Characterizing the structural, biophysical and functional effects of S-Glutathionylation on STIM1 Ca2+ sensing

Christian Michael Sirko, The University of Western Ontario

Abstract

Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca²⁺) sensing protein that initiates cytoplasmic Ca²⁺ influx via store-operated calcium entry (SOCE). STIM1, in conjunction with Orai, a plasma membrane (PM) protein, function as mediators of SOCE through the formation of calcium-release activated calcium (CRAC) channels. S-Glutathionylation of STIM1 at Cys56 has been shown to evoke constitutive Ca²⁺ entry in DT40 cells, however no studies have carefully investigated the biophysical and structural effects of this covalent modification to the luminal domain, which are critical for understanding the molecular mechanism underlying the regulation of STIM1 and SOCE by this modification. By establishing a protocol for site-specific STIM1 S-glutathionylation using reduced glutathione (GSH) and diamide, a thiol-specific oxidant, my data have revealed that modification of STIM1, at either Cys49 or Cys56, induces thermodynamic destabilization in the Ca²⁺ deplete state and structural changes as measured by solvent-exposed hydrophobicity and solution NMR. Furthermore, modification at Cys56 significantly reduces Ca²⁺ binding affinity, as measured via intrinsic fluorescence and far-UV CD spectroscopy. This weakening in Ca²⁺ binding is correlated with structural perturbations localized to common regions of the core EF-SAM domain, which include 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. Excitingly, I also designed a novel glutathione mimic at the residue level that strongly recapitulates the S-glutathionylation structural and biophysical effects within the STIM1 luminal domain and constitutively activates SOCE when introduced into full-length STIM1. Collectively, my research has revealed novel molecular insights into S-glutathionylation-mediated STIM1 structural and functional changes, laying the groundwork for the development of SOCE modulators to be used as research tools and/or therapeutic strategies for myriad Ca²⁺ signaled malignancies.