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.

BioTechMed Graz A 8010 Graz Austria

Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic Nove Hrady CZ 373 33 Czech Republic

College of Biomedical Sciences Larkin University Miami FL 33169 USA

Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London Ontario N6A 5C1 Canada

Faculty of Medicine University of Ottawa Ottawa Ontario K1H 8M5 Canada

Gottfried Schatz Research Center Medical University of Graz A 8010 Graz Austria

Institute of Biophysics JKU Life Science Center Johannes Kepler University Linz A 4020 Linz Austria

Schulich Dentistry Schulich School of Medicine and Dentistry University of Western Ontario London Ontario N6A 5C1 Canada

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