Molecular basis of allosteric Orai1 channel activation by STIM1

Yeung, Priscilla S.-W.; Yamashita, Megumi; Prakriya, Murali

doi:10.1113/jp276550

Cited by 65 publications

(61 citation statements)

References 91 publications

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“…Concerning the latter, the requirement of more than three intact Orai subunits is in line with the non-linear dependence of Orai activation and STIM1 coupling 64 . The transmission of the activation signal across the entire Orai channel complex is likely enforced by the TM2/3 ring which has recently been proposed to function as an individual unit that devolves strong cooperativity between STIM1 coupling at six single subunits and pore opening 58,65,66 .…”

Section: Discussionmentioning

confidence: 99%

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A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening: Clearance and synergy of Orai1 gating checkpoints controls pore opening

Tiffner

Schober

Hoeglinger

et al. 2020

Preprint

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The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of LoF mutations act dominant over most GoF mutations within the same as well as of an adjacent Orai subunit. We further established inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints is required to allow STIM1 coupling and Orai1 pore opening.Graphical Abstract

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Section: Discussionmentioning

confidence: 99%

“…The TIP3p (64) model is used to represent water molecules. The CHARMM36 forcefield was used to treat protein (65) and lipids (66). Calcium and chloride ions were used to neutralize the system with an ionic strength set at 100 mM.…”

Section: Simulation Protocolsmentioning

confidence: 99%

A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening: Clearance and synergy of Orai1 gating checkpoints controls pore opening

Tiffner

Schober

Hoeglinger

et al. 2020

Preprint

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“…It remains to be clarified whether the tendency for a greater degradation rate of SARAF in the presence of TG is associated with the SOCE and/or activation of NEDD4 family E3 ligases. Influx of extracellular Ca 2+ through the SOCE system is achieved by the interplay of ER-transmembrane STIM1 and plasma membrane CRAC channel representatively comprised of Orai1 [ 37 , 38 ]. The SOCE apparatus is regulated by a number of modulators that physically associate with STIM1 or Orai1 [ 39 ].…”

Section: Discussionmentioning

confidence: 99%

The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

Zhang

Muramatsu

Matsuo

et al. 2020

IJMS

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ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

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“…On the channel side, Yeung and colleagues describe the molecular basis of the allosteric process that culminates in the opening of the Orai1 channel pore following STIM1 binding (Yeung et al . 2019). This work reveals a critical role for transmission of the gating signal through the Orai1 transmembrane domains to open a hydrophobic gate.…”

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confidence: 99%