Structural rearrangements underlying the activation of STIM1 by ER calcium depletion.

In resting cells, STIM1, the dimeric endoplasmic reticulum (ER) Ca2+ sensor that controls store-operated Ca2+ entry (SOCE), is held in a Ca2+-bound inactive state by multiple intramolecular restraints, or brakes. Receptor-evoked release of Ca2+ from the ER causes a large conformational change in STIM1 that releases the brakes and exposes the CRAC activation domain (CAD), enabling it to bind and open store-operated Orai1 channels in the plasma membrane. We performed single-molecule Förster resonance energy transfer (smFRET) measurements with purified STIM1 to better understand how Ca2+ release from the luminal domain of STIM1 drives the conformational changes in the cytosolic domain that underlie CAD release. We find that Ca2+ removal releases the CAD from CC1α1 (the "CC1 clamp") without obligatory formation of the CC1 coiled-coil that has been associated with CAD release in cells. Surprisingly, the CAD rearranges dramatically during release, as the two hairpin protomers that create its characteristic V-shaped structure are spread apart. Locking the two protomers together by cysteine crosslinking prevents CAD release, suggesting that the CAD must rearrange to escape the CC1 clamp. Our data support a model in which ER depletion-induced dimerization of the luminal SAM domains drives the cytosolic domain into multiple intermediate states including a 3-helix bundle of CC1α1/2/3, releasing the CC1 clamp and allowing the CAD to escape through a "fold-out" mechanism. Subsequent formation of the CC1 coiled-coil enables the CAD to revert to its original shape and extends it toward the plasma membrane to activate Orai1.