STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation

Abstract

Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca(2+) release-activated Ca(2+) (CRAC) current having an important role in T-cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca(2+) stores are depleted. Here we show, utilizing a novel STIM1-derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C-terminal, intramolecular transition into an extended conformation when activating ORAI1. The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled-coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

Figure 1

Designing a STIM1-derived conformational sensor. (A) Block diagram summarizing intermolecular/intramolecular NFRET of double-labelled YFP–STIM1–CFP fragments: 233–420, 233–430, 233–450, 233–474 (OASF), 233–485, 233–535 and 233–685 (complete STIM1 C-terminus). (B) Time course of constitutive whole-cell inward currents at −86 mV of HEK293 cells expressing YFP–OASF–CFP with ORAI1 (upper panel) and respective I/V curve taken at t=0 s (lower panel). (C) Localization and calculated NFRET life cell image series of selected STIM1 fragments: 233–420, 233–474 (OASF), 233–535 and 233–685. Calibration bar is 5 μm throughout. (D) The YFP–OASF–CFP FRET sensor detected by western blot with an anti-GFP antibody when expressed in HEK293 cells. (E) A cartoon indicating the intramolecular/intermolecular FRET of OASF labelled either with YFP/CFP (left) or CFP/CFP, YFP/YFP (right). Block diagram comparing intermolecular with intramolecular NFRET of double-labelled STIM1 OASF fragments as depicted in the upper panel.

Figure 2

OASF sensor coupling to ORAI1. (Right panel) Localization and calculated NFRET life cell image series of HEK293 cells expressing YFP–OASF–CFP and (A) ORAI1, (B) ORAI1_E106Q (C) ORAI1_L273S. Calibration bar is 5 μm throughout. Magnified section as indicated by the white box highlights the decrease of FRET in regions of the plasma membrane compared with the cytosol in (A, B). (Left panel) Respective block diagram of separately calculated NFRET for regions including the plasma membrane (within the two yellow borders) and the cytosol (within white border). The ‘plasma membrane’ was assumed as 1.5–2 μm of the edge of the cell image.

Figure 3

Engineering OASF head-to-tail proximity by mutations. (A, B) Localization and calculated NFRET life cell image series of YFP–OASF–CFP wild-type and mutants without (A) or with (B) ORAI1 co-expressed. Calibration bar is 5 μm throughout. (C) Block diagram summarizing NFRET of double-labelled OASF mutants: YFP–OASF–CFP (wild type), YFP–OASF L251S–CFP, YFP–OASF A376K–CFP, YFP–OASF L416S L423S–CFP and YFP–OASF R426L–CFP. (D) Intensity plots representing localization of YFP–OASF–CFP wild-type and mutants without (upper panel) and with (lower panel) ORAI1 in regions close to the plasma membrane as indicated by the dashed line.

Figure 4

Dimerization/oligomerization and conformation of OASF and mutants. (A) Block diagram summarizing intermolecular NFRET between double-labelled CFP/CFP and YFP/YFP OASF forms: OASF (wild type), OASF L251S, OASF L416S L423S and OASF R426L. (B) Purity and far-UV CD spectra of OASF-ext (aa 234–491) mutant and wild-type forms. Spectra were acquired at 20°C in 20 mM Tris, 200 mM NaCl, 2 mM DTT, pH 8 using protein concentrations ranging from 0.14 to 0.35 mg ml⁻¹. Protein purity was confirmed using Coomassie-stained SDS–PAGE (inset). (C) SEC with in-line MALS analyses of OASF-ext (aa 234–491) mutant and wild-type forms. SEC experiments were performed at 4°C in 20 mM Tris, 100 mM NaCl, 50 mM L-Arg/L-Glu, 2 mM DTT, pH 8 using 0.85–2.0 mg ml⁻¹ protein.

Figure 5

OASF sensor coupling to ORAI1–R91W. (Left panel) Localization and calculated NFRET life cell image series of HEK293 cells expressing ORAI1_R91W and (A) YFP–OASF–CFP, (B) YFP–OASF L251S–CFP, (C) YFP–OASF L416S L423S–CFP, (D) YFP–OASF V419S–CFP. Calibration bar is 5 μm throughout. (Right panel) Corresponding intensity plots representing localization of YFP–OASF–CFP and mutants in regions close to the cell membrane as indicated by the dashed line.

Figure 6

Controlling full-length STIM1 clustering and coupling efficiency with ORAI1. (Left panel) Life cell image series showing localization and overlay from HEK293 cells expressing CFP–ORAI1 and (A) YFP–STIM1, (B) YFP–STIM1 L251S (C) YFP–STIM1 L416S L423S (D) YFP–STIM1 R426L under resting cell conditions (upper panel) and following 5 min store depletion with 60 μM BHQ in nominally free extracellular Ca²⁺ solutions (lower panel). (Right panel) Respective time courses of whole-cell inward currents at −86 mV activated by passive store depletion and blocked by 10 μM La³⁺ at t=200 s.