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

• Published in: Nature (London) Vol. 502; no. 7472; pp. 575 - 579
• Main Authors: Ring, Aaron M., Manglik, Aashish, Kruse, Andrew C., Enos, Michael D., Weis, William I., Garcia, K. Christopher, Kobilka, Brian K.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24.10.2013
• Subjects: 631/535/1266; 631/92/612/194; Adrenergic beta-2 Receptor Agonists - pharmacology; Benzoxazines - pharmacology; Binding Sites - drug effects; Crystallography, X-Ray; Directed Molecular Evolution; Epinephrine - pharmacology; Humanities and Social Sciences; Humans; Hydrogen Bonding - drug effects; Isoproterenol - analogs & derivatives; Isoproterenol - pharmacology; letter; Ligands; Models, Molecular; multidisciplinary; Protein Engineering; Protein Stability - drug effects; Receptors, Adrenergic, beta-2 - chemistry; Receptors, Adrenergic, beta-2 - drug effects; Receptors, Adrenergic, beta-2 - metabolism; Science; Single-Chain Antibodies - genetics; Single-Chain Antibodies - pharmacology; Tyrosine - chemistry; Tyrosine - metabolism; Water - chemistry; Water - pharmacology
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature12572

Here, by developing a high-affinity camelid antibody fragment that stabilizes the active state of the β 2 -adrenoceptor, the X-ray crystal structures of the receptor in complex with three agonists, including adrenaline, were determined. Structure of the activated β 2 -adrenoceptor This study reports three structures of fully active human β 2 adrenergic receptor (β 2 AR) in complex with diverse agonists: BI167107, hydroxybenzyl isoproterenol, and the endogenous agonist adrenaline. β 2 AR is a G-protein-coupled receptor (GPCR), ubiquitous membrane proteins that are targeted by many clinically used drugs. The molecular processes by which they bind to their endogenous agonists and activate effector proteins remain poorly understood. Despite the chemical diversity of the three agonists examined, all three stabilize highly similar active states in the receptor. Subtle structural differences provide insight into how a single GPCR is activated by multiple agonists, a phenomenon that is critically important to drug development. 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 agonists 1 , 2 or receptor stabilization by mutagenesis 3 , 4 , 5 . Many natural agonists such as adrenaline, which activates the β 2 -adrenoceptor (β 2 AR), 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 β 2 AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β 2 AR 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 β 2 AR and related GPCRs.