We present a new concept of partial agonism at G protein-coupled receptors. We demonstrate the coexistence of two functionally distinct populations of the muscarinic M2 receptor stabilized by one dynamic ligand, which binds in two opposite orientations. The ratio of orientations determines the cellular response. Our concept allows predicting and virtually titrating ligand efficacy, which opens unprecedented opportunities for the design of drugs with graded activation of the biological system.

G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors 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 M 2 receptors to demonstrate the existence and function of such inactive agonist⅐receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist⅐receptor 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) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist⅐receptor 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.Specific and coordinated cell-to-cell communication regulates the flow of information between cells, and proper information processing ensures physiological functions of biological systems. G protein-coupled receptors (GPCRs), 8 constituting the largest class of membrane proteins in mammals, are essential mediators of chemically and light-encoded information (1-4). GPCRs sense a great variety of extracellular stimuli, e.g. neurotransmitters and hormones, and subsequently translate this information into an intracellular response via G proteins, ␤-arrestins, and possibly GPCR-interacting proteins (2-5). Because of their abundance and relevance in regulating the majority of (patho-)physiological processes in humans, GPCRs have for a long time represented the most important drug targets being addressed by at least a third of all currently marketed drugs (6, 7).Agonist binding leads to receptor activation, which is followed by intracellular G protein recruitment and subsequent cell signaling. Breakthroughs in GPCR crystallography have led to inactive and active crystal structures of the same receptor protein. Among these are rhodopsin (8 -10) and more recently the ␤ 2 -adrenergic (11-14), M 2 muscarinic (15, 16), and -opioid receptors (17, 18). These structures most likely represent energetically favored, relatively stable inactive and active receptor⅐ligand complexes. Despite the diversity of crystallized receptors, a common...

G protein-coupled receptors transmit extracellular signals across cell membranes via different G protein classes and β-arrestins. Some pathways may be therapeutically beneficial, whereas others may be detrimental under certain pathophysiological conditions. For many GPCRs, biased agonists are available, which preferentially signal through one pathway or a subset of pathways, and harnessing biased agonism could be a potential novel therapeutic strategy. However, the incomplete mechanistic understanding of biased agonism hampers rational design of biased ligands. Using the muscarinic M receptor as a model system, we have analyzed the relationship between ligand-dependent conformational changes as revealed in all-atom MD simulations and the activation of specific G proteins. We find that the extent of closure of the extracellular, allosteric binding site interferes with the activation of certain G proteins. Our data allow the rational design of G-biased agonists at the M receptor and delineate a simple principle which may be translated to other GPRCs.