An S-glutathiomimetic Provides Structural Insights into Stromal Interaction Molecule-1 Regulation

Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca²⁺) sensing protein that regulates store-operated calcium entry (SOCE). In SOCE, STIM1 activates Orai1-composed Ca²⁺ channels in the plasma membrane (PM) after ER stored Ca²⁺ depletion. S-Glutathionylation of STIM1 at Cys56 evokes constitutive SOCE in DT40 cells; however, the structural and biophysical mechanisms underlying the regulation of STIM1 by this modification are poorly defined. By establishing a protocol for site-specific STIM1 S-glutathionylation using reduced glutathione and diamide, we have revealed that modification of STIM1 at either Cys49 or Cys56 induces thermodynamic destabilization and conformational changes that result in increased solvent-exposed hydrophobicity. Further, S-glutathionylation or point-mutation of Cys56 reduces Ca²⁺ binding affinity, as measured by intrinsic fluorescence and far-UV circular dichroism spectroscopies. Solution NMR showed S-glutathionylated-induced perturbations in STIM1 are localized to the α1 helix of the canonical EF-hand, the α3 and α4 helices of the non-canonical EF-hand and α6 and α8 helices of the SAM domain. Finally, we designed an S-glutathiomimetic mutation that strongly recapitulates the structural, biophysical and functional effects within the STIM1 luminal domain and we envision to be another tool for understanding the effects of protein S-glutathionylation in vitro, in cellulo and in vivo.