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The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

The involvement of inositol trisphosphate receptor (IP3R) in modulating store-operated calcium entry (SOCE) was established many years ago. Nevertheless, the molecular mechanism responsible for this observation has not been elucidated to this date. In the present study we show that IP3R associates to STIM1 upon depletion of the endoplasmic reticulum (ER) by activation of the inositol trisphosphate signaling cascade via G-protein coupled receptors. IP3R-STIM1 association results in enhanced STIM1 puncta formation and larger Orai-mediated whole-cell currents as well as increased calcium influx. Depleting the ER with a calcium ATPase inhibitor (thapsigargin, TG) does not induce IP3R-STIM1 association, indicating that this association requires an active IP3R. The IP3R-STIM1 association is only observed after IP3R activation, as evidenced by FRET experiments and co-immunoprecipitation assays. ER intraluminal calcium measurements using Mag-Fluo-4 showed enhanced calcium depletion when IP3R is overexpressed. A STIM1-GCaMP fusion protein indicates that STIM1 detects lower calcium concentrations near its EF-hand domain when IP3R is overexpressed when compared with the fluorescence reported by a GCaMP homogenously distributed in the ER lumen (ER-GCaMP). All these data together strongly suggest that activation of inositol trisphosphate signaling cascade induces the formation of the IP3R-STIM1 complex. The activated IP3R provides a reduced intraluminal calcium microenvironment near STIM1, resulting in enhanced activation of Orai currents and SOCE.

Voltage-gated sodium channels (NaV) are responsible for the rapid depolarization of many excitable cells. They readily inactivate, a process where currents diminish after milliseconds of channel opening. They are also targets for a multitude of disease-causing mutations, many of which have been shown to affect inactivation. A cluster of disease mutations, linked to Long-QT and Brugada syndromes, is located in a C-terminal EF-hand like domain of NaV1.5, the predominant cardiac sodium channel isoform. Previous studies have suggested interactions with the III-IV linker, a cytosolic element directly involved in inactivation. Here we validate and map the interaction interface using isothermal titration calorimetry (ITC) and NMR spectroscopy. We investigated the impact of various disease mutations on the stability of the domain, and found that mutations that cause misfolding of the EF-hand domain result in hyperpolarizing shifts in the steady-state inactivation curve. Conversely, mutations in the III-IV linker that disrupt the interaction with the EF-hand domain also result in large hyperpolarization shifts, supporting the interaction between both elements in intact channels. Disrupting the interaction also causes large late currents, pointing to a dual role of the interaction in reducing the population of channels entering inactivation and in stabilizing the inactivated state.

The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVβ, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVβ, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.

Acid proteins capable of nucleating Ca2+ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. The helix-loop helix EF-hands are the most common Ca2+-binding motifs in proteins. Calcium is bound by the loop region. These motifs are found in many proteins that are regulated by calcium. Gad m 1, an Atlantic cod β-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca2+ buffer in fish muscle; the neutral and acid apo-forms of this protein can form amyloids. Since Ca2+-nucleating proteins have a propensity to form extended β-strand structures, we wondered whether amyloid assemblies of an EF-hand protein were able to influence calcium carbonate crystallization in vitro. Here, we used the Gad m 1 chain as a model to generate monomeric and amyloid assemblies and to analyze their effect on calcite formation in vitro. We found that only amyloid assemblies alter calcite morphology., This work was supported by grants from the Spanish AEI/EU-FEDER SAF2014-52661-C3 and BFU2015-72271-EXP.

The present study demonstrates cation-driven optical properties of artificial luciferases (ALucs) from copepod luciferases, as an optical readout for bioanalysis. An assignment of the supersecondary structure code (SSC) of ALucs revealed that ALucs carry a helix-loop-helix structure, which appears at the same sites of the EF-hands of typical Ca(2+)-binding proteins. A mutagenesis study shows that the EF-hand-like structure is a pivotal site for enzymatic activity. The effects of 20 kinds of mono- and multivalent cations on ALuc activities were estimated with column-purified ALuc16. High pH values boost the ALuc activities with both the native coelenterazine and an analog called 6-pi-OH-CTZ. Multivalent cations, Ca(II), Mg(II), and Cr(VI), elevate and prolong the ALuc activities, whereas Co(II), Cu(II) and Pb(II) greatly hamper the ALuc activities. Ca(II) greatly prolongs the optical intensities, suggesting a contribution to the structural robustness of ALucs. The inhibitory effect of multivalent cations on the ALuc activities was utilized for creating dose-response curves. The intrinsic cation-driven selectivity and optical intensity of ALucs enable researchers to constitute de novo biosensors for multivalent cations.

SUMMARYFhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.

We have developed a method to align a pair of EF-hand domains, an EF-lobe, using the symmetry axis that is intrinsic to the EF-lobe itself. The coordinate system for the alignment is dependent only on the symmetry of the EF-lobe. The absolute positions of each component in the structure can be described in this coordinate system. Our site provides the foundation for the analyses and comparisons of structural features of EF-lobes.

Plant NADPH oxidases also known as respiratory burst oxidase homologs (Rbohs) are a family of membrane-bound enzymes that play diverse roles in the defense response and morphogenetic processes via regulated generation of reactive oxygen species. Rbohs are associated with a variety of functions, although the reason for this is not clear. To evaluate using bioinformatics, the possible mechanisms for the observed functional diversity within the plant kingdom, 127 Rboh protein sequences representing 26 plant species were analyzed. Multiple clusters were identified with gene duplications that were both dicot as well as monocot-specific. The N-terminal sequences were observed to be highly variable. The conserved cysteine (equivalent of Cys890) in C-terminal of AtRbohD suggested that the redox-based modification like S-nitrosylation may regulate the activity of other Rbohs. Three-dimensional models corresponding to the N-terminal domain for Rbohs from Arabidopsis thaliana and Oryza sativa were constructed and molecular dynamics studies were carried out to study the role of Ca(2+) in the folding of Rboh proteins. Certain mutations indicated possibly affect the structure and function of the plant NADPH oxidases, thereby providing the rationale for further experimental validation.

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca2+, Mg2+, Zn2+, and Cu2+. All OvCaBPs showed mobility shifts with Ca2+ and Zn2+. OvCaBP1 showed also positive results with Mg2+ and Cu2+. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.

Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) signaling pathway facilitating extracellular Ca(2+) influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca(2+) stores. Stromal interaction molecule 1 (STIM1) is the Ca(2+) sensor that activates SOCE following ER/SR Ca(2+) depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca(2+) concentrations. Low ER Ca(2+) levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca(2+) channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys(49) and Cys(56) in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca(2+) depletion–dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys(49) and Cys(56) S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.

Starting with conformations of calcium-binding sites in parvalbumin and integrin (representative structures of EF-hand and calcium blade zones, respectively) we introduce four new different local Ca2+-recognition units in proteins: a one-residue unit type I (ORI); a three-residue unit type I (TRI); a one-residue unit type II (ORII) and a three-residue unit type II (TRII). Based on the amount and nature of variable atoms, the type I and II units theoretically can have four and twelve variants, respectively. Analysis of known “Ca2+-bound functional niches” in proteins revealed presence of almost all possible variants of Ca2+-recognition units in actual structures. Parvalbumin, integrin alpha-IIb and sixteen other proteins with different Ca2+-bound functional niches contain various consecutively joined combinations of OR(I/II) and TR(I/II) units. Such a OR(I/II)+TR(I/II) joint unit forms a tripeptide, which uses three main-chain atoms for metal binding: nitrogenn (Donor), oxygenn (Acceptor) and nitrogenn+2 (Donor). Thus, taken together, the described ORI, TRI, ORII and TRII units can serve as elementary blocks to construct more complex calcium recognizing substructures in a variety of calcium binding sites of unrelated proteins.

Centrin is a member of the EF-hand super family of calcium-binding proteins, which can behave as a part of damage detector initiated the initiation of nucleotide excision repair (NER). Its self-assembly plays a causative role in fiber contraction associated with the cell division cycle and ciliogenesis. To explore the possible role of DNA in the process of centrin self-assembly, the aggregation properties of N-terminal domain of Euplotes octocarinatus centrin (N-EoCen) in the presence of DNA with or without metal ions are investigated. It is verified that metal ions, such as Ca2+ and Tb3+, can bind to N-EoCen with 2:1 stoichiometry by isothermal titration calorimetry (ITC). Importantly, this study reports that double strand DNA (dsDNA) is capable of binding N-EoCen, changing conformation of protein and modulating centrin aggregation, as demonstrated by extensive biophysical assays. Interestingly, the open conformation of protein induced by metal ions may be favour of the interaction of protein with dsDNA. Nevertheless, the randomly coiled single strand DNA (ssDNA) is completely inefficient to the aggregation regulation. Furthermore, results reveal that hydrophobic site could play important role in the process. This finding may link to the potent roles of centrin in the NER process.

We have classified 865 sequences of EF-hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF-lobe (pair of adjacent EF-hands). They have two or more EF-lobes; the relative positions of their EF-lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF-lobes of these groups indicate that the mutual positions of EF-lobes were established soon after divergence of an EF-lobe for each group and before the duplication and fusion of EF-lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF-lobe precursor, even if their conformations are similar to the canonical EF-hand. This is an example of convergent evolution.

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca2+-binding protein that also binds to specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca2+, but detaches from DRE under Ca2+ stimulation, allowing gene expression. The Ca2+-binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca2+ binding properties and the subsequent conformational changes of full length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though EF-1 hand is not capable of binding to Ca2+. Moreover, EF-2 is a weaker affinity site compared to EF-3 and 4 hands. Comparison of HDX profiles between WT DREAM and two EF-1 mutated constructs illustrate that the conformational changes in EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca2+ binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of coactivator, CREB-binding protein (CBP). These long-range interactions strongly suggest how conformational changes transmit the Ca2+ signal to CREB-mediated gene transcription.

Spectraplakins are large molecules that cross-link F-actin and microtubules (MTs). Mutations in spectraplakins yield defective cell polarization, aberrant focal adhesion dynamics, and dystonia. We present the 2.8 A crystal structure of the hACF7 EF1-EF2-GAR MT-binding module and delineate the GAR residues critical for MT binding. The EF1-EF2 and GAR domains are autonomous domains connected by a flexible linker. The EF1-EF2 domain is an EFβ-scaffold with two bound Ca2+ ions that straddle an N-terminal α helix. The GAR domain has a unique α/β sandwich fold that coordinates Zn2+. While the EF1-EF2 domain is not sufficient for MT binding, the GAR domain is and likely enhances EF1-EF2-MT engagement. Residues in a conserved basic patch, distal to the GAR domain's Zn2+-binding site, mediate MT binding.

Tescalcin (TESC, also known as calcineurin-homologous protein 3, CHP3) is a 24-kDa EF-hand Ca2+-binding protein that has recently emerged as a regulator of cell differentiation and growth. The TESC gene has also been linked to human brain abnormalities, and high expression of tescalcin has been found in several cancers. The expression level of tescalcin changes dramatically during development and upon signal-induced cell differentiation. Recent studies have shown that tescalcin is not only subjected to up- or down-regulation, but also has an active role in pathways that drive cell growth and differentiation programs. At the molecular level, there is compelling experimental evidence showing that tescalcin can directly interact with and regulate the activities of the Na+/H+ exchanger NHE1, subunit 4 of the COP9 signalosome (CSN4) and protein kinase glycogen-synthase kinase 3 (GSK3). In hematopoetic precursor cells, tescalcin has been shown to couple activation of the extracellular signal-regulated kinase (ERK) cascade to the expression of transcription factors that control cell differentiation. The purpose of this Commentary is to summarize recent efforts that have served to characterize the biochemical, genetic and physiological attributes of tescalcin, and its unique role in the regulation of various cellular functions.

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a major pest of rice in Asia, is able to successfully puncture sieve tubes in rice with its piercing stylet and then to ingest phloem sap. How BPH manages to continuously feed on rice remains unclear. Here, we cloned the gene NlSEF1, which is highly expressed in the salivary glands of BPH. The NlSEF1 protein has EF-hand Ca2+-binding activity and can be secreted into rice plants when BPH feed. Infestation of rice by BPH nymphs whose NlSEF1 was knocked down elicited higher levels of Ca2+ and H2O2 but not jasmonic acid, jasmonoyl-isoleucine (JA-Ile) and SA in rice than did infestation by control nymphs; Consistently, wounding plus the recombination protein NlSEF1 suppressed the production of H2O2 in rice. Bioassays revealed that NlSEF1-knockdown BPH nymphs had a higher mortality rate and lower feeding capacity on rice than control nymphs. These results indicate that the salivary protein in BPH, NlSEF1, functions as an effector and plays important roles in interactions between BPH and rice by mediating the plant’s defense responses.

Calpains are Ca2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3′s PEF domain and its crystal structure in the presence of Ca2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca2+ bound at the EF5-hand used for homodimer association. Three of the four Ca2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit. Database Structural data are available in the Protein Data Bank database under accession number 4OKH. Structured digital abstract Calpain-3 and Calpain-3 bind by x-ray crystallography (View interaction)

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca(2+). However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca(2+) binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.

Polycystin 2 (PC2) is a calcium-dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C-terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single-channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild-type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium-binding EF hand is intact. These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function.—Kuo, I. Y., Keeler, C., Corbin, R., Ćelić, A., Petri, E. T., Hodsdon, M. E., Ehrlich, B. E. The number and location of EF hand motifs dictates the calcium dependence of polycystin-2 function.

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK. S100A14, one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens, and there is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding. Moreover, we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.

S100A5 is a calcium-binding protein of S100 family, which represents a major ligand to the receptor for advanced glycation end product (RAGE), a pattern recognition receptor engaged in diverse pathological processes. Here we have characterized calcium binding of S100A5 and the complex formation between S100A5 and RAGE using calorimetry and NMR spectroscopy. S100A5 binds to calcium ions in a sequential manner with the equilibrium dissociation constants (K

The mitochondrial calcium uniporter is a Ca2+‐activated Ca2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca2+ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca2+. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca2+‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca2+ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca2+ signals.

The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10-kilodalton, metazoan-specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.

We recently identified several Ca(2+)-binding proteins (CaBP) from the S100 and annexin family to be regulated by TSH in FRTL-5 cells. Here, we study the regulation of S100A4, S100A6 and ANXA2 in primary human thyrocytes (PHT) derived from surrounding tissues (ST), cold benign thyroid nodules (CTN) and autonomously functioning thyroid nodules (AFTN). We investigated the expression and regulation of CaBP and the effect of their expression on Ca(2+) and TSHR signaling. We used an approach that accounts for the potential of an individual PHT culture to proliferate or to express thyroid differentiation features by assessing the expression of FOS and TPO. We found a strong correlation between the regulation of CaBP and the proliferation-associated transcription factor gene FOS. PKA and MEK1/2 were regulators of ANXA2 expression, while PI3-K and triiodothyronine were additionally involved in S100 regulation. The modulated expression of CaBP was reflected by changes in ATP-elicited Ca(2+) signaling in PHT. S100A4 increased the ratio of subsequent Ca(2+) responses and showed a Ca(2+) buffering effect, while ANXA2 affected the first Ca(2+) response to ATP. Overexpression of S100A4 led to a reduced activation of NFAT by TSH. Using S100A4 E33Q, D63N, F72Q and Y75K mutants we found that the effects of S100A4 expression on Ca(2+) signaling are mediated by protein interaction. We present evidence that TSH has the ability to fine-tune Ca(2+) signals through the regulation of CaBP expression. This represents a novel putative cross-regulating mechanism in thyrocytes that could affect thyrocyte signaling and physiology.

Miro is a highly conserved calcium-binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified 'hidden' EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand-cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation-dependent regulation of mitochondrial function by Miro.

A cDNA clone encoding 8 kDa protein was retrieved from an EST pool of Chinese liver fluke Clonorchis sinensis. A deduced polypeptide of the cDNA clone was similar to 8 kDa Ca(2+)-binding proteins from other parasitic trematodes, and, thus, named as CsCa8, containing two EF-hand Ca(2+)-binding sites. Homology models predicted CsCa8 to be a single globular structure having four helices and molecular folds similar to Ca(2+)-binding state of other small Ca(2+)-binding proteins. Recombinant CsCa8 protein showed specific Ca(2+)-binding affinity and shifting in native gel mobility assay. Mouse immune sera raised against recombinant CsCa8 protein recognized native CsCa8 from adult C. sinensis worm extract. CsCa8 was localized in oral and ventral suckers, vitelline follicles and subtegumental tissues. These findings suggest that CsCa8 might be involved in cellular Ca(2+) signal transduction for muscle contraction and egg production.

Lead toxicity is associated with various human diseases. While Ca(2+) binding proteins such as calmodulin (CaM) are often reported to be molecular targets for Pb(2+)-binding and lead toxicity, the effect of Pb(2+) on the Ca(2+)/CaM regulated biological activities cannot be described by the primary mechanism of ionic displacement (e.g., ionic mimicry). The focus of this study was to investigate the mechanism of lead toxicity through binding differences between Ca(2+) and Pb(2+) for CaM, an essential intracellular trigger protein with two EF-Hand Ca(2+)-binding sites in each of its two domains that regulates many molecular targets via Ca(2+)-induced conformational change. Fluorescence changes in phenylalanine indicated that Pb(2+) binds with 8-fold higher affinity than Ca(2+) in the N-terminal domain. Additionally, NMR chemical shift changes and an unusual biphasic response observed in tyrosine fluorescence associated with C-terminal domain sites EF-III and EF-IV suggest a single higher affinity Pb(2+)-binding site with a 3-fold higher affinity than Ca(2+), coupled with a second site exhibiting affinity nearly equivalent to that of the N-terminal domain sites. Our results further indicate that Pb(2+) displaces Ca(2+) only in the N-terminal domain, with minimal perturbation of the C-terminal domain, however significant structural/dynamic changes are observed in the trans-domain linker region which appear to be due to Pb(2+)-binding outside of the known calcium-binding sites. These data suggest that opportunistic Pb(2+)-binding in Ca(2+)/CaM has a profound impact on the conformation and dynamics of the essential molecular recognition sites of the central helix, and provides insight into the molecular toxicity of non-essential metal ions.

The EF-hand motif (helix-loop-helix) is a Ca(2+)-binding domain that is common among many intracellular Ca(2+)-binding proteins. We applied Fourier-transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E-loop-helix F). The 17-residue peptides corresponding to loop-helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D-type amino acid, were investigated to disturb the α-helical conformation of helix F systematically. These D-type-substituted peptides showed no band at about 1555 cm(-1) even in the Ca(2+)-loaded state although the native peptide (L-type only) showed a band at about 1555 cm(-1) in the Ca(2+)-loaded state, which is assigned to the side-chain COO(-) group of Glu at the 12th position, serving as the ligand for Ca(2+) in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca(2+) and the side-chain COO(-) group of Glu at the 12th position. Implications of the COO(-) antisymmetric stretch and the amide-I' of the synthetic peptide analogues of the Ca(2+)-binding sites are discussed.

We recently reported that a computationally designed catalyst nicknamed AlleyCat facilitates C-H proton abstraction in Kemp elimination at neutral pH in a selective and calcium-dependent fashion by a factor of approximately 100,000 (Korendovych et al. in Proc. Natl. Acad. Sci. USA 108:6823, 2011). Kemp elimination produced a colored product that can be easily read out, thus making AlleyCat a catalytically amplified metal sensor for calcium. Here we report that metal-binding EF-hand motifs in AlleyCat could be redesigned to incorporate trivalent metal ions without significant loss of catalytic activity. Mutation of a single neutral residue at position 9 of each of the EF-hands to glutamate results in almost a two orders of magnitude improvement of selectivity for trivalent metal ions over calcium. Development of this new lanthanide-dependent switchable Kemp eliminase, named CuSeCat EE, provides the foundation for further selectivity improvement and broadening the scope of the repertoire of metals for sensing. A concerted effort in the design of switchable enzymes has many environmental, sensing, and metal ion tracking applications.

Ca(2+) binding proteins (CaBPs) are important regulators of neuronal Ca(2+) signaling acting either as buffers that shape Ca(2+) transients and Ca(2+) diffusion and/or as Ca(2+) sensors. The diffusional mobility represents a crucial functional parameter of CaBPs describing their range of action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells and its absence leads to impaired motor control. We quantified the diffusional mobility of dye labelled CR in mouse granule cells using two photon fluorescence recovery after photobleaching (FRAP). We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials which were associated with dendritic Ca(2+) transients the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF hand 5 of CR. Our results suggest long lasting Ca(2+) dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca(2+) buffer and point towards a Ca(2+) sensor function of CR.

Double mutation of Q41L and K75I in the N-domain of calmodulin (N-Cam) stabilizes the closed form of N-Cam such that binding of Ca

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca2+ signaling may play a central role in this process. Free Ca2+ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca2+ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca2+ uniporter machinery in mammals. MICU binds Ca2+ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca2+ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca2+ in the matrix. Furthermore, Ca2+ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca2+ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca2+ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca2+ uptake by moderating influx, thereby shaping Ca2+ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca2+ signaling in plants.

The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase–derived reactive oxygen species (ROS) stimulate a Ca 2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana . We found that Ca 2+ , in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca 2+ can determine cell shape.

Calmodulin is a representative calcium-binding protein comprised of four Ca