Minute-timescale simulations of G Protein Coupled Receptor A2A activation mechanism reveals a receptor pseudo-active state

G protein-coupled receptors (GPCRs) are a most prominent pharmacological target, regulating various biological functions and targeted by over one third of marketed drugs. These receptors are activated by orthosteric ligands and undergo large conformational changes that lead to coupling effector proteins. Despite their relevance, many aspects of their activation mechanism remain unclear, hampering rational drug design studies. In this work, we elucidate the activation mechanism of the adenosine A2A receptor (A2AR), a class A GPCR, using molecular dynamics simulations and free energy calculations. We have explored the entire conformational landscape of A2AR in its basal apo form and in differently ligated conditions, identifying the receptor's lowest energy functional states. Among these is a novel pseudo-active state (pAS) stabilised by specific "microswitch" residues interactions like the R5.66/E6.30 salt bridge. In this form, A2A is able to couple β-arrestin over G proteins, enlightening possible routes for biased signalling. Our findings shed light on GPCR activation dynamics, paving the way to the rational design of drugs for various diseases involving A2AR, including cancer, inflammation, cardiovascular, Parkinson's, and Alzheimer's diseases. ### Competing Interest Statement The authors have declared no competing interest.