The 2β Insert Perturbs Folding, Stability and Hydrophobic Exposure of Stromal Interaction Molecules

Stromal interaction molecule (STIM)1 and 2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels through oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil domains couple to PM Orai1 protein subunits to form Ca2+ released activated Ca2+ (CRAC) channels that facilitate store-operated Ca2+ entry (SOCE). One splice variant of STIM2, STIM2β, contains an extra 8-residue (2β insert) located within the coiled-coils and inhibits SOCE through an unresolved mechanism, adding another layer of complexity to Ca2+ regulation in mammals. I hypothesize that the 2β insert perturbs the coiled-coil conformation and dynamics commensurate with an ability to activate SOCE. My data show the 2β insertion induced an overall reduction in α-helicity, thermal stability, and promoted a conformation of greater exposed hydrophobicity which affected oligomerization. Previous studies show STIM2 more weakly couples to Orai1 compared to STIM1, and STIM2β is completely incapable of binding to Orai1. My functional studies show the 2β insertion in the STIM1 context also significantly inhibits SOCE. Therefore, my data suggests that the 2β insert inhibits STIM function and SOCE through a mechanism which perturbs α-helical levels and destabilizes the cytosolic domain coiled-coil structure, independent of the requirement for weaker STIM2 coupling to Orai1