Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1)

Abstract

Calcium influx through the Ca(2+) release-activated Ca(2+) (CRAC) channel is an essential process in many types of cells. Upon store depletion, the calcium sensor in the endoplasmic reticulum, STIM1, activates Orai1, a CRAC channel in the plasma membrane. We have determined the structures of SOAR from Homo sapiens (hSOAR), which is part of STIM1 and is capable of constitutively activating Orai1, and the entire coiled coil region of STIM1 from Caenorhabditis elegans (ceSTIM1-CCR) in an inactive state. Our studies reveal that the formation of a SOAR dimer is necessary to activate the Orai1 channel. Mutations that disrupt SOAR dimerization or remove the cluster of positive residues abolish STIM1 activation of Orai1. We identified a possible inhibitory helix within the structure of ceSTIM1-CCR that tightly interacts with SOAR. Functional studies suggest that the inhibitory helix may keep the C-terminus of STIM1 in an inactive state. Our data allowed us to propose a model for STIM1 activation.

Fig. 1.

SOAR dimer. (A) Schematic drawing of hSOAR. (B, C) Cartoon representation of the overall structure of the hSOAR monomer and the red-green hSOAR dimer. (D) Stereoview of the detailed interactions between the two monomers. The oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are shown by magenta dotted lines. (E) Western blots of coimmunoprecipitated wild-type and mutant hSOAR with the hOrai1 channel. (F) Localization of wild-type and mutant hSTIM1 transfected alone (first image in each row) and cotransfected with hOrai1. All hSTIM1 constructs were C-terminally tagged with EGFP and all hOrai1 constructs were N-terminally tagged with mCherry. Scale bars are 10 μm. (G) Fura-2 Ca²⁺ measurements of HeLa cells expressing hOrai1 alone or hOrai1 with wild-type or mutant hSTIM1. Inset, immunoblotting with anti-STIM1 antibody.

Fig. 2.

Cluster of positive residues in hSOAR. (A) Electrostatic surface representation of the hSOAR dimer. The surface is colored as follows: negative, red; positive, blue; and neutral, white. The two monomers in the dimer are related by twofold noncrystallographic symmetry. For example, the Arg387 residue is located on the back of the left monomer and thus labeled on the surface of the right monomer. Two lysine residues (Lys384 and Lys385) are only displayed on the surface of the left monomer. (B) Western blots of hSOAR coimmunoprecipitated with the hOrai1 channel. (C) Localization of wild-type and mutant hSTIM1 tagged with EGFP transfected alone (first image in each row) and cotransfected with hOrai1 tagged with mCherry. Scale bars are 10 μm. (D) Fura-2 Ca²⁺ measurements of HeLa cells expressing hOrai1 alone or hOrai1 with wild-type or mutant hSTIM1. Inset, immunoblotting with anti-STIM1 antibody.

Fig. 3.

Structure of ceSTIM1-CCR. (A) Schematic drawing of STIM1 from C. elegans. The ceSTIM1-CCR construct is shown in red. Overall structure of the ceSTIM1-CCR monomer (B) and dimer (C). Helix αA is named the inhibitory helix in this paper. (D) Stereoview of the detailed interactions between αA (green) and ceSOAR (helices αB and αC, magenta). The oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are shown by orange dotted lines.

Fig. 4.

The inhibitory helix maintains hSTIM1 in an inactive state. (A) Schematic drawing of wild-type and mutant STIM1 from H. sapiens. (B) Localization of hSTIM1 mutants transfected alone (first image in each row) and cotransfected with hOrai1 without TG treatment. Scale bars are 10 μm. (C) Fura-2 Ca²⁺ measurements of HeLa cells expressing hOrai1 alone or hOrai1 with wild-type or mutant hSTIM1. Inset, immunoblotting with anti-STIM1 antibody.

Fig. 5.

Model of STIM1 activation. In the resting state, the STIM1 molecule mainly exists as a dimer. The SOAR dimer is likely occluded by the inhibitory helices and the region consisting of amino acids 486–685. Calcium depletion from the ER store induces dimerization or oligomerization of STIM1-N, causing conformational changes of inhibitory helices and thus release of dimeric SOAR. The STIM1 dimer in the active state may directly couple to and activate the Orai1 channel on the PM. Alternatively, STIM1 molecules may oligomerize due to aggregation of STIM1-N and then activate the Orai1 channel.