In silico insights into the activation mechanism of the P2Y12 receptor: Identification of a novel histidine-mediated microswitch

The P2Y₁₂ receptor (P2Y₁₂R), a G protein-coupled receptor (GPCR) expressed predominantly on platelet membranes, is a key therapeutic target for the inhibition of platelet aggregation. Despite its clinical relevance, the molecular mechanism governing its activation remains incompletely understood. Here, we investigated the conformational dynamics underlying P2Y₁₂R activation, focusing on the transition from the agonist-bound inactive state to the active conformation. Extensive all-atom molecular dynamics simulations were performed on P2Y₁₂R complexed with the full agonist 2MeSADP. Essential dynamics analysis revealed that P2Y₁₂R follows a conformational selection mechanism, whereby the receptor samples active-like conformations even in the absence of G-protein binding. This analysis also uncovered a previously uncharacterized microswitch acting as a critical driver of activation. To further dissect the molecular basis of receptor activation, we examined three canonical GPCR microswitches using multiple structural descriptors, elucidating their concerted roles in stabilizing intermediate and active states. The newly identified microswitch was subsequently used as a reaction coordinate in enhanced-sampling simulations, enabling reconstruction of the free energy landscape of the activation pathway. Together, these results provide a comprehensive molecular model of P2Y₁₂R activation, advancing our understanding of GPCR activation and offering mechanistic insights that may guide the design of next-generation selective agonists and antagonists targeting this clinically important receptor.