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

Abstract

Store-operated Ca²⁺ entry (SOCE) is a major Ca²⁺ signaling pathway facilitating extracellular Ca²⁺ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca²⁺ stores. Stromal interaction molecule 1 (STIM1) is the Ca²⁺ sensor that activates SOCE following ER/SR Ca²⁺ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca²⁺ concentrations. Low ER Ca²⁺ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca²⁺ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys⁴⁹ and Cys⁵⁶ in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca²⁺ 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 S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys⁴⁹ and Cys⁵⁶S-nitrosylation 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 S-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.

Keywords: S-nitrosylation; calcium signaling; electrostatics; nitric oxide; nuclear magnetic resonance (NMR); store operated calcium entry; stromal interaction molecule 1 (STIM1); thermodynamics.