Adrenaline-activated structure of β2-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

Structural insights into μ-opioid receptor activation

International audienceActivation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors