Identification of ligand-specific G-protein coupled receptor states and prediction of downstream efficacy via data-driven modeling

Abstract

G protein-coupled receptors (GPCRs) shift between inactive states and active signaling states, to which intracellular binding partners can bind. Extracellular binding of ligands stabilizes different receptor states and modulates the intracellular response via a complex allosteric process, which is not completely understood. Despite the recent advances in structure determination and spectroscopy techniques, a comprehensive view of the ligand-protein interplay remains a challenge. We derived free energy landscapes describing activation of the {beta}2 adrenergic receptor ({beta}2AR) bound to ligands with different efficacy profiles using enhanced sampling molecular dynamics (MD) simulations. The resulting free energy landscapes reveal clear shifts towards active-like states at the G protein binding site for receptors bound to partial and full agonists compared to antagonists and inverse agonists. Not only do the ligands control the population of states, they also modulate the conformational ensemble of the receptor by tuning allosteric protein microswitches. We find an excellent correlation between the conformation of the microswitches close to the ligand binding site and in the transmembrane region and experimentally reported cAMP signaling responses, highlighting the predictive power of our approach. Using dimensionality reduction techniques, we could further assess the similarity between the unique conformational states induced by different ligands. Two distant hotspots governing agonism on transmembrane helices 5 and 7, including the conserved NPxxY motif, formed the endpoints of an allosteric pathway between the binding sites. Our results demonstrate how molecular dynamics simulations can further provide insights into the mechanism of GPCR regulation by ligands, which may contribute to the design of drugs with specific efficacy profiles.