An S-glutathiomimetic Provides Structural Insights into Stromal Interaction Molecule-1 Regulation |
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Affiliation: | Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A5C1, Canada |
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Abstract: | 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. |
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Keywords: | stromal interaction molecule-1 store-operated calcium entry structural biology AM" },{" #name" :" keyword" ," $" :{" id" :" k0045" }," $$" :[{" #name" :" text" ," _" :" acetoxymethyl ester ANS" },{" #name" :" keyword" ," $" :{" id" :" k0055" }," $$" :[{" #name" :" text" ," _" :" 8-anilinonapthalene-1-sulfonic acid C" },{" #name" :" keyword" ," $" :{" id" :" k0065" }," $$" :[{" #name" :" text" ," _" :" carboxyl terminus ionized calcium CC" },{" #name" :" keyword" ," $" :{" id" :" k0085" }," $$" :[{" #name" :" text" ," _" :" coiled coil CD" },{" #name" :" keyword" ," $" :{" id" :" k0095" }," $$" :[{" #name" :" text" ," _" :" circular dichroism midpoint of chemical denaturation CRAC" },{" #name" :" keyword" ," $" :{" id" :" k0115" }," $$" :[{" #name" :" text" ," _" :" calcium release-activated calcium CSP" },{" #name" :" keyword" ," $" :{" id" :" k0125" }," $$" :[{" #name" :" text" ," _" :" chemical shift perturbation DMEM" },{" #name" :" keyword" ," $" :{" id" :" k0135" }," 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