Abstract
The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it toward the plasma membrane to activate Orai and SOCE after store depletion.
Highlights
Store-operated Ca2+ entry (SOCE) is a nearly ubiquitous signaling pathway activated by 38 extracellular stimuli that deplete Ca2+ from the endoplasmic reticulum (ER)
In studies of full-length STIM1 in intact cells, cysteine crosslinking after store depletion suggests that after unbinding from CRAC activation domain (CAD), the two coiled-coil 1 (CC1) domains pack together along their entire length, in contrast to the CC1 crystal and CC1α3-CC2 NMR structures. These findings offer the first structural view of the STIM1 inhibitory clamp and reveal the massive conformational changes evoked by store depletion, which serve to reorient and translocate CAD towards the PM where it can bind Orai1 and activate SOCE
The CC2 domains in ctSTIM1 are oriented in a parallel configuration We initially applied single-molecule Förster resonance energy transfer (smFRET) to test whether the conformation of CAD within the cytosolic domain resembles that of the CC2-CC3 crystal structure (Yang et al, 2012) or the CC1α3-CC2 NMR structure (Stathopulos et al, 2013), referred to below as the ‘crystal structure’ or ‘NMR structure’ respectively
Summary
Store-operated Ca2+ entry (SOCE) is a nearly ubiquitous signaling pathway activated by 38 extracellular stimuli that deplete Ca2+ from the endoplasmic reticulum (ER). Loss-of-function mutations in the core SOCE components lead to serious human pathologies such as severe combined immunodeficiency, autoimmunity, myopathy, ectodermal dysplasia and anhidrosis (Lacruz and Feske, 2015), while gain-of-function mutations cause Stormorken syndrome, miosis, myopathy, thrombocytopenia and excessive bleeding (Böhm and Laporte, 2018). These clinical manifestations underscore the need for precise regulation of SOCE to ensure that it is silent when 46 ER Ca2+ stores are full yet reliably activated by stimuli that drive store depletion. Ca2+ bound to the luminal EF hands of the STIM1 dimer suppresses STIM1 activity by promoting intramolecular association of the coiled-coil 1 (CC1)
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