Drugs targeting the orthosteric, primary binding site of G-protein coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic beta-2 adrenergic receptor in complex with an orthosteric agonist and Compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an alpha helical conformation required to engage the G-protein. Structural comparison explains the selectivity of the compound for beta-2 over the beta-1 adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

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The α1A-adrenergic receptor (α1AAR) belongs to the family of G protein-coupled receptors that respond to adrenaline and noradrenaline. α1AAR is involved in smooth muscle contraction and cognitive function. Here, we present three cryo-electron microscopy structures of human α1AAR bound to the endogenous agonist noradrenaline, its selective agonist oxymetazoline, and the antagonist tamsulosin, with resolutions range from 2.9 Å to 3.5 Å. Our active and inactive α1AAR structures reveal the activation mechanism and distinct ligand binding modes for noradrenaline compared with other adrenergic receptor subtypes. In addition, we identified a nanobody that preferentially binds to the extracellular vestibule of α1AAR when bound to the selective agonist oxymetazoline. These results should facilitate the design of more selective therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the β1 and β2 adrenergic receptors (β1AR and β2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the β2AR over the β1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the β2AR, as well as a less stable binding pocket for constrained epinephrine in the β1AR. The differences in the amino acid sequence of the extracellular vestibule of the β1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the β2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

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G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the process of functional association between G proteins and GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit. In atomic detail, we reveal the temporal progression of structural rearrangements of GDP-bound heterotrimeric Gs protein (GsGDP) upon coupling to the β2-adrenergic receptor (β2AR) using molecular dynamics simulations. The binding of GsGDP to the β2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release, the rate-limiting step during G-protein activation. In particular, the opening of α1-αF helices, displacement of the αG helix, and the opening of the α-helical domain weaken the interaction between GDP and the G protein. Signal transduction to the G protein occurs via a novel receptor interface, confirmed by site-directed mutagenesis and functional assays. From this β2AR-GsGDP intermediate, the G protein must undergo an in-plane rotation along the receptor axis to reach the β2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface interact to transmit the signal over 30Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G protein coupling specificity.