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.
摘要：
基质相互作用分子1（STIM1）是一种内/肌浆网（ER/SR）钙（Ca2）感应蛋白，可调节储存操作的钙进入（SOCE）。在SOCE,在ER存储的Ca2耗尽后，STIM1激活质膜（PM）中由Orai1组成的Ca2通道。STIM1在Cys56处的S-谷胱甘肽酰化在DT40细胞中引起组成型SOCE；然而，通过这种修饰调节STIM1的基础结构和生物物理机制尚不明确.通过建立使用还原型谷胱甘肽和二酰胺进行位点特异性STIM1S-谷胱甘肽酰化的方案，我们已经揭示了STIM1在Cys49或Cys56处的修饰会引起热力学不稳定和构象变化，从而导致溶剂暴露的疏水性增加。Further,Cys56的S-谷胱甘肽酰化或点突变降低了Ca2+结合亲和力，通过固有荧光和远紫外圆二色性光谱测量。溶液NMR显示STIM1中S-谷胱甘肽化诱导的扰动位于标准EF手的α1螺旋上，非规范EF-手的α3和α4螺旋以及SAM结构域的α6和α8螺旋。最后,我们设计了一种S-谷胱甘肽模拟突变，STIM1管腔结构域内的生物物理和功能效应，我们设想成为另一种理解蛋白S-谷胱甘肽酰化体外效应的工具，在细胞和体内。
