G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signaling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs1, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulfide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β2AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method2, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper3) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

The parasympathetic limb of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G protein coupled receptors (GPCRs) that mediate the response to acetylcholine released from parasympathetic nerves.1–5 Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiologic control of cardiovascular function through activation of G protein-coupled inwardly-rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of antagonist-bound M2 receptor, the first human acetylcholine receptor to be characterized structurally. The antagonist QNB binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all 5 muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The M2 receptor structure provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Cost-effective, efficacious therapeutics are urgently needed against the COVID-19 pandemic. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration and determined a structure of one of the most potent in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. We constructed multivalent Nb constructs that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization.

Protease-Activated Receptor-1 (PAR1) is the prototypical member of a family of G protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the N-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the receptor’s heptahelical bundle to effect G protein-activation. Here we report a 2.2Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by PAR1’s tethered ligand. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated GPCRs, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. PARs are important targets for drug development. The structure reported here will aid development of improved PAR1 antagonists and discovery of antagonists to other members of this receptor family.

Salmeterol is a partial agonist for the β 2 adrenergic receptor (β 2 AR), and the first long-acting β 2 AR agonist (LABA) to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol has been controversial both for its safety and mechanism of action. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β 2 AR in complex with an active-state stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β 1 AR and β 2 AR explain the high receptor subtype selectivity. A structural comparison with the β 2 AR bound to the full agonist epinephrine reveals differences in the hydrogen bond network involving residues Ser 204 5.43 and Asn 293 6.55 . Mutagenesis and biophysical studies suggest that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G protein activation and the limited β-arrestin recruitment for salmeterol.