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

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 agonists or receptor stabilization by mutagenesis. 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.

Figure 1. Conformational selection of nanobodies and characterization of high affinity Nb6B9

a, Schematic representation of yeast display of Nb80. Nb80 is fused to the amino terminus of Aga2p, which attaches to the yeast cell wall through a covalent interaction with Aga1p. b, Staining of Nb80-expressing yeast with β₂AR bound to the agonist BI167107 (left) or the inverse agonist carazolol (right). c, Flow-chart summary of conformational selection process. d, Histogram overlays assessing β₂AR staining of the library at each round of selection. The left panel shows staining with 1 μM BI167107-occupied receptor, while the right panel shows staining with 1 μM carazolol-occupied receptor. e, Representative single-cycle kinetics SPR sensorgram of wild-type Nb80 (top) and engineered Nb6B9 binding immobilized β₂AR bound to BI167107. f, ³H-dihydroalprenolol (³H-DHA) competition binding shows a comparable increase in β₂AR affinity for adrenaline in the presence of Nb6B9 as with G protein G_s. ³HDHA affinity is largely unchanged in the presence of Nb6B9 (Supplementary Table 2). Data and error bars represent the mean ± standard error of the mean from three experiments.

Figure 2. Structure of the activated β₂AR in complex with three agonists

a, Chemical structures of the three ligands used for crystallization trials. b, All three active-state structures, showing remarkable similarity in overall receptor conformation. c, The 2.8 Å resolution structure of BI167107-bound β₂AR reveals active-state water molecules: a bridging water molecule participates in a polar network at the ligand binding site (top) and a second water molecule mediates a hydrogen bond between two highly conserved tyrosines. Such an interaction is possible in the active state (orange) but not the inactive state (gray).

Figure 3. Comparison of agonist binding modes

a, Comparison of BI167107-bound receptor (orange) with HBI-bound receptor (green) shows highly conserved agonist binding mode. b, Similarly, adrenaline-bound (cyan) and HBI-bound (green) receptor structures are highly similar. c, An analogous comparison of BI167107-bound β₂AR (orange) with adrenaline-bound receptor (cyan) shows the similar polar networks for the two ligands (black dotted lines) with a notable difference in the hydrogen bonding of Asn293^6.55 to the amide proton in BI167107 (red dotted line) or the meta hydroxyl of adrenaline (blue dotted line) d, Due to this difference, Asn293^6.55 and TM6 shift inward in the adrenaline-bound structure,, leading to a cascade of changes culminating in a rearrangement of ECL3.

Figure 4. Activation by catecholamine agonists

For the first time, structures of catecholamine-bound adrenergic receptors in active and inactive conformations can be compared. a, The structure of β₂AR in an active conformation bound to the agonist adrenaline reveals an extended polar contact network linking the orthosteric site to extracellular loops 2 and 3, while the structure of thermostabilized β₁AR in an inactive conformation bound to a similar catecholamine agonist, isoprenaline, shows a far more limited polar network. c, Likewise, a surface view of the active state structure shows a substantial contraction of the binding site compared with d, the inactive β₁AR structure.