Conserved and species-specific STIM autoinhibition revealed by AlphaFold3-guided dissection

Store-operated Calcium (Ca ²⁺ ) entry (SOCE), mediated by dynamic STIM–Orai coupling, is crucial for cellular signaling, yet the molecular architecture of STIM autoinhibition remains poorly defined. Using an AlphaFold3-guided functional validation strategy combining FRET-based biosensors, mutagenesis, and Ca ²⁺ imaging, we systematically dissected the CC1–SOAR autoinhibitory interface in both human STIM1 and C. elegans STIM (cSTIM). Our results reveal a conserved hydrophobic core essential for maintaining the resting state in both species. Interestingly, cSTIM possesses an extended polar interaction network that strengthens autoinhibition and correlates with slower activation kinetics. Species-specific polar networks differentially modulate interface stability, providing an additional layer of regulatory tuning. This integrated approach not only validates AlphaFold3 as a powerful tool for studying dynamic regulatory interfaces but also establishes a structural framework for understanding STIM activation and disease-associated mutations, offering mechanistic insights with therapeutic implications for SOCE-related disorders.