The inhibitory helix controls the intramolecular conformational switching of the C-terminus of STIM1

Abstract

Store-operated Ca(2+) entry (SOCE) is a critical Ca(2+) signaling pathway in many cell types. After sensing Ca(2+) store depletion in the endoplasmic reticulum (ER) lumen, STIM1 (STromal Interaction Molecule 1) oligomerizes and then interacts with and activates the Orai1 calcium channel. Our previous research has demonstrated that the inhibitory helix (IH) adjacent to the first coiled-coil region (CC1) of STIM1 may keep the whole C-terminus of STIM1 in an inactive state. However, the specific conformational change of CC1-IH that drives the transition of STIM1 from the resting state to the active state remains elusive. Herein, we report the structural analysis of CC1-IH, which revealed that the entire CC1-IH molecule forms a very long helix. Structural and biochemical analyses indicated that IH, and not the CC1 region, contributes to the oligomerization of STIM1. Small-angle X-ray scattering (SAXS) analysis suggested that the C-terminus of STIM1 including the IH region displays a collapsed conformation, whereas the construct without the IH region has an extended conformation. These two conformations may correspond to the conformational states of the C-terminus of STIM1 before and after activation. Taken together, our results provide direct biochemical evidence that the IH region controls the conformational switching of the C-terminus of STIM1.

Figure 1. Structure of CC1-IH.

(A) Schematic layout of the human STIM1 molecule with its coiled-coil regions CC1, IH and SOAR shown in cyan, dark blue and pink, respectively. Below the schematic are the various constructs utilized in our research. (B) Left, cartoon of the asymmetric unit, which contains four CC1-IH molecules. In the crystal structure, each CC1-IH molecule forms an elongated α-helix from its N-terminus to its C-terminus. Right, 90° rotation of the image at left. (C) Sequence alignment of CC1-IH (amino acids 237-340) from Homo sapiens, Mus musculus, Sus scrofa, Bos taurus, Gallus gallus, Xenopus laevis and Danio rerio. Residues that are conserved in all species are highlighted by red boxes; residues that are conserved in all species but one or two are in red. The residue numbers shown in black are based on the human STIM1 sequence.

Figure 2. Oligomeric state of CC1-IH in solution.

(A) Sedimentation velocity analytical ultracentrifugation analysis of wild-type CC1-IH (residues 237-340) and CC1 with the inhibitory helix removed (residues 237-310). The curves shown in blue and orange represent wild-type CC1-IH and CC1, respectively, and their calculated molecular weights are marked in blue and orange, respectively. (B) Sedimentation velocity of MBP-tagged IH (residues 310-340). (C) A CC1-IH dimer mediated by each inhibitory helix at the extreme C-terminus of each monomer. (D, E) Detailed view of the interactions within the interface of the CC1-IH dimer. Their interactions are not fully described here because the two helices interacted to form the dimer in a reciprocal manner. Residues are colored based on atom type: carbon is shown in green/cyan (based on the corresponding monomer), oxygen is red, and nitrogen is blue.

Figure 3. The IH domain keeps SOAR inactive.

(A) Molecular layout of the simulated inactive and active fragments used in the SAXS analysis and the corresponding fragments used in the intracellular calcium measurement experiments. (B) Fura-2 Ca²⁺ measurements of HeLa cells expressing Orai1 alone, Orai1 plus STIM1-N-CIS, Orai1 plus STIM1-N-CIS-DelIH and Orai1 plus STIM1-FL. (C) Quantitative analysis of Ca²⁺ fluorescence at different stages.

Figure 4. Different conformational states of STIM1-Ccyto in solution as revealed by SAXS.

(A, B) Oligomeric states of STIM1-CIS (A) and STIM1-CIS-DelIH (B) in solution determined using multi-angle laser scattering. (C, D) Experimental scattering curves for STIM1-CIS (C) and STIM1-CIS-DelIH (D) in solution: 1, experimental SAXS curve; 2, scattering patterns calculated from the DAMMIN model; 3, smooth curve back transformed from p(r) and extrapolated to a zero scattering angle for STIM1-CIS. Upper-right, the distance distribution function p(r) computed using the program GNOM. (E) Hypothetical molecular shape of STIM1-CIS in solution. STIM1-CIS may adopt a collapsed conformation, which represents the resting state of STIM1-Ccyto. The SOAR and CC1-IH dimers are shown in light pink and cyan, respectively. (F) Superimposition of an STIM1-CIS bead model (shown as white dots) developed based on the SAXS data and the known SOAR structure (α-helices shown in light pink). (G) Hypothetical molecular shape of STIM1-CIS-DelIH in solution. STIM1-CIS-DelIH may adopt a stretched and elongated conformation when IH is removed (or when IH releases the SOAR dimer), which represents the activated state of STIM1-Ccyto. The SOAR and CC1-IH dimers are shown in light pink and cyan, respectively. (H) Superimposition of the STIM1-CIS-DelIH bead model (shown as white dots) developed based on the SAXS data and the known SOAR and CC1 structures (α-helices shown in light pink and cyan, respectively).