Abstact

The 5-HT(1F) receptor is a serotonin receptor subtype highly expressed in trigeminal sensory neurons, where it modulates neuropeptide release and nociceptive signaling without inducing vasoconstriction. This makes it an important therapeutic target for migraine. LY334370 was developed as a first-generation selective 5-HT(1F)R agonist and demonstrated efficacy in clinical studies. However, the molecular mechanism underlying 5-HT(1F)R activation by LY334370 remains poorly understood. Here, we determined a 3.13 A cryo-EM structure of the LY334370-bound 5-HT(1F)R-miniGalpha(oA) complex. Combined with functional analyses, this structure delineates the molecular determinants underlying LY334370 recognition. Comparison with BRL54443 indicates that LY334370 selectivity for 5-HT(1F)R is driven by its optimal accommodation within the receptor-specific extended binding pocket. Furthermore, comparative analysis with the lasmiditan-bound 5-HT(1F)R-Galpha(i1) complex reveals distinct agonist binding modes and provides mechanistic insight into Galpha(i/o) subtype-specific coupling. Collectively, these findings elucidate the structural basis of 5-HT(1F)R activation, ligand selectivity, and G protein coupling, providing a structural framework for the rational design of safer and more effective anti-migraine drugs.