Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

In resting cells, STIM1, the dimeric 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 FRET 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 CC11 (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 stabilizes an intermediate 3-helix bundle structure arising from helical interactions of CC12 and CC13 with CC11, thereby releasing the CC1 clamp and allowing the CAD to escape through a fold-out mechanism, with subsequent formation of the CC1 coiled-coil enabling the CAD to revert to its original shape to activate Orai1 in vivo.