Domain interactions controlling STIM1 activation of Orai1 channels

Store-operated Ca2+ entry, essential for the adaptive immune system, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain and STIM1/Orai1 co- clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of domain interactions in controlling STIM1 quiescent as well as active conformation, and cluster formation. New homo- and hetero-meric interactions will be identified focusing on the first a-helix of coiled-coil 1 (CC1 a1) and the CC3 domains. While the heteromeric CC1 a1-CC3 interaction may lock STIM1 in a tightly-packed, quiescent conformation, CC1 a1 homomerization induced via ER store depletion seems to uncouple this CC1 a1-CC3 interaction. This in turn leads to the extended STIM1 C-terminal conformation where CAD/SOAR exposure mediates cluster formation via CC3 assembly. Residues contributing to this higher-order CC3 oligomerization will be additionally identified. Our mechanistic analysis will be extended to resolve the molecular mechanism of a disease-related CC1 point mutation associated with the Stormorken syndrome. In the end, we aim at unravelling the key STIM1 activation steps that control both the exposure and cluster formation of the CAD/SOAR domain essential for the coupling to and activation of Orai1 channels.

Store-operated Ca2 entry, essential for the adaptive immune system, is initiated by the endoplasmic reticulum (ER) Ca2 sensor STIM1. Ca2 entry occurs through the plasma membrane resident Ca2 channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2 store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of domain interactions in controlling STIM1 quiescent as well as active conformation, and cluster formation. New homo- and hetero-meric interactions have been identified focusing on the first a-helix of coiled-coil 1 (CC1 a1) and the CC3 domains. While the heteromeric CC1 a1-CC3 interaction locks STIM1 in a tightly-packed, quiescent conformation, CC1 a1 homomerization induced via ER store depletion uncouples this CC1 a1-CC3 interaction. This in turn leads to the extended STIM1 C-terminal conformation where CAD/SOAR exposure mediates cluster formation via CC3 assembly. Our mechanistic analysis has been extended to resolve the molecular mechanism of a disease-related CC1 point mutation associated with the Stormorken syndrome providing a novel mechanism for the CC1 domain controlling CAD/SOAR exposure. In the end, we have unravelled the key STIM1 activation steps that control both the exposure and cluster formation of the CAD/SOAR domain essential for the coupling to and activation of Orai1 channels.