Revealing molecular determinants of ligand efficacy and affinity at the D₂dopamine receptor through molecular dynamics simulations

G protein-coupled receptors (GPCRs) control numerous physiological processes and are important therapeutic targets. Despite major research efforts, rational design of drugs that stimulate GPCR signaling is challenging because the molecular basis of activation remains poorly understood. Here, a combination of molecular dynamics simulations and pharmacological assays was used to study the activation mechanism of the D₂dopamine receptor (D₂R), a major drug target for central nervous system diseases. Enhanced sampling simulations were performed to identify the key conformational changes involved in D₂R activation by dopamine, and a computational platform for ligand profiling based on free energy calculations was developed. Simulations and experimental characterization of a series of dopamine derivatives showed that free energy calculations can predict the effect of small chemical modifications on ligand affinity and efficacy. Furthermore, simulations of D₂dopamine and β₂adrenergic receptor activation revealed that ligand-induced activation of these GPCRs is driven by different molecular mechanisms despite recognizing chemically similar catecholamine agonists. Whereas dopamine interactions with the sixth transmembrane helix primarily drive activation of the D₂R, hydrogen bonding with the fifth helix is the key interaction for activation of the β₂adrenergic receptor. Our results highlight the complexity of GPCR activation and illustrate how molecular simulations can provide mechanistic insight and quantitative predictions of ligand activity, enabling structure-based drug design.