Structural Mechanisms of Store-Operated and Mitochondrial Calcium Regulation: Initiation Points for Drug Discovery

Calcium (Ca2+) is a universal signaling ion that is essential for the life and death processes of all eukaryotes. In humans, numerous cell stimulation pathways lead to the mobilization of sarco/endoplasmic reticulum (S/ER) stored Ca2+, resulting in the propagation of Ca2+ signals through the activation of processes, such as store-operated Ca2+ entry (SOCE). SOCE provides a sustained Ca2+ entry into the cytosol; moreover, the uptake of SOCE-mediated Ca2+ by mitochondria can shape cytosolic Ca2+ signals, function as a feedback signal for the SOCE molecular machinery, and drive numerous mitochondrial processes, including adenosine triphosphate (ATP) production and distinct cell death pathways. In recent years, tremendous progress has been made in identifying the proteins mediating these signaling pathways and elucidating molecular structures, invaluable for understanding the underlying mechanisms of function. Nevertheless, there remains a disconnect between using this accumulating protein structural knowledge and the design of new research tools and therapies. In this review, we provide an overview of the Ca2+ signaling pathways that are involved in mediating S/ER stored Ca2+ release, SOCE, and mitochondrial Ca2+ uptake, as well as pinpoint multiple levels of crosstalk between these pathways. Further, we highlight the significant protein structures elucidated in recent years controlling these Ca2+ signaling pathways. Finally, we describe a simple strategy that aimed at applying the protein structural data to initiating drug design.


Mitochondrial Calcium Uptake and SOCE Crosstalk
The first link between mitochondrial Ca 2+ uptake and SOCE was identified well before the molecules that are involved in mediating these processes. It was demonstrated that the pharmacological disruption of the inner mitochondrial membrane (IMM) potential or depletion of sodium (Na + ) could inhibit SOCE [137]. Indeed, the physiological relevance of this mitochondria-SOCE link was shown in studies comparing nuclear factor of activated T-cells (NFAT) translocation after T-cell activation in the presence and absence of mitochondrial Ca 2+ uptake diminishers [138]. Mitochondrial Ca 2+ uptake near store-operated Ca 2+ channels is believed to prevent Ca 2+ -dependent inactivation by limiting the formation of high Ca 2+ level domains. Interestingly, one study showed that the distance between mitochondria and PM SOCE channels decreases upon SOCE activation, allowing for a more sustained Ca 2+ entry due to reduced CDI [139]. In contrast, follow-up work suggested that mitochondrial Ca 2+ uptake, but not mitochondrial translocation/motility, was required for STIM1 and Orai1-mediated SOCE [140]. Furthermore, respiring mitochondria were independently shown to be required for SOCE activation and were found to regulate slow CDI of SOCE by buffering cytosolic Ca 2+ levels [141].
Specific links between SOCE and MCU have also been established with MCU knockdown studies showing reduced mitochondrial Ca 2+ uptake following SOCE [142]. MCU is not the only recently identified mitochondrial protein that has been directly linked to SOCE regulation. The Na + /Ca 2+ /lithium (Li + ) exchanger (NCLX) on the IMM has been shown to be vital for clearance of mitochondrial Ca 2+ (reviewed in [143]). SOCE is accompanied by a rise in cytosolic Na + , which is used by NCLX in order to drive Ca 2+ efflux from the matrix. The removal of extracellular Na + during SOCE activation inhibits NCLX and SOCE [144]. The activity of NCLX is tightly linked to Orai1 channel inactivation. In the absence of NCLX activity, the loss of Ca 2+ efflux from the matrix enhances ROS-dependent oxidation of a Cys residue on Orai1, which inhibits SOCE [144]. It is becoming clear that the crosstalk between SOCE and mitochondria involve several different molecules that can both positively and negatively regulate Orai1 channel function [145].

Mitochondrial Calcium Uniporter (MCU)
Mitochondria are primarily recognized for their role in adenosine triphosphate (ATP) production, a process that requires an electrochemical gradient across the IMM [146]. Mitochondrial Ca 2+ uptake plays a central role in regulating this energy production and it is also instrumental in regulating apoptosis and shaping cytosolic Ca 2+ transients [147]. While Ca 2+ can readily move through the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane, Ca 2+ uptake into the mitochondrial matrix is more precisely controlled [148]. MCU plays a major role in mediating Ca 2+ uptake into the matrix, functioning as a multimeric protein complex that forms a Ca 2+ selective pore through the IMM [12,13]. Under resting conditions, the MCU complex minimally permits the movement of Ca 2+ into the matrix, despite the highly negative IMM potential (~−180 mV); following a rise in cytosolic Ca 2+ levels, the MCU channel open probability increases, allowing for Ca 2+ to move into the matrix, being driven by the IMM potential [146,149,150]. The MCU complex is composed of the channel-forming protein, MCU, which tetramerizes to form a functional pore [151][152][153][154][155], and several additional regulatory protein components. The best studied protein regulators include a family of EF-hand containing proteins, mitochondrial Ca 2+ uptake 1, 2, and 3 (MICU1, MICU2 and MICU3) [149,156,157]; a small protein that is involved in bridging MICU proteins to the MCU pore, essential MCU regulator (EMRE) [158]; an MCU paralog, MCU dominant negative beta subunit (MCUb) [159]; and a complex stabilizing protein, MCU regulator 1 (MCUR1) [160].
The MCU pore forming subunit is an~40 kDa protein (including the mitochondrial targeting sequence; NCBI accession NP_612366.1) composed of two transmembrane domains spanning the IMM, with both the N-and carboxyl (C)-termini localized in the mitochondrial matrix [12,13,161] ( Figure 3A). An Asp-Ile-Met-Glu (DIME) motif confers Ca 2+ selectivity and controls permeability through the channel due to binding of Ca 2+ to the acidic residues [12,13]. Amino acid substitutions at D261 and E264 lead to a loss of Ca 2+ uptake, which is likely due to the removal of these negative charges [12,13]. MCU subunits homo-oligomerize to form a functioning Ca 2+ -permeable pore [13]. Initially, a solution nuclear magnetic resonance (NMR)-driven model of Caenorhabditis elegans MCU was suggested to exist as a pentamer, with the DIME motifs forming an unstructured loop at the opening of the channel [162]. Several resolved structures since the C. elegans NMR model have established metazoan MCU as a tetramer with the DIME motifs lining the pore as part of the helical transmembrane regions [151,152,154] ( Figure 3B).  Interestingly, the C-terminal domain of MCU alone can form a channel, because it contains the TM1 and TM2 helices that create the IMM channel pore [151,154,162,163]; however, the N-terminal domain (NTD) of human MCU, which resides in the matrix, has been demonstrated to be an important hub for inputs that regulate the channel function ( Figure 3C). For example, the self-association of MCU-NTD promotes channel assembly and activation [164], oxidative modification of C97 within MCU-NTD alters the channel architecture and function [165], and the S92A mutation within MCU-NTD dominant negatively disrupts mitochondrial Ca 2+ uptake [163]. It is noteworthy that MCU-NTD from lower order organisms assembles as a dimer of dimers directly under the pore [151][152][153]155] and a dimer of crescent-arranged tetramers across two human MCU channels, as revealed by cryo-electron microscopy (cryo-EM) [154].

Essential Mitochondrial Calcium Uniporter Regulator (EMRE)
EMRE is a metazoan-specific~11 kDa (including the mitochondrial targeting sequence; (NCBI accession NP_201575.3) single-pass membrane protein that spans the IMM [158]. EMRE interacts with MCU at the IMM and with the MICU1/MICU2 heterodimer in the intermembrane space (IMS) [158,166]. Thus, EMRE acts to bridge the MICU1/MICU2 Ca 2+ -sensing properties to MCU channel activity. The presence of an Asp-rich C-terminus is thought to be important in MCU complex regulation [158,166]; however, the specific topology of EMRE has been debated in the past [166,167]. The most recent human MCU complex structure has shown that the EMRE C-terminus is positioned in the IMS [154] (Figure 3B). EMRE is ubiquitously expressed in all tissues, and the knockdown of EMRE leads to the loss of MCU-mediated Ca 2+ uptake in mammals; subsequent overexpression of MCU is unable to recover the channel function [158,166]. The knockdown of MCU concomitantly reduces EMRE expression levels, indicating a mechanism of co-stabilization [158]. It is important to note that EMRE also plays an integral role in gating MCU by stabilizing the position of an MCU juxta-membrane loop away from the pore exit [154] (Figure 3B).

Mitochondrial Calcium Uptake (MICU) Proteins
MICU1 was the first component of the MCU complex discovered [156]. MICU1 is an~55 kDa (including the mitochondrial targeting sequence; NCBI accession NP_001350442.1) membrane associated protein, containing two conserved EF-hand Ca 2+ -binding domains [156]. MICU1 is localized to the IMS and indirectly modulates MCU activity via interactions with EMRE [156,158,168]. The initial findings suggested MICU1 functions independently to alter MCU-facilitated Ca 2+ uptake; however, two additional MICU isoforms, MICU2 and MICU3, have since been discovered to play a role in this Ca 2+ -dependent regulation [157,169]. MICU2 and MICU3 contain the EF-hand Ca 2+ -binding domains that are similarly found in MICU1, despite only sharing 25% sequence identity [157]. MICU1 and MICU2 are ubiquitously expressed in all tissues, while MICU3 appears to be more enriched in the nervous system and skeletal muscle [157,170,171]. MICU1 and MICU2 play synergistic roles in MCU regulation and, together in a complex, function as gatekeepers of the channel [170]. It has been proposed that, at low cytosolic Ca 2+ levels, MICU1 and MICU2 form a loose dimer, which inactivates MCU [172][173][174][175][176]. Following a rise in cytosolic and IMS Ca 2+ concentrations, Ca 2+ binds to the MICU EF-hands and triggers a conformational rearrangement that promotes tighter MICU1/MICU2 interactions and relieves MCU inhibition [172][173][174][175][176]. MICU3 appears to have a specific role in regulating neuronal function and synaptic activity. Specifically, MICU3 is believed to dimerize with MICU1 via disulfide bonds, functioning as a potent enhancer of mitochondrial Ca 2+ uptake that is controlled by MCU in skeletal muscle and the central nervous system [171].

Mitochondrial Calcium Uniporter Dominant Negative Beta (MCUb) Subunit
MCUb, an MCU paralog, is an~39 kDa protein (including the mitochondrial targeting sequence; NCBI accession NP_060388.2) that shares~50% sequence identity with MCU [159] (Figure 3A). Much like MCU, MCUb has two transmembrane domains, two coiled-coil domains, and matrix-oriented Nand C-termini. Although MCUb contains a similar DIME motif as MCU, MCUb has two key amino acid substitutions (i.e., R251W and E256V) in the pore lining region, which might underlie the inability of MCUb to function as a Ca 2+ -permeable pore [159]. Interestingly, MCU pore subunits have been shown to hetero-oligomerize with MCUb [159]. Mechanistically, MCUb might displace an MCU subunit from the complex to dominant negatively inhibit the channel function [180]. Once incorporated into the MCU pore, MCUb leads to a drastic decrease in mitochondrial Ca 2+ uptake by diminishing the open probability of the channel and Ca 2+ permeability [159,180]. Nevertheless, the precise atomic basis by which MCUb inhibits MCU function remains elusive.

Mitochondrial Calcium Uniporter Regulator-1 (MCUR1)
MCUR1 has been identified as a positive regulator of MCU, functioning as a scaffold factor that binds to both MCU and EMRE [149,181]. MCUR1 is an~40 kDa protein (including the mitochondrial targeting sequence; NCBI accession NCBI: NP_001026883.1), which has two transmembrane domains, two conserved coiled-coil domains, and N-and C-termini residing in the IMS. The interaction between MCU and MCUR1 likely occurs in the mitochondrial matrix, stabilizing the MCU complex [181]. MCUR1 knockdown decreases mitochondrial Ca 2+ uptake, reduces the IMM potential, and disrupts oxidative phosphorylation [181,182]. Collectively, these data suggest that MCUR1 not only regulates MCU-dependent Ca 2+ uptake, but also plays a key role in controlling cellular bioenergetics.

MCU and Disease
Mitochondrial Ca 2+ signaling has been implicated in a variety of pathophysiological disorders. Aberrant mitochondrial Ca 2+ uptake not only affects processes reliant on maintaining tight cytosolic Ca 2+ concentrations, but also impacts oxidative metabolism and can trigger cell death pathways [146,147,183]. Under cell stress conditions, mitochondrial Ca 2+ overload and/or oxidative stress results in an opening of the mitochondrial permeability transition pore (mPTP) [183,184]. High conductance mPTP allows the free movement of ions and small molecules less than~1.5 kDa, dissipating the IMM potential and aberrating ATP production [185,186]. Ultimately, this type of mPTP opening leads to the swelling and rupture of mitochondria and release of larger pro-apoptotic factors [183,184]. Thus, dysregulated mitochondrial Ca 2+ uptake has dire implications for cell survival and has been associated with various diseases, including cancer, neurodegenerative pathologies, diabetes, and learning and muscular disorders, among others [187][188][189][190][191].
Enhanced mitochondrial Ca 2+ uptake can suppress cytosolic Ca 2+ signals in fibroblasts from MICU1-deficient patients [193], which is consistent with past studies showing mitochondria can suppress cytosolic Ca 2+ signals [197][198][199][200]. Further, this enhanced mitochondrial Ca 2+ uptake may be related to work showing deletion of MICU1 in mouse hepatocytes causes sensitization to Ca 2+ -overload-induced mPTP opening [201]. The identification of heritable mutations in MCU complex components that lead to disease underscore the importance of not only the MCU channel, but also the diverse regulatory controls of MCU function.

Leucine Zipper EF-Hand Containing Transmembrane Protein-1 (LETM1)
LETM1 is an essential IMM protein linked to mitochondrial ion transport, regulation of cell cycle, mitochondrial oxidative stress and bioenergetic function [202]. Interestingly, LETM1 has been shown to play a role in mitochondrial Ca 2+ and K + ion homeostasis, regulating key facets of mitochondrial physiology, such as osmotic balance and ATP production [14,[203][204][205][206][207][208]. While the molecular mechanisms by which LETM1 functions remain incompletely understood, it is clear that LETM1 is pivotal in mitochondrial function and cellular health. The deletion of the LETM1 homologue in yeast, MDM38, results in mitochondrial swelling, loss of cristae, and disruption of cellular respiration [205]. Homozygous LETM1 deletion is embryonically lethal within~6 days in mice [204]. Clinically, LETM1 haploinsufficiency in humans is thought to be responsible for seizures in patients with Wolf-Hirschhorn syndrome (WHS) [209,210] (see below). Consistent with these findings, two independent studies identified LETM1 as one of~2000 essential genes in human cell cultures [211,212].
Human LETM1 is an~83 kDa transmembrane protein (including the mitochondrial targeting sequence; NCBI accession NP_036450.1) that localizes in the IMM [206,207] (Figure 4A). LETM1 is thought to topologically orient with the N-terminus facing the IMS. This IMS region includes a putative coiled-coil domain and a notable PTEN-induced kinase 1 (PINK1) mediated threonine phosphorylation site [217]. LETM1 contains a single sequence-identifiable transmembrane domain and a large matrix-oriented C-terminal region, which includes a putative ribosome binding domain, coiled-coil domains, and a Ca 2+ -binding EF-hand motif [15]. This topology for LETM1 is consistent with restricted proteinase K digestion studies [204,206,207]. However, a recent study using probes targeting tyrosine residues has proposed a new topology for LETM1, where two transmembrane domains exist and both the N-and C-termini are located in the matrix [218] (Figure 4A).
Human LETM1 is an ~83 kDa transmembrane protein (including the mitochondrial targeting sequence; NCBI accession NP_036450.1) that localizes in the IMM [206,207] (Figure 4A). LETM1 is thought to topologically orient with the N-terminus facing the IMS. This IMS region includes a putative coiled-coil domain and a notable PTEN-induced kinase 1 (PINK1) mediated threonine phosphorylation site [217]. LETM1 contains a single sequence-identifiable transmembrane domain and a large matrix-oriented C-terminal region, which includes a putative ribosome binding domain, coiled-coil domains, and a Ca 2+ -binding EF-hand motif [15]. This topology for LETM1 is consistent with restricted proteinase K digestion studies [204,206,207]. However, a recent study using probes targeting tyrosine residues has proposed a new topology for LETM1, where two transmembrane domains exist and both the N-and C-termini are located in the matrix [218] (Figure 4A). LETM1 self-oligomerizes and directly facilitates selective Ca 2+ /H + exchange within liposomes without any accessory proteins [204,206,208]. A low-resolution EM study suggests human LETM1 forms a hexamer, and pH may modulate the conformation [206]. Interestingly, the crystal structure of the C-terminal domain of the yeast homologue MDM38 has been solved and exhibits a 14-3-3 like protein interaction domain, which might indicate potential sites for inter-or intra-protomer binding [219,220] (Figure 4B,C). It is tempting to speculate that these interfaces could be targeted by drugs and small molecules aimed at modulating LETM1 assembly and function. . The predicted/putative location of four coiled-coil domains (red), two transmembranes helices (green), EF-hand Ca 2+ -binding motif (yellow), mitochondrial targeting sequence (blue), ribosome binding domain (magenta) and 14-3-3 like domain (cyan) are indicated with residue ranges shown above the respective segment based on the Uniprot annotation. (B) Yeast MDM38 ribosome binding domain crystal structure exhibiting a 14-3-3 like fold. The amino (N)terminal and carboxyl (C) terminal helices are coloured blue and red, respectively. The N-terminal helix has been previously implicated in mediating protein-protein interactions of the structurally homologous human 14-3-3 epsilon protein [219]. The conserved residue positions necessary for functional human LETM1 assembly and growth complementation of yeast deficient in MDM38 [220] are coloured magenta (i.e., D295, R318, G319, M320 in yeast MDM38; shown as sticks). (C) Surface representation of the yeast MDM38 ribosome binding domain showing a putative substrate/protein interaction cavity (orange), which may interact with phosphorylated proteins akin to human 14-3-3 structural homologues [219]. CC1 -2, -3, 4, coiled-coil-1, -2, -3; TM1, -2, transmembrane-1, -2; EF, EFhand; RBD, ribosome binding domain; MTS, mitochondrial targeting sequence. The structure figures were made using the 3SKQ [219] pdb coordinate file for yeast MDM38 ribosome binding domain. . The predicted/putative location of four coiled-coil domains (red), two transmembranes helices (green), EF-hand Ca 2+ -binding motif (yellow), mitochondrial targeting sequence (blue), ribosome binding domain (magenta) and 14-3-3 like domain (cyan) are indicated with residue ranges shown above the respective segment based on the Uniprot annotation. (B) Yeast MDM38 ribosome binding domain crystal structure exhibiting a 14-3-3 like fold. The amino (N)-terminal and carboxyl (C) terminal helices are coloured blue and red, respectively. The N-terminal helix has been previously implicated in mediating protein-protein interactions of the structurally homologous human 14-3-3 epsilon protein [219]. The conserved residue positions necessary for functional human LETM1 assembly and growth complementation of yeast deficient in MDM38 [220] are coloured magenta (i.e., D295, R318, G319, M320 in yeast MDM38; shown as sticks). (C) Surface representation of the yeast MDM38 ribosome binding domain showing a putative substrate/protein interaction cavity (orange), which may interact with phosphorylated proteins akin to human 14-3-3 structural homologues [219]. CC1 -2, -3, 4, coiled-coil-1, -2, -3; TM1, -2, transmembrane-1, -2; EF, EF-hand; RBD, ribosome binding domain; MTS, mitochondrial targeting sequence. The structure figures were made using the 3SKQ [219] pdb coordinate file for yeast MDM38 ribosome binding domain.
LETM1 self-oligomerizes and directly facilitates selective Ca 2+ /H + exchange within liposomes without any accessory proteins [204,206,208]. A low-resolution EM study suggests human LETM1 forms a hexamer, and pH may modulate the conformation [206]. Interestingly, the crystal structure of the C-terminal domain of the yeast homologue MDM38 has been solved and exhibits a 14-3-3 like protein interaction domain, which might indicate potential sites for inter-or intra-protomer binding [219,220] (Figure 4B,C). It is tempting to speculate that these interfaces could be targeted by drugs and small molecules aimed at modulating LETM1 assembly and function.

LETM1 and Disease
Located on the chromosome band 4p16.3, LETM1 is included in one of two critical regions lost in almost all patients exhibiting the full phenotype of WHS, which causes delayed growth and development, microcephaly, distinctive facial abnormalities, and seizures [204,209]. Deficiency in mitochondrial Ca 2+ transport, decreased ATP levels, and particularly increased ROS may be key determinants of the neurodegeneration linked to seizures in this disease [210]. Given that loss of LETM1 suppresses ATP production, enhances ROS, and inhibits cell proliferation [221], it is no surprise the overexpression of LETM1 has been found in many cancers, including ovarian, rectal, stomach, esophagus, breast, colon, and lung non-small cell carcinoma [222][223][224].

Generating New Therapeutics and Diagnostics from Protein Structures
Over the past~15 years, a number of protein structures involved in regulating cytosolic and stored Ca 2+ have been elucidated. This surge of structural information has not only been fueled by high throughput proteomic and genomic approaches that have solved decades old mysteries regarding the molecular identities of proteins that are involved in mediating ubiquitous cellular processes, such as SOCE [30,31] and mitochondrial Ca 2+ uptake [12,13], but also by improving technologies aimed at revealing protein structures, such as cryo-EM, solution NMR, and X-ray crystallography. For example, several high-resolution structures of the molecular machinery involved in mediating SOCE have been elucidated, including the STIM Ca 2+ -sensing domains by solution NMR [75,113], the cytosolic coiled-coil domains by solution NMR [107], and X-ray crystallography [106,108] (Figure 1), as well as the Orai1 Ca 2+ channel itself by cryo-EM and X-ray crystallography [120,225,226]. Cryo-EM has driven medium/high-resolution structures of MCU from lower order and metazoan organisms, including humans [151][152][153][154][155] (Figure 3). Importantly, X-ray crystallography has also contributed to understanding the underlying mechanisms of MCU regulation with elucidations of the important gatekeeping proteins [173,[175][176][177][178], environment-specific conformations [163,164,227], and scaffolds [154,228]. The first elucidations of IP 3 Rs by crystallography dates back almost two decades with structures of the N-terminal domain [229,230]. The analogous N-terminal region in RyR has also been studied by both crystallography and NMR [231,232]. In fact, crystal structures of the full N-terminal domains of both IP 3 Rs and RyRs revealed a remarkable structural and functional conservation between these receptor cousins at the apical positions of the receptors [233,234]. These tetrameric channels have also been excellent candidates for cryo-EM, due to their inherently large size [235][236][237]. Other Ca 2+ toolkit components, such as Na + /Ca 2+ exchangers (NCX) [238], S/ER Ca 2+ ATPase (SERCA) pumps [239,240], and VGICs, have also been structurally resolved [241,242]. This list of structures only scratches the surface of the available Ca 2+ toolkit structural data, yet reinforces a major question at the nexus of the structural biology, cell biology, and clinical research fields. How can we use this wealth of protein structural information to design new diagnostic tools and drugs for research and treatment of disease?
Indeed, while obtaining structural information has been invaluable for informing the broad scientific community on the molecular and structural basis for the function of hundreds or thousands of Ca 2+ signaling proteins, there is a disconnect between applying this protein structural knowledge and rationally developing research tools or drugs. Perhaps this disconnect exists due to the relatively static insights provided by many of the resolved structures. Most (if not all) protein function involves dynamic changes in conformation and stability. Rationalizing effective drugs without a detailed understanding of the conformational ensembles present in the inactive, transition, and active states of proteins is practically challenging.

Store-Operated and Mitochondrial Ca 2+ Entry Proteins as Drug Targets
The SOCE and mitochondrial Ca 2+ uptake protein machinery are appealing drug targets for several reasons. First, mutations causing disease have been identified in the proteins mediating these processes (see above). Second, complexes of multiple different proteins regulate SOCE and mitochondrial Ca 2+ uptake; hence, the modulation of function, in some cases, could be achieved by targeting native protein interaction partner components. Third, drugs targeting ion channels are demonstrated to be effective at treating a wide range of human diseases and acute conditions [243][244][245]. Fourth, clinically approved immunosuppressive drugs already exist that block signaling processes that are directly mediated by SOCE (e.g., cyclosporin [246,247]). Finally, the acute administration of MCU inhibitors is known to protect cells from hypoxia/ischemia-induced injury [248][249][250][251]. The specific structures available for the SOCE and mitochondrial Ca 2+ uptake molecular machinery provide a unique opportunity for the development of new diagnostics and/or therapeutics that can biasedly target different aspects of these pathways.
Small molecule screens targeting MCU function have already been performed. An initial study employed Saccharomyces cerevisiae as an MCU-null organism to screen for small molecule modulators of MCU function. Human MCU, human EMRE, and a mitochondrial-targeted Ca 2+ indicator (i.e., aequorin) were expressed in these yeasts to establish a reporter system for the screening of~600 clinically approved drugs [252]. In a second approach, HEK293A cells expressing mitochondrial aequorin were used to screen a 120,000 compound library, successfully identifying an MCU inhibitor [253]. In the most recently published screen, HeLa cells expressing mitochondrially-targeted aequorin were used to identify two MCU inhibitors from a library of 44,000 compounds [254]. All of these in cellulo screens used genetically encoded aequorin, reliably targeted to the matrix, for luminescence readout of changes in Ca 2+ .
Since the identification of STIM1 and Orai1, published drug screens targeting SOCE have largely involved the over-expression of STIM1 and Orai1 in mammalian cells or the use of cells with endogenous levels of STIM1 and Orai1, in combination with indicator dyes or proteins monitoring cytosolic Ca 2+ or NFAT translocation to the nucleus [255][256][257][258]. One in vitro study used recombinant functional domains to screen for binding to immobilized small molecules by immunofluorescence [259]. In silico tools have also been used, but only to identify drug candidates through small molecule homology to known SOCE inhibitors [260,261].
Thus, while drug screens targeting the protein machinery that mediates both store-operated and mitochondrial Ca 2+ uptake have been performed, these published approaches have not used the available protein structure information as initiation points to drug discovery.

Initiating Protein Structure-based Drug Discovery
A starting point for drug development could be initiated by a simple high-throughput small molecule in silico screen of millions of compounds. This computational screen would use available protein structures and knowledge of the vital interfaces modulating activity. The structures of the small molecules/drugs would also need to be known. Such small molecule databases and computational docking programs exist and are freely available (e.g., see ZINC15 [262] and AUTODOCK [263]). The major bottleneck for this initial step is computing the resources that are required to dock millions of compounds in a relatively short time. Nevertheless, once top leads are identified, subsequent wet laboratory experiments are required in order to validate the interaction and physiological effect(s). Downstream structure-activity-relationship studies could be subsequently used to better understand the small molecule chemical groups that are required for binding and eliciting a cellular effect and improving the compound [264].
Of course, there are limitations to these large-scale computational screens. For example, the protein structures are generally treated as rigid bodies, unless using higher level molecular dynamics simulations. Water and/or lipid molecules are typically not modeled in these fast docking schemes, even though these molecules play integral roles in biological interactions. Most critically, default scoring schemes may not be optimal for a system under study, since not all of the interactions equally rely on specific stabilization forces (e.g., H-bonding, hydrophobic, charge, shape complementarity).
High resolution structural information can also be used to develop tools for high-throughput drug discovery. For example, fluorescence-resonance energy transfer (FRET)-based biosensors can be rationalized based on proximity information that is revealed by isolated and co-complexed structures. With respect to Ca 2+ signaling, these types of FRET-based sensors have been engineered and used in screens for the identification of SERCA and RyR modulators. A green fluorescent protein (GFP) and red fluorescent protein (RFP) FRET pair was fused into the cytosolic headpiece of SERCA, which undergoes large scale structural changes during pump activity. This intramolecular FRET sensor was successfully used to identify the small molecule modulators of SERCA function [265,266]. Since the sensor is genetically encoded, the cell lines stably expressing this sensor could be used in the screening. An analogous approach was used by the same group to identify modulators of RyR function. However, the RyR screen relied on FRET between FK binding protein 12.6 (FKBP12) and calmodulin, which co-complex with RyR and show different FRET signals that are based on RyR conformation [267,268]. In these RyR experiments, Alexa Fluors were chemically linked to FKBP12 and calmodulin in vitro, and vesicles containing RyRs were prepared from muscle tissues. Two aspects of STIM/Orai and MCU signaling make this approach to identifying drugs particularly appealing: (i) these proteins function in large complexes that are amenable to intermolecular FRET monitoring and (ii) STIMs are known to undergo large conformational changes, making them amenable to intramolecular FRET monitoring.

Concluding Remarks
Not discussed herein are the countless additional experimental (involving molecules, cells, animals, and humans), logistical, and administrative hurdles that are required to move any small molecule lead into clinical use. Nevertheless, we provide a simple strategy for applying high-resolution structural knowledge of proteins as a starting point to drug development. Excitingly, several key protein structures are available that inform on the mechanisms regulating the SOCE-mitochondrial Ca 2+ signaling axis, providing several possible initiating points to protein structure-based drug design. The elucidation of Ca 2+ signaling protein structures is vital for understanding the fundamental mechanisms underlying cellular processes that are integral to life and death. The structural information can also provide an entry point for the development of tools and therapeutics targeting these pathways.