Energy Landscape Sampling Reveals Ligand-Dependent Structural Dynamics of the Protease-Activated Receptor 1

Protease-activated receptor 1 (PAR1) is a class A G protein-coupled receptor activated by proteolytic cleavage and exposure of a tethered agonist. While existing structures have revealed inactive and active conformations, the ligand-dependent conformational energy landscape governing PAR1 activation remains poorly understood. Here, we employed enhanced-sampling molecular dynamics simulations using the string method with swarms of trajectories to quantify how extracellular ligands and G-protein binding modulate PAR1 activation. Free-energy landscapes were computed for five receptor states (apo, tethered agonist, thrombin receptor-activating peptide (TRAP), vorapaxar (VPX), and VPX + Na+) using five transmembrane (TM) helix distance metrics as collective variables. In the apo state, PAR1 samples inactive, intermediate, and active-like conformations, indicating substantial intrinsic receptor flexibility. VPX increases the free energy of active conformations and restricts TM helix 5 and 6 rearrangements, while VPX + Na+ eliminates intermediate basins and stabilizes the inactive state. In contrast, the tethered agonist stabilizes the active state as the global minimum and promotes TM6 displacement of up to ∼1 nm. The simulations reveal a continuous activation pathway linking extracellular ligand engagement to intracellular motifs, including the connector, DRF, intracellular loop 3 (ICL3), and KRK motifs, with intracellular chloride ion coordination contributing to inactive-state stabilization. Gαq binding reduces TM6 flexibility and shifts the free-energy minimum toward more active-like conformations. Alanine mutagenesis and Gq-CASE BRET assays validate predicted roles of key residues in activation and G-protein coupling. Together, these results provide a detailed understanding of ligand- and transducer-dependent PAR1 signaling and inform the design of pathway-selective PAR1 modulators.