Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

CERM University of Florence 50019 Sesto Fiorentino Italy

Department of Biomaterials Institute of Clinical Dentistry University of Oslo 0455 Oslo Norway

Department of Chemistry Faculty of Science University of South Bohemia Branišovská 1645 31A 370 05 České Budějovice Czech Republic

Department of Chemistry Ugo Schiff University of Florence 50019 Sesto Fiorentino Italy

Department of Medical Genetics Oslo University Hospital and University of Oslo 0450 Oslo Norway

Faculty of Veterinary Medicine Norwegian University of Life Sciences 1430 Ås Norway

Institut für Analytische Chemie University of Vienna Währinger Straße 38 1090 Wien Austria

Institute for Experimental Medical Research Oslo University Hospital and University of Oslo 0450 Oslo Norway

Institute of Biochemistry Johannes Kepler University Linz Altenbergerstrasse 69 4040 Linz Austria

Institute of Biophysics Johannes Kepler University Linz Gruberstrasse 40 4020 Linz Austria

Institute of Organic Chemistry and Institute of Inorganic Chemistry Johannes Kepler University Linz Altenbergerstrasse 69 4040 Linz Austria

Institute of Theoretical Physics Johannes Kepler University Linz Altenbergerstrasse 69 4040 Linz Austria

Magnetic Resonance Center University of Florence and Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine 50019 Sesto Fiorentino Italy

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