Abstract
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1–CC3 clamp strength to control the activation of STIM1.
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Data availability
The 20 lowest total energy structures and the NOE constraints of WT CC1 were deposited at the PDB database under accession code 6YEL. The chemical shifts assignments of the WT STIM1 CC1 and its Stormorken mutant were deposited in the BMRB database under IDs 50114 and 50118, respectively. All other relevant data are available in this article and its Supplementary Information files, or from the corresponding authors upon reasonable request.
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Acknowledgements
This work has been supported by iNEXT, grant number 653706, funded by the Horizon 2020 program of the European Commission and by the Austrian Science Fund (FWF) PhD program W1250 ‘NanoCell’, P32947 to M. Fahrner as well as P27263 to C.R., by the Fondazione Cassa di Risparmio di Firenze and the Italian Ministero dell’Istruzione, dell’Università e della Ricerca through the ‘Progetto Dipartimenti di Eccellenza 2018-2022’ to the Department of Chemistry ‘Ugo Schiff’ of the University of Florence. L.C., M. Fragai, C.L. and E.R. also acknowledge the support of the University of Florence and the Recombinant Proteins JOYNLAB. Instruct-ERIC, a Landmark ESFRI project and specifically the CERM/CIRMMP Italy Center is also acknowledged. The NMR experiments were performed at the NMR laboratory of the Upper Austrian—South Bohemian Research Infrastructure Center in Linz, ‘RERI-uasb’, supported by the European Union through the ERDF INTERREG IV (RU2-EU-124/100–2010) program (ETC Austria-Czech Republic 2007–2013, project M00146, ‘RERI-uasb’ for N.M.) and at the CERM institute in Sesto Fiorentino (iNEXT NMR HEDC infrastructure, PID 6162 and 3428). Inspiring discussions with members of the European Cooperation in Science and Technology Action CA15209 EURELAX are acknowledged.
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C.L., C.R. and N.M. conceived and oversaw the study. P.R., M. Fahrner, H.G., C.R. and N.M. wrote the manuscript. M. Fahrner carried out molecular biology experiments. P.R., L.C., A.G., E.R., M. Fragai and M.B. conducted and analyzed NMR experiments. H.G. performed and analyzed electrophysiological as well as fluorescence microscopy experiments. F.H., H.K. and T.R. contributed to writing and mechanistic concepts of protein function. All authors reviewed and approved the final version of the manuscript.
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Extended data
Extended Data Fig. 1 CC1 crystallographic structure and corresponding patch‐ clamp‐data.
a, Top: Comparison of the published crystallographic structure fragment (PDB: 4O9B, blue) to the STIM1 CC1 NMR model. The positions of the residues mutated in the X‐ray structure are labelled. The first five N‐terminal residues (G229‐F233) of the recombinant fragment, the remainder of the thrombin cleavage sequence, are not part of the native sequence. Bottom: Partial sequence alignment of the fragments used for NMR (top) and crystallography (bottom) with mutations indicated by red boxes. The molecular graphics were created by PyMOL (v. 2.3, Schrödinger, LLC). b, Depiction of a patch clamp experiment using the whole cell configuration. The entry of Ca2+ ions through Orai1 channels generates an electrical current that is registered by an Ag/AgCl electrode. This electrode is inserted into a glass pipette that is sealed to the plasma membrane of a target cell. c, Orai1 current activation shown by patch clamp recordings of N‐terminally tagged CFP‐Orai1 co‐expressed with YFP‐STIM1 M244L + L321M (pink). HEK293 cells were exclusively used for all recordings. The patch clamp experiment was replicated on two different days using independent transfections with the indicated number of cells (n). Data represent mean values ± SEM.
Extended Data Fig. 2 The solution NMR structure of STIM1 CC1.
Side a and top b views of the solution structure of STIM1 CC1. Blue color represents residues that are in close NOE contact with the SDS detergent (the residues are listed in the table insert). The NMR experiments were recorded in presence of 7.0 mM SDS to avoid non‐specific CC1 homo‐oligomerization occurring in absence of the detergent. We note that at this concentration below the CMC (critical micelle concentration) of 8.2 mM (25 °C), SDS does not cause any secondary structure changes, as proven by CD spectra (Supplementary Fig. 1). The intermolecular NOEs observed between the surface‐exposed residues of STIM1 CC1 and SDS molecules are consistent with protection of the highlighted (blue) residues listed in the table insert by the detergent, thus preventing CC1 homo‐oligomerization while leaving intramolecular coiled‐coil contacts intact.
Extended Data Fig. 3 Spectra of STIM1 CC1 wild‐type and Stormorken mutant.
Assigned 700 MHz 1H‐15N HSQC spectra of 0.3 mM 15N‐STIM1 CC1 wild‐type a and Stormorken mutant b.
Extended Data Fig. 4 Secondary structure prediction of STIM1 CC1 wild‐type and Stormorken mutant.
Secondary structure prediction for STIM1 CC1 wild‐type a and Stormorken mutant b from Talos‐N36. Green lines represent the order parameter S2 predicted from the chemical shifts. Red bars indicate the probability (in %*100) for residue to adapt a helical secondary structure.
Extended Data Fig. 5 STIM1 homomerization and Orai1 activation by STIM1 mutants.
a, STIM1 homomerization experiments of N‐terminally tagged CFP‐ and YFP‐STIM1 I290S + A293S ± R304W. Ca2+ store depletion was induced by perfusion with 1 μM thapsigargin in Ca2+ free solution. b, Orai1 current activation shown by patch clamp recordings of N‐terminally tagged CFP‐Orai1 co‐ expressed with YFP‐STIM1 L251S ± I290S + A293S. Color code: WT (black), L251S (purple), R304W (red), L251S + I290S + A293S (gray), I290S + A293S + R304W (blue), and I290S + A293S (magenta). HEK293 cells were exclusively used for all experiments. Experiments were replicated on at least two different days using independent transfections with the indicated number of cells (n). Data represent mean values ± SEM.
Extended Data Fig. 6 Graphical representation of NOE distance and hydrogen bond restraints.
Graphical representation of NOE distance and hydrogen bond restraints used for the structure calculation. The parallel closely spaced lines indicate intra‐helical (i to i+4) restraints from CS‐Rosetta31. The lines crossing each other near the center are characteristic of anti‐parallel alignment of helices. The secondary structure ranges are indicated, as well as the non‐native residues (G1‐F5), by color coding corresponding to the Figures in the main text.
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Rathner, P., Fahrner, M., Cerofolini, L. et al. Interhelical interactions within the STIM1 CC1 domain modulate CRAC channel activation. Nat Chem Biol 17, 196–204 (2021). https://doi.org/10.1038/s41589-020-00672-8
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DOI: https://doi.org/10.1038/s41589-020-00672-8
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