Letter | Published:

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

Nature volume 502, pages 575579 (24 October 2013) | Download Citation


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.

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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.


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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

    • Aaron M. Ring
    • , Aashish Manglik
    •  & Andrew C. Kruse

    These authors contributed equally to this work.


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

    • Aaron M. Ring
    • , Michael D. Enos
    • , William I. Weis
    •  & K. Christopher Garcia
  3. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA

    • K. Christopher Garcia


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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.

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.

Corresponding authors

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

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    Supplementary Information

    This file contains Supplementary Figures 1-10, Supplementary Tables 1-3 and an additional reference.

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