Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Photocrosslinking-induced CRAC channel-like Orai1 activation independent of STIM1.

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Swing-out opening of stromal interaction molecule 1.

Stromal interaction molecule 1 (STIM1) resides in the endoplasmic reticulum (ER) membrane and senses luminal calcium (Ca2+ ) concentration. STIM1 activation involves a large-scale conformational transition that exposes a STIM1 domain termed "CAD/SOAR", - which is required for activation of the calcium channel Orai. Under resting cell conditions, STIM1 assumes a quiescent state where CAD/SOAR is suspended in an intramolecular clamp formed by the coiled-coil 1 domain (CC1) and CAD/SOAR. Here, we present a structural model of the cytosolic part of the STIM1 resting state using molecular docking simulations that take into account previously reported interaction sites between the CC1α1 and CAD/SOAR domains. We corroborate and refine previously reported interdomain coiled-coil contacts. Based on our model, we provide a detailed analysis of the CC1-CAD/SOAR binding interface using molecular dynamics simulations. We find a very similar binding interface for a proposed domain-swapped configuration of STIM1, where the CAD/SOAR domain of one monomer interacts with the CC1α1 domain of another monomer of STIM1. The rich structural and dynamical information obtained from our simulations reveals novel interaction sites such as M244, I409, or E370, which are crucial for STIM1 quiescent state stability. We tested our predictions by electrophysiological and Förster resonance energy transfer experiments on corresponding single-point mutants. These experiments provide compelling support for the structural model of the STIM1 quiescent state reported here. Based on transitions observed in enhanced-sampling simulations paired with an analysis of the quiescent STIM1 conformational dynamics, our work offers a first atomistic model for CC1α1-CAD/SOAR detachment.

Science CommuniCa2+tion Developing Scientific Literacy on Calcium: The Involvement of CRAC Currents in Human Health and Disease.

All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells' membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca2+ and its role in human health and disease with focus on CRAC channels.

Calcium Signals during SARS-CoV-2 Infection: Assessing the Potential of Emerging Therapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). This respiratory illness was declared a pandemic by the world health organization (WHO) in March 2020, just a few weeks after being described for the first time. Since then, global research effort has considerably increased humanity's knowledge about both viruses and disease. It has also spawned several vaccines that have proven to be key tools in attenuating the spread of the pandemic and severity of COVID-19. However, with vaccine-related skepticism being on the rise, as well as breakthrough infections in the vaccinated population and the threat of a complete immune escape variant, alternative strategies in the fight against SARS-CoV-2 are urgently required. Calcium signals have long been known to play an essential role in infection with diverse viruses and thus constitute a promising avenue for further research on therapeutic strategies. In this review, we introduce the pivotal role of calcium signaling in viral infection cascades. Based on this, we discuss prospective calcium-related treatment targets and strategies for the cure of COVID-19 that exploit viral dependence on calcium signals.

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins.

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions.

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the 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 with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified 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 are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

Orai channels: key players in Ca2+ homeostasis.

Maintaining a precise calcium (Ca2+) balance is vital for cellular survival. The most prominent pathway to shuttle Ca2+ into cells is the Ca2+ release activated Ca2+ (CRAC) channel. Orai proteins are indispensable players in this central mechanism of Ca2+ entry. This short review traces the latest articles published in the field of CRAC channel signalling with a focus on the structure of the pore-forming Orai proteins, the propagation of the binding signal from STIM1 through the channel to the central pore and their role in human health and disease.