Sequential activation of STIM1 links Ca ²⁺ with luminal domain unfolding

Disease-associated mutations in the Ca2+ sensor STIM1 alter its conformation and disrupt Ca2+ ion binding. Keeping STIM1 unSTIMulated Upon ER Ca2+ depletion, the ER-localized Ca2+ sensor STIM1 activates the Orai family of plasma membrane–localized Ca2+ channels to replenish Ca2+ stores. Schober et al. described the conformational changes that occur during the initial steps of STIM1 activation. The authors used biochemical and electrophysiological analyses and molecular dynamics simulations to characterize constitutively active STIM1 mutants associated with tubular aggregate myopathy or cancer, as well as naturally occurring STIM1 variants. Their results indicated that although STIM1 was stabilized by the binding of a single Ca2+ ion, it could bind to multiple Ca2+ ions, a property that was disrupted by disease-associated mutations. Furthermore, these mutations caused STIM1 to adopt an unfolded conformation similar to that caused by Ca2+ depletion in wild-type STIM1. 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.