A charge-sensing region in the stromal interaction molecule 1 luminal domain confers stabilization-mediated inhibition of SOCE in response to -nitrosylation.

Store-operated Ca entry (SOCE) is a major Ca signaling pathway facilitating extracellular Ca influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca stores. Stromal interaction molecule 1 (STIM1) is the Ca sensor that activates SOCE following ER/SR Ca depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca concentrations. Low ER Ca levels trigger STIM1 destabilization and oligomerization， culminating in the opening of Orai1-composed Ca channels on the plasma membrane. NO-mediated nitrosylation of cysteine thiols regulates myriad protein functions， but its effects on the structural mechanisms that regulate SOCE are unclear. Here， we demonstrate that nitrosylation of Cys and Cys in STIM1 enhances the thermodynamic stability of its luminal domain， resulting in suppressed hydrophobic exposure and diminished Ca depletion-dependent oligomerization. Using solution NMR spectroscopy， we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the nitrosylated nonconserved region of STIM1. Finally， using live cells， we found that the enhanced luminal domain stability conferred by either Cys and Cysnitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively， our results suggest that nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core， which modulates the thermodynamic stability of the STIM1 luminal domain.