The many states of STIM1 Fahrner, Marc; Romanin, Christoph
eLife,
12/2021, Volume:
10
Journal Article
Peer reviewed
Open access
Harnessing single-molecule FRET illuminates the structural changes necessary for a protein to fine-tune the influx of calcium when reserves inside a cell run low.
Molecular mechanisms of STIM/Orai communication Derler, Isabella; Jardin, Isaac; Romanin, Christoph
American Journal of Physiology: Cell Physiology,
04/2016, Volume:
310, Issue:
8
Journal Article
Peer reviewed
Open access
Ca(2+)entry into the cell via store-operated Ca(2+)release-activated Ca(2+)(CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, ...secretion, and cell death. These store-operated Ca(2+)channels open after depletion of intracellular Ca(2+)stores, and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an endoplasmic reticulum-located Ca(2+)sensor, while Orai forms a highly Ca(2+)-selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography, thereby establishing a portrait of key mechanistic steps in the CRAC channel signaling cascade. The focus is on the activation of the STIM proteins, the subsequent coupling of STIM1 to Orai1, and the consequent structural rearrangements that gate the Orai channels into the open state to allow Ca(2+)permeation into the cell.
STIM Proteins: An Ever-Expanding Family Grabmayr, Herwig; Romanin, Christoph; Fahrner, Marc
International journal of molecular sciences,
12/2020, Volume:
22, Issue:
1
Journal Article
Peer reviewed
Open access
Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in ...the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel ...Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.
Background: STIM1 and Orai1 are key players in the store-operated Ca2+ entry.
Results: The activation state of STIM1 is precisely controlled by heteromeric interaction between coiled-coil domains.
Conclusion: A coiled-coil clamp provides control over STIM1 conformation and clustering.
Significance: Understanding of the STIM1 C-terminal switching mechanism is crucial for the control of Orai1 activation.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The ...cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1-CC2 fragment in the apo and Orai1-bound states. Each CC1-CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2' helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM-Orai association pocket. STIM1 mutants disrupting CC1:CC1' interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices.
Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion ...channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation-deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical 'lipid clamp' identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.
In immune cells, generation of sustained Ca2+ levels is mediated by the Ca2+ release-activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 ...(STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca2+ stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233–450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50–74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca2+ currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233–420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233–485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
STIM1 and Orai1 are key components of the Ca
-release activated Ca
(CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca
sensor ...STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.
The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca
2+
sensor in the endoplasmic reticulum (ER), and Orai1, the Ca
2+
ion channel in the plasma membrane. Ca
2+
store ...depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners.
Graphic abstract
Legend: Upon intracellular Ca
2+
store depletion of the endoplasmic reticulum (ER), Ca
2+
dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca
2+
influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca
2+
-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca
2+
store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca
2+
store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).
Full text
Available for:
EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an ...active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α
and CC1α
helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.
PDF
