Skip to main content
SpringerLink
Log in

Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody

  • Letter
  • Published:

From Nature

View current issue Submit your manuscript

Abstract

G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists1,2 or receptor stabilization by mutagenesis3,4,5. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Conformational selection of nanobodies and characterization of high-affinity Nb6B9.
Figure 2: Structure of the activated β2AR in complex with three agonists.
Figure 3: Comparison of agonist-binding modes.
Figure 4: Activation by catecholamine agonists.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the β2AR–Nb6B9 complexes with BI167107, HBI and adrenaline ligands are deposited in the Protein Data Bank under accession codes 4LDE, 4LDL and 4LDO, respectively.

References

  1. Rosenbaum, D. M. et al. Structure and function of an irreversible agonist–β2 adrenoceptor complex. Nature 469, 236–240 (2011)

    Article  ADS  CAS  Google Scholar 

  2. Xu, F. et al. Structure of an agonist-bound human A2A adenosine receptor. Science 332, 322–327 (2011)

    Article  ADS  CAS  Google Scholar 

  3. White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012)

    Article  ADS  CAS  Google Scholar 

  4. Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474, 521–525 (2011)

    Article  CAS  Google Scholar 

  5. Warne, T. et al. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469, 241–244 (2011)

    Article  ADS  CAS  Google Scholar 

  6. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013)

    Article  ADS  CAS  Google Scholar 

  7. Nygaard, R. et al. The dynamic process of β2-adrenergic receptor activation. Cell 152, 532–542 (2013)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Deupi, X. et al. Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proc. Natl Acad. Sci. USA 109, 119–124 (2012)

    Article  ADS  CAS  Google Scholar 

  12. Schneider, E. H., Schnell, D., Strasser, A., Dove, S. & Seifert, R. Impact of the DRY motif and the missing “ionic lock” on constitutive activity and G-protein coupling of the human histamine H4 receptor. J. Pharmacol. Exp. Ther. 333, 382–392 (2010)

    Article  CAS  Google Scholar 

  13. Elgeti, M. et al. Conserved Tyr2235.58 plays different roles in the activation and G-protein interaction of rhodopsin. J. Am. Chem. Soc. 133, 7159–7165 (2011)

    Article  CAS  Google Scholar 

  14. Goncalves, J. A. et al. Highly conserved tyrosine stabilizes the active state of rhodopsin. Proc. Natl Acad. Sci. USA 107, 19861–19866 (2010)

    Article  ADS  CAS  Google Scholar 

  15. Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Sci. USA 105, 877–882 (2008)

    Article  ADS  CAS  Google Scholar 

  16. Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S. & Dixon, R. A. Identification of two serine residues involved in agonist activation of the β-adrenergic receptor. J. Biol. Chem. 264, 13572–13578 (1989)

    CAS  PubMed  Google Scholar 

  17. Liapakis, G. et al. The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the β2-adrenergic receptor. J. Biol. Chem. 275, 37779–37788 (2000)

    Article  CAS  Google Scholar 

  18. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008)

    Article  ADS  CAS  Google Scholar 

  19. 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)

    Article  CAS  Google Scholar 

  20. Wang, Z., Mathias, A., Stavrou, S. & Neville, D. M., Jr A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities. Protein Eng. Des. Sel. 18, 337–343 (2005)

    Article  Google Scholar 

  21. Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nature Protocols 1, 755–768 (2006)

    Article  CAS  Google Scholar 

  22. 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. USA 104, 7682–7687 (2007)

    Article  ADS  CAS  Google Scholar 

  23. Zou, Y., Weis, W. I. & Kobilka, B. K. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor. PLoS ONE 7, e46039 (2012)

    Article  ADS  CAS  Google Scholar 

  24. Otwinowski, Z. & Minor, W. in Methods in Enzymology Vol. 276 (ed. Carter, C. W. Jr) 307–326 (Academic, 1997)

    Google Scholar 

  25. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  26. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    Article  CAS  Google Scholar 

  27. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

Download references

Acknowledgements

We thank D. Hilger for critical reading of the manuscript. We acknowledge support from the Stanford Medical Scientist Training Program (A.M. and A.M.R.), the American Heart Association (A.M.), the National Science Foundation (A.C.K.), the Ruth L. Kirschstein National Research Service Award (A.M.R.), National Institutes of Health grants NS02847123 and GM08311806 (B.K.K.), from the Mathers Foundation (B.K.K., W.I.W. and K.C.G.), and from the Howard Hughes Medical Institute (K.C.G.).

Author information

Author notes

  1. Aaron M. Ring, Aashish Manglik and Andrew C. Kruse: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, 94305, California, USA

    Aaron M. Ring, Aashish Manglik, Andrew C. Kruse, Michael D. Enos, William I. Weis, K. Christopher Garcia & Brian K. Kobilka

  2. Department of Structural Biology, Stanford University, Stanford, 94305, California, USA

    Aaron M. Ring, Michael D. Enos, William I. Weis & K. Christopher Garcia

  3. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, 94305, California, USA

    K. Christopher Garcia

Authors
  1. Aaron M. Ring
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aashish Manglik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew C. Kruse
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael D. Enos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William I. Weis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K. Christopher Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Brian K. Kobilka
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.M.R. designed and performed yeast display staining and selection experiments, nanobody expression, purification and characterization on yeast and by SPR. A.M. and A.C.K. designed and performed receptor expression, purification, radioligand-binding experiments and crystallography experiments. M.D.E. performed crystallization experiments with adrenaline-bound β2AR under the supervision of A.M. and A.C.K. The manuscript was written by A.M.R., A.M. and A.C.K. W.I.W. supervised structure refinement. K.C.G. and B.K.K. supervised experiments, and B.K.K. supervised manuscript preparation.

Corresponding authors

Correspondence to K. Christopher Garcia or Brian K. Kobilka.

Ethics declarations

Competing interests

A.M.R., A.M., A.C.K. and B.K.K. have filed a patent application describing methods of generating conformationally selective affinity reagents for transmembrane proteins presented here.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10, Supplementary Tables 1-3 and an additional reference. (PDF 8205 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ring, A., Manglik, A., Kruse, A. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013). https://doi.org/10.1038/nature12572

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12572

  • Springer Nature Limited

This article is cited by

Search

Navigation