A Molecular Switch Governing Piezo1 Gating beyond Symmetry Breaking

A single substituent can flip a ligand from an agonist to an inhibitor, yet the underlying conformational dynamics remain elusive. Here, multiscale simulations calibrated against Yoda1 binding data resolve the ligand-dependent gating free-energy landscapes of the mechanosensitive channel Piezo1. In the unliganded state, the free-energy landscape supports a gating mechanism involving axial pore dislocation, lateral dilation, and realignment. The agonist Yoda1 stabilizes a widely dilated pore with persistent axial asymmetry, whereas the inhibitor Dooku1 arrests pore expansion after initial dislocation. This divergence reveals a geometrically selective relay between the intracellular Beam and transmembrane helical unit 9 (THU9). Productive coupling to pore dilation, rather than Beam rotation alone, dictates the pharmacological response. Guided by this mechanism, we designed Yoda1 analogs bearing an amino or nitro substitution at the same position, whose free-energy landscapes predicted contrasting functions. Intracellular calcium fluorescence assays were consistent with these predictions: the amino-substituted analog 188 elevated intracellular calcium levels, whereas the nitro-substituted analog 187 attenuated the Yoda1-evoked calcium response. These findings link single-site chemical modifications to divergent gating outcomes via symmetry breaking and pore dilation, identifying productive conformational coupling as a key molecular design parameter alongside binding affinity.