Minute-timescale free-energy calculations reveal a pseudo-active state in the adenosine A_2A receptor activation...

G protein-coupled receptors (GPCRs) are membrane proteins targeted by over one-third of marketed drugs. Understanding their activation mechanism is essential for precise regulation of drug pharmacological response. In this work, we elucidate the conformational landscape of the adenosine A_2A receptor (A_2AR) activation mechanism in its basal apo form and under different ligand-bound conditions through minute-timescale free-energy calculations. We identified a pseudo-active state (pAs) of the A_2AR apo form, stabilized by specific "microswitch" residues, including a salt bridge established between the conserved residues R^5.66 and E^6.30. The pAs enables A_2AR to couple with Gs protein upon rearrangement of the intracellular end of transmembrane helix 6, providing unprecedented structural insights into receptor function and signaling dynamics. Our simulation protocol is versatile and can be adapted to study the activation of any GPCRs, potentially making it a valuable tool for drug design and "biased signaling" studies.