Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to GPCRs induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist-receptor complexes on a molecular level. Employing all-atom molecular dynamics (MD) simulations, dynophores (i.e. a combination of static 3D-pharmacophores and MD-based conformational sampling), ligand design and receptor mutagenesis, we show that inactive agonist-receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) vs. dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist-receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists, where binding modes will be only subtly different and confined to only one binding site.