Computational design of constitutively active mutants of Dopamine D2 receptor inspired by ligand-independent activation mechanisms

G protein-coupled receptors (GPCRs) can signal in the absence of agonists through constitutive activity. This activity can be enhanced by mutations, resulting in receptors known as constitutively active mutants (CAMs). Such receptors can be implicated in various physiological and pathophysiological conditions, and also offer significant therapeutic potential. However, the molecular basis of their constitutive activity remains unknown. To investigate how CAMs affect receptor activation, we employed enhanced sampling simulations to study the dopamine D2 receptor (D2R), a key target in central nervous system therapies. Free energy landscape analyses revealed that CAMs promote a conformational shift favoring an active state similar to the agonist-bound receptor. To then identify novel CAMs, we developed a comprehensive strategy combining structural comparison, in-silico residue scanning, and free energy calculations, validated by luminescence-complementation-based assays. Applied to D2R, this approach uncovered a new single-point CAM, D2R-I481.46W, which was functionally validated. Further investigation revealed that this mutation activates allosteric communication pathways primarily involving transmembrane helix 5, particularly Ser1945.43, underscoring its role in transmitting activation signals to the intracellular domain. These findings deepen our understanding of constitutive GPCR activity and demonstrate the utility of this framework for identifying CAMs as ligand-independent models for structural, cellular, and physiological studies.