Adrenaline-activated structure of the β(2)-adrenoceptor stabilized by an engineered nanobody

G protein-coupled receptors (GPCRs) are integral membrane proteins that play 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. Thus 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(3-5). 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 active-state β(2)AR bound to three chemically distinct agonists: the ultra high-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. The adrenaline-bound receptor structure is overall similar to the others, but shows 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.