Ligand-specific conformational dynamics of the α2A-adrenergic receptor revealed by hydrogen-deuterium exchange mass spectrometry

Biased agonists targeting the α_2A-adrenergic receptor (α_2AAR) hold therapeutic promise by selectively engaging G protein over β-arrestin pathways, yet their structural mechanisms remain unclear. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we investigated α_2AAR conformational dynamics with full agonists (norepinephrine and dexmedetomidine) or the Gi/o-biased partial agonist PS75, in the absence or presence of heterotrimeric GoA (GoA). Without GoA, agonists induced only subtle and localized differences, whereas GoA-bound states revealed broader, agonist-specific conformational differences, suggesting that agonists pre-configure distinct pre-active receptor states. Intracellular loop 2 (ICL2) emerged as a common molecular switch for GoA binding, while TM2/5/7, ICL3, and helix 8 underwent agonist-specific conformational changes. Notably, PS75 induces an α_2AAR-GoA complex that differs from those formed by full agonists, characterized by insufficient α5 C-terminal insertion into the receptor cytosolic core, likely due to limited TM6 outward movement. These findings provide mechanistic insight into partial agonism, with possible implications for biased signaling.

HDX-MS; biased ligand; conformational dynamics; partial agonism; α(2A)-adrenergic receptor.