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.

基质相互作用分子 1 (STIM1) 有两个重要功能：内质网内的 Ca ²⁺感应和激活钙池操纵的 Ca ²⁺通道 Orai1，从而实现质膜 Ca ²⁺内流。我们将分子动力学 (MD) 模拟与活细胞记录相结合，并确定了激活后管腔 STIM1 结构域的顺序 Ca ²⁺依赖性构象。此外，我们还鉴定了规范和非规范 EF-hand 结构域内可以结合多个 Ca ²⁺离子的残基。在MD模拟中，单个Ca ²⁺离子足以稳定管腔STIM1复合物。 Ca ²⁺储存耗尽使两个 EF 手不稳定，引发它们与稳定 SAM 结构域一起形成的疏水裂缝的分解。与针对典型 EF 手的管状聚集性肌病或癌症相关的点突变，以及疏水性裂口产生组成型聚集的 STIM1，这与通过 Orai1 通道激活 Ca ²⁺进入相关。根据我们的结果，我们提出了 STIM1 Ca ²⁺结合模型，并在分子水平上完善了目前已知的 STIM1 激活的初始步骤。