Sequential activation of STIM1 links Ca²⁺ with luminal domain unfolding

The stromal interaction molecule 1 (STIM1) has two important functions, Ca²⁺ sensing within the endoplasmic reticulum and activation of the store-operated Ca²⁺ channel Orai1, enabling plasma-membrane Ca²⁺ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca²⁺-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca²⁺ ions. In MD simulations, a single Ca²⁺ ion was sufficient to stabilize the luminal STIM1 complex. Ca²⁺ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca²⁺ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca²⁺ binding and refine the currently known initial steps of STIM1 activation on a molecular level.