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Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca2+-binding superfamily. NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GG A U A U loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro. [ABSTRACT FROM AUTHOR]

• Ca2+ is a ubiquitous intracellular secondary messenger regulating the signal transduction pathway through signals encoded by different Ca2+ signatures. • Ca2+-binding proteins have a conserved EF-hand motif, which plays key role in plant growth and development. • EF-hand motif containing proteins plays significant role in the regulation of stress responses in plants. Calcium (Ca2+) is an essential element for the growth and development and provides rigidity to plant cells in calcium pectate form, and also involved in microtubule formation, cell proliferation, and stress tolerance. Ca2+ is a ubiquitous intracellular secondary messenger regulating the signal transduction pathway through signals encoded by different Ca2+ signatures like Ca2+ sensors, Calmodulins (CaM), Calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs) in plants. A fluctuation in response to an external or extracellular stimulus increases the cytoplasmic calcium [Ca2+] cyt concentration. This increase in Ca2+ concentration is sensed by proteins present in the cell, and these proteins interact with increasing Ca2+ ions and lead to change in the conformation of proteins from closed to open and allow other signal transduction molecules for binding and activation. The proteins which bind with Ca2+ are called Ca2+-binding proteins or Ca2+ sensors (with or without EF-hand motif). Ca2+ sensors like CDPK regulate the expression of genes under various abiotic and biotic stresses. Ca2+ regulates the cell cycle by participating in nuclear envelope breakdown and reformation, cleavage furrow formation, and cell plate formation. Ca2+ signaling also cross-talk with other signaling molecules like reactive oxidative species (ROS), MAPKs, ABA, nitric oxide (NO), and osmolytes, results in massive stress response. External stimuli like light, heat, drought, salinity, cold, chemical, or elicitor released after the pathogenic attack, etc. are responsible for activation of Ca2+ signaling. Ca2+ influx is usually a diffusion process through channels, so there is no need for energy, but through the transporter, the influx of Ca2+ requires energy in the form of ATP. Active transport of Ca2+ takes place via Ca2+-ATPase and H+-Ca2+ antiporter. This review describes the importance of Ca2+ signaling, Ca2+ channels, Ca2+ transporters, Ca2+ sensors, and EF-hand motif in plants to cope with various environmental cues. [Display omitted] [ABSTRACT FROM AUTHOR]

Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca2+-binding superfamily. NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGAUAU loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro.

Proteins' fuzziness are features for communicating changes in cell signaling instigated by binding with secondary messengers, such as calcium ions, associated with the coordination of muscle contraction, neurotransmitter release, and gene expression. Binding with the disordered parts of a protein, calcium ions must balance their charge states with the shape of calcium-binding proteins and their versatile pool of partners depending on the circumstances they transmit, but it is unclear whether the limited experimental data available can be used to train models to accurately predict the charges of calcium-binding protein variants. Here, we developed a chemistry-informed, machine-learning algorithm that implements a game theoretic approach to explain the output of a machine-learning model without the prerequisite of an excessively large database for high-performance prediction of atomic charges. We used the ab initio electronic structure data representing calcium ions and the structures of the disordered segments of calcium-binding peptides with surrounding water molecules to train several explainable models. Network theory was used to extract the topological features of atomic interactions in the structurally complex data dictated by the coordination chemistry of a calcium ion, a potent indicator of its charge state in protein. With our designs, we provided a framework of explainable machine learning model to annotate atomic charges of calcium ions in calcium-binding proteins with domain knowledge in response to the chemical changes in an environment based on the limited size of scientific data in a genome space.

Centrin-1, a Ca2+ sensor protein of the centrin family is a crucial player for cell division in eukaryotes and plays a key role in the microtubule organising centre. Despite being regarded as a calcium sensor with a matched structure to calmodulin/troponin C, the protein undergoes mild changes in conformation and binds Ca2+ with moderate affinity. We present an in-depth analysis of the Ca2+ sensing by individual EF-hand motifs of centrin-1 and address unsolved questions of the rationales for moderate affinity and conformational transitions of the protein. Employing the more sensitive approach of Trp scanning of individual EF-hand motif, we have undertaken an exhaustive investigation of Ca2+ binding to individual EF-hand motifs, named EF1 to EF4. All four EF-hand motifs of centrin-1 are structural as all of them bind both Ca2+ and Mg2+. EF1 and EF4 are the most flexible sites as they undergo drastic conformational changes following Ca2+ binding, whereas EF3 responds to Ca2+ minimally. On the other hand, EF2 moves towards the protein surface upon binding Ca2+. The independent filling mode of Ca2+ to EF-hand motifs and lack of intermotif communication explain the lack of cooperativity of binding, thus constraining centrin-1 to a moderate affinity binding protein. Thus, centrin-1 is distinct from other calcium sensors such as calmodulin. • Centrin-1, a "calmodulin-like" protein lacks regulatory or Ca2+ specific EF-hand motifs. • EF1 and EF4 motifs are most responsive sites for Ca2+ binding, hence form the sites for target binding. • Intermotif communication is absent in centrin-1. [ABSTRACT FROM AUTHOR]

Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn2+ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 106 M-1) and one low-affinity (2 × 105 M-1) Zn2+-binding sites, whereas Mg2+-loaded and Ca2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn2+-binding sites of apo- and Mg2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn2+ concentrations displace Mg2+ from the EF-hands and decrease the stoichiometry of Ca2+ binding. Meanwhile, one of the EF-hands of Zn2+-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn2+ binding to Ca2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca2+-free NCS-1 and favours the interaction of its Ca2+-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.

The helix-loop-helix (i.e., EF-hand) is the most common motif in the superfamily of Ca2+-binding proteins. Parvalbumins, as the classical EF-hand proteins, are associated with the muscle relaxation/contration, the calcium buffering etc. The previous researches focus more of interest on the ion coupled/decoupled mechanism using molecular dynamics (MD) computations. We developed the novel approach instead of MD, in which the Landau free energy was introduced to describe the protein in term of the skeletal Cα chain. The unfolding and folding processes were simulated by the Glauber algorithm under the repeated heating and cooling cycles. The high-quality crystal structure (2PVB) was as the trigger of non-equilibrium dynamics simulation for parvalbumin-β (Parv). The evolution of three dimensional structure during the folding was displayed for the residues 8-64 fragment in Parv. The phenomenon of helix nucleation was observed, that was, the 310 helix at residues 35-37 and the front of α-helix at residues 40-50 were firstly formed in the folding process. We also found two potential misfoldings which were caused by the distortion of the local structures at residues 35-37, 45-50. [ABSTRACT FROM AUTHOR]

Mitochondrial calcium uptake ( MICU ) plays a vital role in the regulation of mitochondrial calcium homeostasis, and, consequently, influences calcium signaling transduction. Although genes involved in mitochondrial calcium uptake have been well studied in animals, less is known about their ubiquity and function in plants. In this study, we identified 96 MICU genes in land plants. On the basis of phylogenetic analysis of MICU proteins, they were classified into three clades: MICU from eudicots (Clade I), from monocots (Clade II), and from a basal angiosperm, a bryophyte, and a lycophyte (Clade III). Pairwise identity analysis across all MICU proteins showed that they are highly conserved among land plants at the protein level. Conserved motif analysis showed that most MICU proteins contained three EF-hands, and an additional EF-hand motif first identified in the MICU of Arabidopsis thaliana but not mammals was found in all 96 putative MICU proteins. This suggests that a cellular pathway of calcium uptake and signaling that requires three EF-hand motifs is evolutionarily conserved in plants. In addition, we discovered that MICU -defective mutants of Arabidopsis thaliana exhibited longer roots than wild-type under high calcium stress. Concurrently, the mRNA transcription levels of MICU were decreased under high calcium conditions. These results suggest that loss-of-function mutations of MICU may have potential roles in helping plants resist high calcium stress. This study provides clues to the possible role of plant MICU in mitochondrial calcium uptake, as well as useful information to support further studies on MICU function in plants. [ABSTRACT FROM AUTHOR]

cf3 -Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3 -CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3 -aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3 -aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3- CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p -hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3 -aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3 -CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3 -aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3 -aequorin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Calcium ion (Ca2+) is a universal second messenger that plays a critical role in plant responses to diverse physiological and environmental stimuli. The stimulus-specific signals are perceived and decoded by a series of Ca2+ binding proteins serving as Ca2+ sensors. The majority of Ca2+ sensors possess the EF-hand motif, a helix-loop-helix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPK-related protein kinases, 2 Ca2+- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related cis-elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca2+-binding ability of these EF-hand proteins, four CMLs (CML1, CML13, CML39, and CML95) were randomly selected for SDS–PAGE mobility-shift assay in the presence and absence of Ca2+. Results showed that all of them have the ability to bind Ca2+. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca2+ signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses.

Mus musculus centrin 1 ( MmCen1) is located at the cilium of photoreceptor cells connecting the outer segment through signal transduction components to the metabolically active inner segment. In the cilium, MmCen1 is involved in the translocation of transducin between compartments as a result of photoreceptor activation. In this study, we report the crystal structure of wild-type MmCen1 and its Ca-binding properties using structure-based mutagenesis. The crystal structure exhibits three structural features, i.e. four Ca equally occupied at each EF-hand motif, structural changes accompanying helix motion at the N- and C-lobes, and adoption of N-C type dimerization when Ca binds to EF-hand I and II in the N-lobe. The presence of MmCen1 dimers was confirmed in solution by native PAGE. Isothermal titration calorimetry data showed sequential binding of Ca at four independent sites. Mutations S45A and D49A in EF-hand I alone disrupted the Ca-binding property of the wild-type protein. Based on the crystal structure of MmCen1, we suggest that a dimerization mode between the N- and C-lobes may be required by Ca binding at the N-lobe. [ABSTRACT FROM AUTHOR]

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.

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca2+ concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca2+/Mg2+ association to DREAM. Our data indicate a minor impact of Ca2+ association on the overall structure of the N- and C-terminal domains, although Ca2+ binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca2+ bound forms of the protein. Time-resolved fluorescence data indicate that Ca2+binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca2+ association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca2+ and Mg2+ suggesting that, under physiological conditions, Ca2+ free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions. [ABSTRACT FROM AUTHOR]

Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca2+. hRCN1 is a member of the CREC family localized in the secretory pathway, and its cellular function remains unclear. In this study, we established a new bacterial expression and purification procedure for hRCN1. We observed that hRCN1 binds Ca2+ in a cooperative manner and the Ca2+ binding caused an increase in the α-helix content of hRCN1. On the other hand, hRCN1 did not change the structure with Mg2+ loading. hRCN1 is a monomeric protein, and its overall structure became more compact upon Ca2+ binding, as revealed by gel-filtration column chromatography and small-angle X-ray scattering. This is the first report of conformational changes in the CREC family upon Ca2+ binding. Our data suggest that CREC family member interactions with target proteins are regulated in the secretory pathway by conformational changes upon Ca2+ binding. [ABSTRACT FROM AUTHOR]

The FK506-binding protein (FKBP) family consists of proteins with a variety of protein-protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl-prolyl cis-trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 ( FKBP7), FKBP60 ( FKBP9), and FKBP65 ( FKBP10), are unique among all FKBPs as they contain the EF-hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP-type domain as well as of the EF-hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand-containing FKBPs. The EF-hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP-type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF-hand motifs. [ABSTRACT FROM AUTHOR]

The EF-hand motif (helix-loop-helix) is a Ca2+-binding domain that is common among many intracellular Ca2+-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 Ca2+-loaded state although the native peptide ( L-type only) showed a band at about 1555 cm−1 in the Ca2+-loaded state, which is assigned to the side-chain COO− group of Glu at the 12th position, serving as the ligand for Ca2+ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca2+ 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 Ca2+-binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342-347, 2013. [ABSTRACT FROM AUTHOR]

Abstract: Plastins are Ca2+-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells. Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca2+, but the sensitivity to Ca2+ is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca2+ ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca2+ and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca2+-dependent activation, while these regions are not required for phosphorylation-induced ROS production. [ABSTRACT FROM PUBLISHER]

Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them . Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques. [ABSTRACT FROM AUTHOR]

Abstract: Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca2+ -binding proteins. As predicted, NcSP84 exhibited Ca2+-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca2+ tended to decline discretely, depending on the concentration of CaCl2 with which it was mixed for 1h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca2+ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes. [Copyright &y& Elsevier]

The article presents a study which analyzed the molecular components that intercede the response to hypoxia in rice and tomato through the detection of low-oxygen stress early response genes. The study examined plant materials through various procedures including stress treatment, treatment with cycloheximide and ruthenium red, and technical procedures. Results revealed the early transcriptional response to hypoxia of rice genes encoding EF-hand proteins.

Abstract: Calneuron I (CalnI) is a calmodulin-like protein that contains two functional EF-hand motifs at the N-terminal and a hydrophobic segment at the C-terminal. CalnI was cloned from the adult rat cortex and fused with GFP at its N-terminal. When expressed in bovine chromaffin cells, wild-type CalnI was localized at the plasma membrane. However, a mutant that lacked the hydrophobic segment was localized in the cytosol and nucleus, while a Ca2+-binding-deficient mutant was found in the cytosol and at the plasma membrane. Evaluation using the whole-cell patch-clamp technique revealed that Ca2+ currents were inhibited by both wild-type CalnI and the Ca2+-binding-deficient mutant. When the bovine N-type Ca2+ channel was expressed in 293T cells, Ca2+ currents were mostly inhibited by co-expression of CalnI, but not by the mutant without the hydrophobic tail. These results suggest that CalnI attenuates Ca2+ channel activity and that its subcellular localization is important for this effect. [Copyright &y& Elsevier]

Abstract: We performed comparative proteomic analyses of pituitary tumor-derived cell lines, and found a new protein, preliminarily called hydrophobestin, which was produced only in somatotrophic cells, MtT/S, but not in non-hormone-producing cells, MtT/E. Hydrophobestin is encoded by the cell growth regulatory gene, Cgr11, which is known to have growth-suppressive potential in several cell lines. We have now sought to investigate the underlying events responsible for cell growth inhibition by hydrophobestin. Immunocytochemisty revealed that hydrophobestin is localized in the Golgi apparatus of MtT/S cells and Cgr11-transfected MtT/E cells. The apparent molecular mass of the protein was determined by Westerm blot analysis of conditioned culture medium of MtT/S cells. Our data show that hydrophobestin is a secretory protein localized in the pituitary gland, adrenal gland, digestive tract, reproductive organs, and kidney. We also found that hydrophobestin promotes compact monolayer cell aggregates in PC12 cells transfected with Cgr11, however, non-transfected, vector- or EF-hand motif-deleted (ΔEF) Cgr11-transfected PC12 cells cannot form compact cell colonies. An antibody recognizing EF-hand motifs showed strong staining in the intercellular space of both Cgr11-transfected PC12 cells and MtT/S cells (Cgr11-expressing cells). Our data suggest that hydrophobestin-mediated cell adhesion may regulate cell growth through compact cell attachment. [Copyright &y& Elsevier]

Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (~30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue “Stains-all” dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli. [ABSTRACT FROM AUTHOR]

Abstract: The GUCA1A gene encodes a guanylate cyclase activating protein (GCAP1) that is involved in regulation of phototransduction in the vertebrate retina. We discovered a novel C312A transversion in exon 2 of the human GUCA1A gene, replacing Asn-104 (N104) in GCAP1 with Lys (K), in two affected members of a family with dominant cone dystrophy. The mutation N104K is located in the third EF-hand motif (EF3) shown previously to be instrumental in converting Ca2+-free GCAP1 to a GC inhibitor in the Ca2+-bound form. In one patient, rod ERGs were fairly stable over a 12-year-period whereas 30Hz flicker ERG and single-flash cone ERGs declined. In both patients, double-flash ERGs showed that rod recovery from an intense test flash was significantly delayed. The EC50 for GC stimulation shifted from ∼250nM in wild-type GCAP1 to ∼800nM in the GCAP1(N104K) mutant suggesting inability of the mutant to assume an inactive form under physiological conditions. The replacement of N104 by K in GCAP1 is the first naturally occurring mutation identified in the EF3 loop. The rod recovery delays observed in double-flash ERG of affected patients suggest a novel dominant-negative effect that slows GC stimulation. [Copyright &y& Elsevier]

Abstract: Human EFHC1 is a member of the EF-hand superfamily of Ca2+-binding proteins with three DM10 domains of unclear function. Point mutations in the EFHC1 gene are related to juvenile myoclonic epilepsy, a fairly common idiopathic generalized epilepsy. Here, we report the first structural and thermodynamic analyses of the EFHC1C-terminus (residues 403–640; named EFHC1C), comprising the last DM10 domain and the EF-hand motif. Circular dichroism spectroscopy revealed that the secondary structure of EFHC1C is composed by 34% of α-helices and 17% of β-strands. Size exclusion chromatography and mass spectrometry showed that under oxidizing condition EFHC1C dimerizes through the formation of disulfide bond. Tandem mass spectrometry (MS/MS) analysis of peptides generated by trypsin digestion suggests that the Cys575 is involved in intermolecular S–S bond. In addition, DTNB assay showed that each reduced EFHC1C molecule has one accessible free thiol. Isothermal titration calorimetry (ITC) showed that while the interaction between Ca2+ and EFHC1C is enthalpically driven (ΔH =−58.6 to −67kJ/mol and TΔS =−22.5 to −31kJ/mol) the interaction between Mg2+ and EFHC1C involves an entropic gain, and is ∼5 times less enthalpically favorable (ΔH =−11.7 to −14kJ/mol and TΔS =21.9 to 19kJ/mol) than for Ca2+ binding. It was also found that under reducing condition Ca2+ or Mg2+ ions bind to EFHC1C in a 1/1 molar ratio, while under oxidizing condition this ratio is reduced, showing that EFHC1C dimerization blocks Ca2+ and Mg2+ binding. [Copyright &y& Elsevier]

Abstract: Nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2) are multifunctional proteins that interact with Ca2+, nucleic acids and various regulatory proteins in different signaling pathways. So far, our understanding of the regulation of the biological functions of nucleobindins remains limited. In our proteome-wide selection for downstream caspase substrates, both NUCB1 and NUCB2 are found to be the downstream substrates of caspases. We report here the detailed analyses of the cleavage of nucleobindins by caspases. Significantly, the caspase cleavage sites are located exactly at one of the Ca2+-binding EF-hand motifs. Our results suggest that the functions of nucleobindins could be modulated by caspase-mediated cleavage in apoptosis. [Copyright &y& Elsevier]

Abstract: BNIP-2 and BNIP-XL are BCH domain-containing proteins that are implicated in programmed cell death. It has been reported that overexpression of BNIP-2 in neuroblastoma cell lines resulted in massive cell death, whereas BNIP-XL was upregulated during NGF-depletion-induced apoptosis in neuroblastoma and was involved in the regulation of differentiation, survival, and aggressiveness of tumor cells. Despite their importance in apoptosis, our understanding of BNIP-2 containing proteins is limited. In this communication, we demonstrate that both BNIP-2 and BNIP-XL are cleaved by caspases during apoptosis. Significantly, the caspase cleavage sites on BNIP-2 are located on its N-terminal EF-hand motif, while that on BNIP-XL is located upstream of the C-terminal BCH domain. Our results suggest that the caspase-mediated cleavage of BNIP-2 and BNIP-XL could result in the release of the BCH domain or smaller fragments that are crucial for their proapoptotic activities. [Copyright &y& Elsevier]

Abstract: Calmodulin (CaM) is a 16.8-kDa calcium-binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helix–loop–helix calcium-binding motif. CaM is composed of N- and C-terminal globular domains (N-CaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3-Å structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single-wavelength anomalous diffraction phasing of a selenomethionyl N-CaM. Our zinc-bound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-calmodulin (apo-CaM), despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a cross-linked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggest that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either stepwise conformational transition or flexible, dynamic energetic state sampling-based transition. [Copyright &y& Elsevier]

Abstract: The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix–loop–helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its α-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant ΔcabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera. [Copyright &y& Elsevier]

United-residue potentials are derived for interactions of the calcium cation with polypeptide chains in energy-based prediction of protein structure with a united-residue (UNRES) force-field. Specific potentials were derived for the interaction of the calcium cation with the Asp, Glu, Asn, and Gln side chains and the peptide group. The analytical expressions for the interaction energies for each of these amino acids were obtained by averaging the electrostatic interaction energy, expressed by a multipole series over the dihedral angles not considered in the united-residue model, that is, the side-chain dihedral angles χ and the dihedral angles λ for the rotation of peptide groups about the Cα•••Cα virtual-bond axes. For the side-chains that do not interact favorably with calcium, simple excluded-volume potentials were introduced. The parameters of the potentials were obtained from ab initio quantum mechanical calculations of model systems at the Restricted Hartree-Fock (RHF) level with the 6-31G(d,p) basis set. The energy surfaces of pairs consisting of Ca2+-acetate, Ca2+-propionate, Ca2+-acetamide, Ca2+-propionamide, and Ca2+-N-methylacetamide systems (modeling the Ca2+-Asp−, Ca2+-Glu−, Ca2+-Asn, Ca2+-Gln, and Ca2+-peptide group interactions) at different distances and orientations were calculated. For each pair, the restricted free energy (RFE) surfaces were calculated by numerical integration over the degrees of freedom lost when switching from the all-atom model to the united-residue model. Finally, the analytical expressions for each pair were fitted to the RFE surfaces. This force-field was able to distinguish the EF-hand motif from all potential binding sites in the crystal structures of bovine α-lactalbumin, whiting parvalbumin, calbindin D9K, and apo-calbindin D9K. [ABSTRACT FROM AUTHOR]

Calcium binding proteins of the S100 family play a central role in many intra- and extracellular processes and abnormal expression was observed in various tumors and human diseases. We have identified a unique new member of this gene family: S100A16 which is the S100 protein widest distributed in human and which is highly conserved in mammals. Up-regulation of S100A16 was found in many tumors implying a central cellular function related to malignant transformation. The gene was composed of four exons, two of which alternatively initiated transcription. Three different transcripts suggested a complex regulation of the S100A16 gene. Moreover, CAG repeats were identified in the transcribed region which might be associated with diseases of the nervous system. All human transcripts encoded the same, typically small S100 protein of 103 amino acids containing the S100-specific motif of two distinct EF-hands. S100A16 was mapped within the S100 gene cluster on human chromosome 1q21, a region that is frequently rearranged in tumors. [Copyright &y& Elsevier]

We attempted to isolate genes involved in neuronal differentiation from a cell line 2Y-3t newly established from a mouse cerebellum. 2Y-3t cells proliferate in serum-containing medium and differentiate into neurons in serum-free medium. We took a subtraction method to isolate genes differerentially expressed in differentiated cells and 17 cDNA clones were isolated. Functions of 6 cDNA clones are unknown. No. 60 cDNA clone has 723 nucleotides encoding 240 amino acid residues. It contains two putative EF-hand motifs and a coiled-coil region at C terminal end. Expression of the clone was undetectable at embryonic stage and was increased in brain during development. In situ hybridization showed that the expression was observed predominantly in neurons, suggesting that the protein may play roles in the neuronal differentiation and function. [Copyright &y& Elsevier]

The apoptosis linked gene (ALG-2) is a 22 kDa Ca2+-binding protein of the penta EF-hand motif family. ALG-2 was discovered in a "death trap" assay using T-cell receptor-mediated apoptosis. Depletion of ALG-2 using an anti-sense ALG-2 construct inhibits apoptosis that is induced by several stimuli, such as staurosporin, dexamethason, Fas, and glucocorticoid. The Ca2+ dependent function of ALG-2 is consistent with the observation that the cytoplasmic Ca2+ level is elevated in apoptotic cells. We found that ALG-2 interacted specifically with the carboxy-terminal region of HEED using a yeast two-hybrid assay system. The mutants of HEED were constructed by deleting five WD repeat motifs one by one from the C-terminus of the protein. These mutants of ALG-2 were made by combining the EF hand Ca2+ binding motifs in various ways. Mapping of the interaction sites, using each of the mutants, revealed that the interaction between HEED and a third EF-hand motif of ALG-2 was stronger than the other combination. [ABSTRACT FROM AUTHOR]

Recent very rapid developments in genome and EST projects have identified an increasing number of gene products homologous to those that were previously identified by other methods. Calpain is no exception. At the time this review is written, 83 genes from 23 living organisms have been identified in the database to encode amino acid sequences showing significant similarities to the protease domain of “conventional” calpain, which was first purified as a homogeneous protein in 1978. Progress in genome/ EST projects has occurred so quickly that there seems to be some confusion as to the identity of each calpain molecule. This review will attempt to clarify all calpain homo-logues, to describe the common and differing features of calpain homologues in terms of structure-function relationship, and to discuss the evolutionary process of calpain. [ABSTRACT FROM AUTHOR]

Calcium–binding proteins contain a variable number of motifs, termed EF–hands, which consist of two perpendicularly placed α–helices and an interhelical loop forming a single calcium–binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium–dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF–hand fish allergen, calcium–binding allergens were discovered in pollens of trees, grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium–binding allergens, these allergens may be important because of their ability to cross–sensitize allergic individuals. Conformation and stability as well as IgE recognition of calcium–binding allergens greatly depend on the presence of protein–bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium–binding allergens can be engineered by recombinant DNA technology for immunotherapy of sensitized patients. [ABSTRACT FROM AUTHOR]

TSD2, a gene necessary for DNA synthesis in Ustilago maydis, was cloned by complementation of the temperature sensitive growth defect of a mutant known previously as pol1-1 and renamed here tsd2-1. Linkage analysis established that the cloned fragment contained an allele of tsd2-1 and not a suppressor. DNA sequence determination of the cloned DNA fragment indicated the presence of a single large uninterrupted open reading frame capable of encoding a protein of 845 amino acids without homology to any known gene involved in DNA synthesis. TSD2 was found to be cell cycle-regulated and mRNA levels peaked in early S or G1 phase. [ABSTRACT FROM AUTHOR]

Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and β = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules. [ABSTRACT FROM AUTHOR]

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo -mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin‑calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers. • Determination of the secondary-structure distribution of mnemiopsin 2 in solution with or without calcium by SR-FTIR spectroscopy • Detection of more stable secondary structure in mutant apo -mnemiopsin 2 • Dependence of conformational changes at the secondary structures of mnemiopsin 2 on calcium concentrations. • Occurrence of the most noticeable structural changes of wild-type and mutant mnemiopsin 2 at optimum calcium concentrations 6 and 2 mM, respectively [ABSTRACT FROM AUTHOR]

Selenoprotein N (SEPN1) is critical to the normal muscular physiology. Mutation of SEPN1 can raise congenital muscular disorder in human. It is also central to maturation and structure of skeletal muscle in chicken. However, human SEPN1 contained an EF-hand motif, which was not found in chicken. And the biochemical and molecular characterization of chicken SEPN1 remains unclear. Hence, protein domains, transcription factors, and interactions of Ca2+ in SEPN1 were analyzed in silico to provide the divergence and homology between chicken and human in this work. The results showed that vertebrates' SEPN1 evolved from a common ancestor. Human and chicken's SEPN1 shared a conserved CUGS-helix domain with function in antioxidant protection. SEPN1 might be a downstream target of JNK pathway, and it could respond to multiple stresses. Human's SEPN1 might not combine with Ca2+ with a single EF-hand motif in calcium homeostasis, and chicken SEPN1 did not have the EF-hand motif in the prediction, indicating the EF-hand motif malfunctioned in chicken SEPN1., (© The Author(s) 2021. Published by Oxford University Press.)

Calcium (Ca2+) signaling is involved in the regulation of diverse biological functions through association with several proteins that enable them to respond to abiotic and biotic stresses. Though Ca2+-dependent signaling has been implicated in the regulation of several physiological processes in Chlamydomonas reinhardtii, Ca2+ sensor proteins are not characterized completely. C. reinhardtii has diverged from land plants lineage, but shares many common genes with animals, particularly those encoding proteins of the eukaryotic flagellum (or cilium) along with the basal body. Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is an important effector of Ca2+ signaling in animals, while calcineurin B-like proteins (CBLs) play an important role in Ca2+ sensing and signaling in plants. The present study led to the identification of 13 novel CBL-like Ca2+ sensors in C. reinhardtii genome. One of the archetypical genes of the newly identified candidate, CrCBL-like1 was characterized. The ability of CrCBL-like1 protein to sense as well as bind Ca2+ were validated using two-step Ca2+-binding kinetics. The CrCBL-like1 protein localized around the plasma membrane, basal bodies and in flagella, and interacted with voltage-gated Ca2+ channel protein present abundantly in the flagella, indicating its involvement in the regulation of the Ca2+ concentration for flagellar movement. The CrCBL-like1 transcript and protein expression were also found to respond to abiotic stresses, suggesting its involvement in diverse physiological processes. Thus, the present study identifies novel Ca2+ sensors and sheds light on key players involved in Ca2+signaling in C. reinhardtii, which could further be extrapolated to understand the evolution of Ca2+ mediated signaling in other eukaryotes., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The S100 family proteins possess a variety of intracellular and extracellular functions. They interact with multiple receptors and signal transducers to regulate pathways that govern inflammation, cell differentiation, proliferation, energy metabolism, apoptosis, calcium homeostasis, cell cytoskeleton and microbial resistance. S100 proteins are also emerging as novel diagnostic markers for identifying and monitoring various diseases. Strategies aimed at targeting S100-mediated signaling pathways hold a great potential in developing novel therapeutics for multiple diseases. In this chapter, we aim to summarize the current knowledge about the role of S100 family proteins in health and disease with a major focus on their role in inflammatory conditions., (© 2020 Elsevier Inc. All rights reserved.)