.J.L. is an investigator with the Howard Hughes Medical Institute.

Author information




X.L. expressed and purified β2AR–T4L, performed crystallization, data collection, data processing, structure determination and refinement. S.A. designed and performed in vitro radio ligand binding and mutagenesis studies. A.W.K. designed the chemical synthetic route for Cmpd-15PA. B.P. designed and performed the in vitro radio ligand binding experiments. A.M. assisted in the design of mutagenesis studies and the development of Cmpd-15PA. K.-C.M. synthesized Cmpd-15PA and analysed the spectral data. X.C. designed the chemical structure and synthetic route for Cmpd-15PA, analysed the spectral data and assisted in manuscript preparation. N.R.L. performed and analysed molecular dynamics simulations, with assistance from A.J.V. R.O.D. oversaw molecular dynamics simulations and analysis. W.I.W. oversaw data processing, structure determination and refinement. The manuscript was written by B.K.K. with assistance from X.L. R.J.L. and B.K.K. coordinated the experiments and supervised the overall research. All authors contributed to the editing of the manuscript.

Corresponding authors

Correspondence to Robert J. Lefkowitz or Brian K. Kobilka.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

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

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Effects of Cmpd-15PA on agonist binding to the β2AR.

a, The level of 3H-Fenoterol (Fen) high-affinity binding to the β2AR was measured after pretreatment with the vehicle control (0.5% DMSO), Cmpd-15 or Cmpd-15PA at various concentrations as indicated in the presence of transducers, either trimeric Gαβγ protein or β-arrestin 1 (β-arr1) together with Fab30. Values were expressed as percentages of the maximal 3H-Fen binding level promoted by each transducer in the vehicle control (0.5% DMSO) and represent mean ± s.e.m. obtained from four independent experiments done in duplicate. b, Isoproterenol (ISO)-125I-CYP competition binding curves were obtained using wild-type (WT) β2AR or β2AR–T4L (T4L) in the absence (vehicle alone) or the presence of Cmpd-15PA at 32 μM. Values are expressed as percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding-log IC50 curve fit and represent mean ± s.e.m. obtained from four independent experiments done in duplicate.

Extended Data Figure 2 Bromine atom of Cmpd-15PA was sensitive to radiation damage.

ac, Clear differences were observed around the bromine atom of Cmpd-15PA between simulated annealing omit maps of the first half of diffraction data (a, green density, 2.3σ) and maps of the second half of diffraction data (b, green density, 2.3σ), resulting in a strong difference electron density centred around the bromine atom (c, purple density, 4σ).

Extended Data Figure 3 Two-dimensional schematic depiction of the Cmpd-15PA binding pocket.

Carbon atoms are coloured in black, oxygen atoms in red, nitrogen atoms in blue, bromine atom in green. Hydrogen bonds are represented as green dashed lines with distances labelled. The figure is generated using LIGPLOT40.

Extended Data Figure 4 Crystallographic evidence support modelling of R isomer of Cmpd-15PA.

a, b, The R isomer fits better into the simulated annealing omit map (green density map, 2.3σ) than S isomer (b). c, When refined with R isomer, the Cmpd-15PA model fits well into the 2Fo − Fc density (grey map, 1.5σ) with only very weak negative Fo − Fc density (red map, 3.0σ). d, When refined with S isomer, the Cmpd-15PA model does not fit as well to the 2Fo − Fc density (grey map, 1.5σ). The negative density Fo − Fc map (red map, 3.0σ) and positive density Fo − Fc map (green map, 3.0σ) suggest that the S isomer is not supported by crystallographic data.

Extended Data Figure 5 Comparison of modulator binding pocket before and after Cmpd-15PA binding.

a, b, The binding pocket is occluded before Cmpd-15PA binding (a), which would be an obstacle for in silico docking because the shape of the pocket is markedly different after Cmpd-15 binding (b).

Extended Data Figure 6 Comparison of the structures of the β2AR (cyan, PDB, 2RH1), β2AR–Nb60 (green, PDB, 5JQH) and β2AR–Cmpd-15PA (orange).

a, Cmpd-15PA binding pocket overlaps with Nb60 binding pocket. bd, Different views of superimposed structures of the β2AR, the β2AR–Nb60 and the β2AR–Cmpd-15PA revealing very little structural difference associated with binding of Nb60 or Cmpd-15PA.

Extended Data Figure 7 Relative activities of Cmpd-15 analogues in β2AR-mediated responses.

For each analogue, only the modified region relative to the parent Cmpd-15 is indicated. Values for ‘inhibition Emax (%)’ are expressed as percentages of analogue-induced inhibition relative the Cmpd-15-induced level. Values for ‘EC50 shift (fold)’ are expressed as rightward fold-shifts compared to the EC50 value obtained in the vehicle (DMSO)-treated control curve. All values represent mean ± s.e.m. obtained from at least four independent experiments done in duplicate. Statistical analyses were performed using ‘one-way ANOVA’ with ‘Dunnetts’ post-tests for comparison to the control. ***P < 0.001. cAMP, G protein-mediated cAMP accumulation; βarr, β-arrestin recruitment to the β2AR.

Extended Data Figure 8 Alignment of residues that form the Cmpd-15PA binding pocket in β2AR with those from β1AR, V2R and AT1R.

There are 21 residues from β2AR that form the Cmpd-15 binding pocket (highlighted green). The identical residues from β1AR, V2R and AT1R are also highlighted in green. 10 out of the 21 residues are located at TM1, ICL1 and TM2 (a), while 11 of the 21 residues are located at TM6, TM7 and helix 8 (b). The top numbering refers to protein sequences in β2AR. The bottom numbering refers to Ballesteros–Weinstein numbering. c, Cmpd-15 has no effect on orthosteric agonist binding to the β1AR. Dose–response curves of isoproterenol (ISO)-competition binding to the β1AR with 125I-CYP were obtained in the presence of various concentrations of Cmpd-15 as indicated. Values were expressed as percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding-log IC50 curve fit and represent mean ± s.e.m. obtained from five independent experiments done in duplicate.

Extended Data Figure 9 Comparison of Cmpd-15PA pocket with intracellular allosteric antagonist pockets of CCR2 and CCR9.

a, Cmpd-15PA pocket is formed by residues from TM1, TM2, TM6, TM7, ICL1 and helix 8 in β2AR. b, c, The binding pocket of CCR2-RA-(R) in CCR2 (b) and the pocket of vercirnon in CCR9 (c) are similar to Cmpd-15PA (blue lines) pocket but involve more interactions with TM3. (Protein Data Bank accession numbers, 5T1A for CCR2/CCE2-RA-(R) and 5LWE for CCR9/vercirnon.)

Extended Data Table 1 Crystallography data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains supplementary text and figures S1-S6. (PDF 1499 kb)

Reporting Summary (PDF 67 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, X., Ahn, S., Kahsai, A. et al. Mechanism of intracellular allosteric β2AR antagonist revealed by X-ray crystal structure. Nature 548, 480–484 (2017).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.