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Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found that the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn 2+ -promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca 2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

Lanmodulin is the first natural, selective macrochelator for f elements-a protein that binds lanthanides with picomolar affinity at 3 EF hands, motifs that instead bind calcium in most other proteins. Here, we use sensitized terbium luminescence to probe the mechanism of lanthanide recognition by this protein as well as to develop a terbium-specific biosensor that can be applied directly in environmental samples. By incorporating tryptophan residues into specific EF hands, we infer the order of metal binding of these three sites. Despite lanmodulin's remarkable lanthanide binding properties, its coordination of approximately two solvent molecules per site (by luminescence lifetime) and metal dissociation kinetics ( k off = 0.02-0.05 s -1 , by stopped-flow fluorescence) are revealed to be rather ordinary among EF hands; what sets lanmodulin apart is that metal association is nearly diffusion limited ( k on ≈ 10 9 M -1 s -1 ). Finally, we show that Trp-substituted lanmodulin can quantify 3 ppb (18 nM) terbium directly in acid mine drainage at pH 3.2 in the presence of a 100-fold excess of other rare earths and a 100 000-fold excess of other metals using a plate reader. These studies not only yield insight into lanmodulin's mechanism of lanthanide recognition and the structures of its metal binding sites but also show that this protein's unique combination of affinity and selectivity outperforms synthetic luminescence-based sensors, opening the door to rapid and inexpensive methods for selective sensing of individual lanthanides in the environment and in-line monitoring in industrial operations.

Voltage-gated sodium channels (Na v s) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Na v EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the Na V IQ motif. The EFL domain and IQ motif are contiguous regions of Na V cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown. Titrations monitored by steady-state fluorescence spectroscopy, structural studies with solution NMR, and computational modeling demonstrated for the first time that both domains of (Ca 2+ ) 4 -CaM (but not apo CaM) directly bind two sites in the N-terminal domain (NTD) of A-type FGF splice variants (FGF11A, FGF12A, FGF13A, and FGF14A) with high affinity. The weaker of the (Ca 2+ ) 4 -CaM-binding sites was known via electrophysiology to have a role in long-term inactivation of the channel but not known to bind CaM. FGF12A binding to a complex of CaM associated with a fragment of the Na V 1.2 CTD increased the Ca 2+ -binding affinity of both CaM domains, consistent with (Ca 2+ ) 4 -CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with Na V IQ motifs for (Ca 2+ ) 4 -CaM. During spikes in the cytosolic Ca 2+ concentration that accompany an action potential, CaM may translocate from the Na V IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Mutations of the voltage-gated sodium channel gene (SCN4A), which encodes Nav1.4, cause nondystrophic myotonia that occasionally is associated with severe apnea and laryngospasm. There are case reports of nondystrophic myotonia due to mutations in the C-terminal tail (CTerm) of Nav1.4, but the functional analysis is scarce., Methods: We present two families with nondystrophic myotonia harboring a novel heterozygous mutation (E1702del) and a known heterozygous mutation (E1702K)., Results: The proband with E1702K exhibited repeated rhabdomyolysis, and the daughter showed laryngospasm and cyanosis. Functional analysis of the two mutations as well as another known heterozygous mutation (T1700_E1703del), all located on EF hand-like motif in CTerm, was conducted with whole-cell recording of heterologously expressed channel. All mutations displayed impaired fast inactivation., Discussion: The CTerm of Nav1.4 is vital for regulating fast inactivation. The study highlights the importance of accumulating pathological mutations of Nav1.4 and their functional analysis data., (© 2020 Wiley Periodicals, Inc.)

Store operated calcium (Ca 2+ ) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca 2+ store depletion causes Orai1-composed Ca 2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca 2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca 2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca 2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca 2+ concentrations; however, no direct structural view of the Ca 2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca 2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca 2+ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca 2+ . Remarkably, we also detected an α-helix (+Ca 2+ ) to β-strand (-Ca 2+ ) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca 2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

STIM and ORAI proteins play a fundamental role in calcium signaling, allowing for calcium influx through the plasma membrane upon depletion of intracellular stores, in a process known as store-operated Ca 2+ entry. Point mutations that lead to gain-of-function activity of either STIM1 or ORAI1 are responsible for a cluster of ultra-rare syndromes characterized by motor disturbances and platelet dysfunction. The prevalence of these disorders is at present unknown. In this study, we describe the generation and characterization of a knock-in mouse model (KI - STIM1 I115F ) that bears a clinically relevant mutation located in one of the two calcium-sensing EF-hand motifs of STIM1. The mouse colony is viable and fertile. Myotubes from these mice show an increased store-operated Ca 2+ entry, as predicted. This most likely causes the dystrophic muscle phenotype observed, which worsens with age. Such histological features are not accompanied by a significant increase in creatine kinase. However, animals have significantly worse performance in rotarod and treadmill tests, showing increased susceptibility to fatigue, in analogy to the human disease. The mice also show increased bleeding time and thrombocytopenia, as well as an unexpected defect in the myeloid lineage and in natural killer cells. The present model, together with recently described models bearing the R304W mutation (located on the coiled-coil domain in the cytosolic side of STIM1), represents an ideal platform to characterize the disorder and test therapeutic strategies for patients with STIM1 mutations, currently without therapeutic solutions.This article has an associated First Person interview with Celia Cordero-Sanchez, co-first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I ('black' cluster) and cluster II ('grey' cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these 'black' and 'gray' clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1-2 strong calcium binding sites (K 1  = 4 × 106 M -1 ) and two cooperative low affinity (K 2  = 4 × 104 M -1 ) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The mitochondrial uniporter is a Ca 2+ -channel complex resident within the organelle's inner membrane. In mammalian cells the uniporter's activity is regulated by Ca 2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca 2+ -binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca 2+ -binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1-EF3 interface likely accounts for the structural and functional coupling between the Ca 2+ -binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca 2+ -binding cooperativity and dominant negative effect of mutation of the Ca 2+ -binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2's C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2's function in cells. We propose that in the MICU1-MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter., Competing Interests: Conflict of interest statement: V.K.M. is a paid advisor to Janssen Pharmaceuticals, Raze Therapeutics, and 5AM Ventures.

Recently we found two highly conserved structural motifs in the members of the EF-hand protein family, which provide a supporting scaffold for their Ca 2+ binding loops. Each structural motif is formed by a cluster of three amino acids. These clusters were called 'black' cluster (cluster I) and 'gray' cluster (cluster II). In the present work, we studied the relationship between the location of the 'black' and 'gray' structural clusters in parvalbumins and the location of the amino acid sequence regions with a tendency for intrinsic disorder. This analysis revealed that in parvalbumins, the residues in the vicinity of the conserved structural clusters constitute parts of the conserved motifs enriched in the disorder-promoting residues. Therefore, the clusters found in parvalbumins are characterized not only by the presence of conserved amino acid residues, but also by the conserved distribution of the intrinsic disorder predisposition within their sequences, suggesting the presence of conserved structural dynamics in the apo-forms of parvalbumins, where the black cluster appears to have greater mobility than the gray cluster., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Giardia intestinalis is a parasite that inhabits the small intestine of humans and other mammals, causing a disease that can manifest itself with acute diarrhea. This parasite is an early divergent eukaryote with a compact genome and a life cycle composed of two distinct cell types: the trophozoite, the replicative form, and the cyst, the infectious form. Signal transduction pathways implicated in differentiation processes of G. intestinalis are largely unknown. Calcium, considered an essential messenger in cell signaling, has been shown to regulate a myriad of key cell processes including metabolism, motility, and exocytosis, among other important functions, through calcium-binding proteins (CaBPs). The most important and largest family of CaBPs is the EF-hand protein family. To investigate the nature of calcium signaling pathways present in this protozoan, an in silico analysis of the genome to identify genes encoding EF-hand proteins was undertaken. Twenty-eight sequences containing EF-hand domains were found; most of which have only a pair of domains, and half of the sequences were divergent or unique to Giardia. In addition, the transcription pattern for eight genes encoding EF-hand proteins was assessed during encystation. It was found that all the genes were differentially transcribed suggesting a different function in this process. The in silico results suggest that in G. intestinalis, calcium is involved in the regulation of protein phosphorylation through kinases and phosphatases.

EF hand (EFh) domain-containing proteins have been implicated in malignant progression, but their precise functional contributions are uncertain. Here, we report evidence that the EFh protein IBA2 promotes the proliferation of breast cancer cells by facilitating their transit through the G1-S cell-cycle transition. Mechanistic investigations revealed that IBA2 acted at the transcriptional level to promote the expression of the critical cell-cycle regulator cyclin D1. Clinically, we found that levels of IBA2 were significantly upregulated in breast cancer specimens, where its expression correlated positively with histologic grade. Our results suggest a key role for IBA2 in mammary tumorigenesis. Cancer Res; 76(15); 4535-45. ©2016 AACR., (©2016 American Association for Cancer Research.)

Calcium, as the most widely accepted messenger, plays an important role in plant stress responses through calcium-dependent signaling pathways. The calmodulin-like family genes (CMLs) encode Ca2+ sensors and function in signaling transduction in response to environmental stimuli. However, until now, the function of plant CML proteins, especially soybean CMLs, is largely unknown. Here, we isolated a Glycine soja CML protein GsCML27, with four conserved EF-hands domains, and identified it as a calcium-binding protein through far-UV CD spectroscopy. We further found that expression of GsCML27 was induced by bicarbonate, salt and osmotic stresses. Interestingly, ectopic expression of GsCML27 in Arabidopsis enhanced plant tolerance to bicarbonate stress, but decreased the salt and osmotic tolerance during the seed germination and early growth stages. Furthermore, we found that ectopic expression of GsCML27 decreases salt tolerance through modifying both the cellular ionic (Na+, K+) content and the osmotic stress regulation. GsCML27 ectopic expression also decreased the expression levels of osmotic stress-responsive genes. Moreover, we also showed that GsCML27 localized in the whole cell, including cytoplasm, plasma membrane and nucleus in Arabidopsis protoplasts and onion epidermal cells, and displayed high expression in roots and embryos. Together, these data present evidence that GsCML27 as a Ca2+-binding EF-hand protein plays a role in plant responses to bicarbonate, salt and osmotic stresses.

Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of (45)Ca(2+), heat stability and Ca(2+) dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca(2+) binding transcriptional regulators.

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF-GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF-GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in "equivalent-light" scenarios.

Caldendrin, L- and S-CaBP1 are CaM-like Ca2+-sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca2+-signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca2+ and Mg2+ with a global Kd in the low µM range. Interestingly, the Mg2+-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg2+-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca2+-binding to Mg2+-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca2+-binding, high Mg2+- and low Ca2+-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca2+ of Mg2+-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca2+-sensors.

Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca(2+) (stimulated by ∼1 µM free Ca(2+) ) suggesting that it can amplify transient Ca(2+) signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca(2+) -binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[(1) H,(1) H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca(2+) in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca(2+) -binding. To determine whether the EF-hand is responsible for Ca(2+) -sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na(+) /H(+) antiporter that increases intracellular Ca(2+) ) induced a 4- to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca(2+) . The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca(2+) -sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca(2+) -sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A, and E304A mutants. Interestingly, the monensin responses and Ca(2+) -sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca(2+) -sensing., (© 2013 Wiley Periodicals, Inc.)

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,andthere 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.

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.

It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca(2+) per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca(2+) were monitored using fluorescence technique.

In trematodes, there is a family of proteins which combine EF-hand-containing domains with dynein light chain (DLC)-like domains. A member of this family from the liver fluke, Fasciola hepatica-FhCaBP4-has been identified and characterised biochemically. FhCaBP4 has an N-terminal domain containing two imperfect EF-hand sequences and a C-terminal dynein light chain-like domain. Molecular modelling predicted that the two domains are joined by a flexible linker. Native gel electrophoresis demonstrated that FhCaBP4 binds to calcium, manganese, barium and strontium ions, but not to magnesium or zinc ions. The hydrophobic, fluorescent probe 8-anilinonaphthalene-1-sulphonate bound more tightly to FhCaBP4 in the presence of calcium ions. This suggests that the protein undergoes a conformational change on ion binding which increases the number of non-polar residues on the surface. FhCaBP4 was protected from limited proteolysis by the calmodulin antagonist W7, but not by trifluoperazine or praziquantel. Protein-protein cross-linking experiments showed that FhCaBP4 underwent calcium ion-dependent dimerisation. Since DLCs are commonly dimeric, it is likely that FhCaBP4 dimerises through this domain. The molecular model reveals that the calcium ion-binding site is located close to a key sequence in the DLC-like domain, suggesting a plausible mechanism for calcium-dependent dimerisation.

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

The B cell receptor (BCR)-elicited calcium flux results in activation of mature B cells. We have recently shown that the adaptor protein Swiprosin-1/EFhd2 (EFhd2) amplifies the BCR-induced calcium flux in B cell lines. EFhd2 is a calcium binding adaptor protein with two predicted EF-hands. Here we asked whether these domains are functional and control its function. Using a blot-overlay assay with radioactive calcium we show that both EF-hands of EFhd2 have an intrinsic capacity to bind calcium. Equilibrium centrifugation confirmed that EFhd2 binds 2 calcium ions, with an apparent Kd of 110 μM. Point mutations revealed that the conserved residues E116 and E152, which reside in the canonical calcium binding loop in EF-hands 1 and 2, are essential for calcium binding by EFhd2. These mutations as well as deletion of the EF-hands, in particular EF-hand 1, abolished the ability of EFhd2 to restore BCR-induced calcium signaling in EFhd2-deficient WEHI231 cells. N-terminal deletions, but not C-terminal deletions, acted similarly. Thus, the N-terminal part of EFhd2 as well as calcium binding to its EF-hands control the intracellular calcium concentration in response to BCR stimulation in WEHI231 cells. Hence, EFhd2 regulates the BCR-elicited calcium flux through a calcium-dependent positive feedback mechanism in WEHI231 cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The SPARC family of proteins represents a diverse group of proteins that modulate cell interaction with the extracellular milieu. The eight members of the SPARC protein family are modular in nature. Each shares a follistatin-like domain and an extracellular calcium binding E-F hand motif. In addition, each family member is secreted into the extracellular space. Some of the shared activities of this family include, regulation of extracellular matrix assembly and deposition, counter-adhesion, effects on extracellular protease activity, and modulation of growth factor/cytokine signaling pathways. Recently, several SPARC family members have been implicated in human disease pathogenesis. This review discusses recent advances in the understanding of the functional roles of the SPARC family of proteins in development and disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Calcium (Ca⁺²) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca⁺²-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca⁺² binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca⁺² binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca⁺² binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca⁺²/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca⁺²-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca⁺² is likely to be key player in Caenorhabditis cell signalling.

The calpains are physiologically important Ca(2+)-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+)](i). We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+) dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover., Competing Interests: The authors have declared that no competing interests exist.

Axotomized central neurons of most invertebrate species demonstrate a strong regenerative capacity, and as such may provide valuable molecular insights and new tools to promote axonal regeneration in injured mammalian neurons. In this study, we identified a novel molluscan protein, caltubin, ubiquitously expressed in central neurons of Lymnaea stagnalis and locally synthesized in regenerating neurites. Reduction of caltubin levels by gene silencing inhibits the outgrowth and regenerative ability of adult Lymnaea neurons and decreases local α- and β-tubulin levels in neurites. Caltubin binds to α- and/or β-tubulin in both Lymnaea and rodent neurons. Expression of caltubin in PC12 cells and mouse cortical neurons promotes NGF-induced axonal outgrowth and attenuates axonal retraction after injury. This is the first study illustrating that a xenoprotein can enhance outgrowth and prevent degeneration of injured mammalian neurons. These results may open up new avenues in molecular repair strategies through the insertion of molecular components of invertebrate regenerative pathways into mammalian neurons.

Calcium ions and calcium-binding proteins play a major role in many cellular processes, in particular skeletogenesis and bone formation. We report here the discovery of a novel S100 protein in fish and the analysis of its gene expression patterns. A 648-bp full-length cDNA encoding an 86-amino acid S100-like calcium-binding protein was identified through the subtractive hybridization of a gilthead seabream (Sparus aurata) cDNA library constructed to identify genes associated with in vitro mineralization. Deduced protein lacks an identifiable signal peptide and exhibits two EF-hand motifs characteristic of S100 proteins. Phylogenetic and bioinformatic analyses of S100 sequences suggested that gilthead seabream protein represents a novel and fish-specific member of the S100 protein family. Expression of S100-like gene was up-regulated during the in vitro mineralization of bone-derived cell lines and during seabream development, from larvae throughout adulthood, reflecting skeletogenesis. Restriction of S100-like gene expression to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish further confirmed the mineralogenic role of the protein in fish and emphasized the potential of S100-like as a marker of mineralizing cartilage in developing fish., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Calcium is a ubiquitous and essential secondary messenger in eukaryotic signal transduction pathways. Calcium binding protein, as a component of pathways, plays various roles in response to biotic and abiotic stresses, as well as in developmental processes in plants. In this study, a calcium binding protein gene, designated as TaCab1 (Triticum aestivum calcium binding EF-hand protein 1), was isolated and characterized from wheat leaves (cv. Suwon 11) infected by Puccinia striiformis f. sp. tritici by in silico cloning and reverse transcription PCR (RT-PCR). TaCab1 did not have an intron and was predicted to encode a 216 amino acid protein which possesses an N-terminal region with a signal peptide, a transmembrane domain, an EF-hand motif and a caleosin domain. The results of transient assays with constructs of TaCab1 with green fluorescent protein (GFP) gene indicated that TaCab1 encodes a transmembrane protein. Quantitative real-time PCR (qRT-PCR) analyses revealed that TaCab1 was highly expressed in leaves than roots and stems. Although up-regulated expression profiles of TaCab1 were quite similar in both incompatible and compatible interactions, its transcript accumulation in the compatible interaction was much higher than in the incompatible interaction. The transcription of TaCab1 was also up-regulated at different degrees after treated by phytohormones [abscisic acid, benzyl adenine, ethylene, methyl jasmonate and salicylic acid (SA)] and stress stimuli [wounding, low temperature, polyethylene glycol and high salinity]. These results suggest that TaCab1 is involved in the plant-pathogen recognition, symptom development, and the basal tolerance to biotic and abiotic stresses through the SA signaling pathway.

The interaction between calcineurin B homologous protein 2 (CHP2) and Na(+) /H(+) exchanger 1 (NHE1), two membrane proteins, is essential for protecting cells from serum deprivation-induced death. Although four putative EF-hands in CHP2 had been predicted for years, Ca²(+) -binding activities of these motifs have not been tested yet, their role in this process remain poorly understood. To identify Ca²(+) -binding motifs required for the stable formation of CHP2/NHE1 complexes, we developed a mutagenesis-based assay in PS120 cells. We found that (45) Ca²(+) bond to two EF-hand motifs (EF3 and 4) of CHP2 proteins with high affinity. Complex formation between CHP2 and the CHP2 binding domain of NHE1 resulted in a marked increase in the Ca²(+) -binding affinity of CHP2. Co-immunoprecipitation and distribution of GFP-tagged CHP2-EF3m/4m also indicated that Ca²(+) affected the membrane location of CHP2 to interact with NHE1. The C-terminal region of CHP2 contains a nuclear export sequence (NES). When the six leucines of NES were mutated to alanines, the resulting CHP2 protein was predominantly localized to the nucleus. Furthermore, mutation of the NES resulted in enhanced proliferation and oncogenic potential of HeLa cells. Together, these results show that calcium and NES control the subcellular distribution of CHP2 and then distinctively regulate cell proliferation., (© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca(2+)-binding sites, and mediates the effects of cytosolic Ca(2+). Minute changes in free intracellular Ca(2+) are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca(2+)/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca(2+)/CaMKs comes mostly from studies of the mammalian enzymes. Thus far very few fungal homologues of NCS-1 and Ca(2+)/CaMKs have been characterized and little is known about their cellular roles. In this minireview, we describe the known sequences, interactions with target proteins and cellular roles of NCS-1 and Ca(2+)/CaMKs in fungi., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: S100A2, a Ca(2+) -binding protein with two EF-hands, is a tumor suppressor in oral cancer. Helix III flanking the C-terminal EF-hand is implicated to participate in the interaction of S100A2 and its target(s). The aim of this study was to examine if the coding sequence polymorphism S100A2_185G>A, leading to the peptide 62 substitution of asparagine (AAC, A allele) for serine (AGC, G allele) in helix III, had modulation effects on S100A-mediated tumor suppression., Subjects and Methods: We sequenced the coding sequence of S100A2 gene in normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), eight oral cancer lines, and 54 pairwise oral cancer specimens. We also compared the in vitro anti-tumor effect of wildtype (G allele) and variant (A allele) S100A2 expression using cell proliferation, migration, invasion, and colony formation assays., Results: With the exception of CAL27 and SCC-15 cancer lines being heterozygotes of A and G alleles, the remaining oral cells were homozygotic in G alleles. No alterations of anti-growth, anti-migration, anti-invasion, and anti-colony formation were observed between variant and wildtype cells. Moreover, no minor S100A2_185A allele was detected in 54-pairwise clinical specimens., Conclusion: The coding sequence polymorphism S100A2_185G>A had no regulatory role in S100A2-mediated tumor suppression in oral cancer., (© 2010 John Wiley & Sons A/S.)

The function of protein phosphatases with EF-hand domains (PPEF) in mammals is not known. Large-scale expression profiling experiments suggest that PPEF expression may correlate with stress protective responses, cell survival, growth, proliferation, or neoplastic transformation. Apoptosis signal regulating kinase-1 (ASK1) is a MAP kinase kinase kinase implicated in cancer, cardiovascular and neurodegenerative diseases. ASK1 is activated by oxidative stress and induces pro-apoptotic or inflammatory signalling, largely via sustained activation of MAP kinases p38 and/or JNK. We identify human PPEF2 as a novel interacting partner and a negative regulator of ASK1. In COS-7 or HEK 293A cells treated with H(2)O(2), expression of PPEF2 abrogated sustained activation of p38 and one of the JNK p46 isoforms, and prevented ASK1-dependent caspase-3 cleavage and activation. PPEF2 efficiently suppressed H(2)O(2)-induced activation of ASK1. Overexpessed as well as endogenous ASK1 co-immunoprecipitated with PPEF2. PPEF2 was considerably more potent both as a suppressor of ASK1 activation and as its interacting partner as compared to protein phosphatase 5 (PP5), a well-known negative regulator of ASK1. PPEF2 was found to form complexes with endogenous Hsp70 and to a lesser extent Hsp90, which are also known interacting partners of PP5. These data identify, for the first time, a possible downstream signalling partner of a mammalian PPEF phosphatase, and suggest that, despite structural divergence, PPEF and PP5 phosphatases may share common interacting partners and functions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The complete amino acid sequence of a calcium-regulatory gene (denoted as Ab-CaReg I) was identified from the disk abalone Haliotis discus discus cDNA library. The Ab-CaReg I is composed of 176 amino acids and the calculated molecular mass and isoelectric point were 20 and 4.2, respectively. The sequence homology of Ab-CaReg I was 28-30 and 18-27% of known calmodulin and troponin C, respectively. Four characteristic calcium-binding EF hand motifs with some modifications at conserved positions of known homologous calmodulin genes were observed in the sequence. The tissue-specific transcription analysis and variation of mRNA transcription level of Ab-CaReg I in gills and mantle after animals were immersed in seawater containing 2000 ppm CaCl(2) was quantified by SYBR Green real-time PCR analysis. Transcription variation of Ab-CaReg I in hemocytes and gills followed by bacteria challenge (Vibrio alginolyticus, Vibrio parahaemolyticus and Listeria monocytogenes) was used to investigate Ab-CaReg I in immune responses. Transcripts of Ab-CaReg I mRNA were mainly detected in hemocytes, mantle, muscle, gills, digestive tract and hepatopancreas with highest expression in hemocytes. The CaCl(2) immersion significantly altered the Ab-CaReg I mRNA transcription level by 3 h, compared to animals in normal seawater (control). The mRNA expression of Ab-CaReg I in gills and hemocytes was upregulated significantly to 11-fold and 4-fold in 3 h compared to control (uninfected), respectively, in bacteria-challenged abalones. The results suggest that Ab-CaReg I could be effectively induced to maintain internal Ca(2+) homeostasis of the animal due to influx of Ca(2+) in the cells by external stimuli such as a high dose of Ca(2+) and pathogens like bacteria., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca(2+)-permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720-797), a flexible linker (798-827), and an oligomeric coiled coil domain (828-895). We found that PC2-EF binds Ca(2+) at a single site and undergoes Ca(2+)-dependent conformational changes, suggesting it is an essential element of Ca(2+)-sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca(2+)-bound PC2-EF. Human PC2-EF contains a divergent non-Ca(2+)-binding helix-loop-helix (HLH) motif packed against a canonical Ca(2+)-binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R(1) and R(2) relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca(2+)-bound states, consistent with a role for PC2-EF as a Ca(2+)-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca(2+)-dependent protein interactions. We propose that Ca(2+)-sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca(2+) dependence of PC2 channel activity by PC2-EF.

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.

The affinities of Ca(2+)-saturated and Ca(2+)-free calmodulin for a fluorescent reporter construct containing the PEP19 IQ domain differ by a factor of approximately 100, with K(d) values of 11.0 +/- 1.2 and 1128.4 +/- 176.5 muM, respectively, while the affinities of a reporter containing the neuromodulin IQ domain are essentially identical, with K(d) values of 2.9 +/- 0.3 and 2.4 +/- 0.3 muM, respectively. When Ca(2+) is bound only to the C-terminal pair of Ca(2+)-binding sites in calmodulin, the K(d) value for the PEP19 reporter complex is decreased approximately 5-fold, while the value for the neuromodulin reporter complex is increased by the same factor. When Ca(2+) is bound only to the N-terminal pair of Ca(2+)-binding sites, the K(d) value for the PEP19 reporter complex is unaffected, but the value for the complex with the neuromodulin reporter is increased approximately 12-fold. These functional differences are largely ascribed to three differences in the CaM-binding sequences of the two reporters. Replacement of a central Gly in the neuromodulin IQ domain with a Lys at this position in PEP19 almost entirely accounts for the distinctive patterns of Ca(2+)-dependent stability changes exhibited by the two complexes. Replacement of a Lys immediately before the "IQ" amino acid pair in the neuromodulin sequence with the Ala in PEP19 accounts for the remaining Ca(2+)-dependent differences. Replacement of an Ala in the N-terminal half of the neuromodulin sequence with the Gln in PEP19 accounts for approximately half of the Ca(2+)-independent difference in the stabilities of the two reporter complexes, with the Ca(2+)-independent effect of the Lys replacement accounting for most of the remainder. Since the central Gly in the neuromodulin sequence is conserved in half of all known IQ domains, these results suggest that the presence or absence of this residue defines two major functional classes.

Ca2+ ions play a vital role as second messengers in plant cells during various developmental processes and in response to environmental stimuli. Plants have evolved a diversity of unique proteins that bind Ca2+ using the evolutionarily conserved EF-hand motif. The currently held hypothesis is that these proteins function as Ca2+ sensors by undergoing conformational changes in response to Ca2+-binding that facilitate their regulation of target proteins and thereby co-ordinate various signalling pathways. The three main classes of these EF-hand Ca2+sensors in plants are CaMs [calmodulins; including CMLs (CaM-like proteins)], CDPKs (calcium-dependent protein kinases) and CBLs (calcineurin B-like proteins). In the plant species examined to date, each of these classes is represented by a large family of proteins, most of which have not been characterized biochemically and whose physiological roles remain unclear. In the present review, we discuss recent advances in research on CaMs and CMLs, CDPKs and CBLs, and we attempt to integrate the current knowledge on the different sensor classes into common physiological themes.

Parvalbumins (PVs) are calcium-buffer proteins that belong to the EF-hand family. Their N-terminal domain consists of two antiparallel helices A and B that make up a flat hydrophobic surface that is associated with the opposite side of the CD and EF binding sites. A single conserved Arg75-Glu81 salt bridge is buried in this hydrophobic interface. The structure of a rat PV mutant in which Arg75 was replaced by alanine was solved by molecular replacement. Unexpectedly, a large distance deviation of 7.8 A was observed for the AB loop but not for the residues that flank the R75A mutation. The thermal stability of the calcium-loaded form is lower (T(m) = 352.0 K; DeltaT(m) = -11.4 K) than that of the wild-type protein and the apo mutant is unfolded at room temperature. Weaker calcium or magnesium affinities were also measured for the R75A mutant (Ca(2+): K(1) = 4.21 x 10(7) M(-1), K(2) = 6.18 x 10(6) M(-1); Mg(2+): K(1) = 2.98 x 10(4) M(-1), K(2) = 3.09 x 10(3) M(-1)). Finally, comparison of the B factors showed an increase in the flexibility of the AB loop that is consistent with this region being more exposed to solvent in the mutant. The mutant structure therefore demonstrates the role of the salt bridge in attaching the nonbinding AB domain to the remaining protein core. Normal-mode analysis indeed indicated an altered orientation of the AB domain with regard to the CD-EF binding domains.

The affinity between the 1-157 and 158-238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D(9k) to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1-157 of GFP are fused to residues 1-43 of calbindin, and in EF2-GFPC, residues 44-75 of calbindin are fused to residues 158-238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca(2+)-dependent.

We explore how inherent flexibility of a protein molecule influences the mechanism controlling allosteric transitions by using a variational model inspired from work in protein folding. The striking differences in the predicted transition mechanism for the opening of the two domains of calmodulin (CaM) emphasize that inherent flexibility is key to understanding the complex conformational changes that occur in proteins. In particular, the C-terminal domain of CaM (cCaM), which is inherently less flexible than its N-terminal domain (nCaM), reveals "cracking" or local partial unfolding during the open/closed transition. This result is in harmony with the picture that cracking relieves local stresses caused by conformational deformations of a sufficiently rigid protein. We also compare the conformational transition in a recently studied even-odd paired fragment of CaM. Our results rationalize the different relative binding affinities of the EF-hands in the engineered fragment compared with the intact odd-even paired EF-hands (nCaM and cCaM) in terms of changes in flexibility along the transition route. Aside from elucidating general theoretical ideas about the cracking mechanism, these studies also emphasize how the remarkable intrinsic plasticity of CaM underlies conformational dynamics essential for its diverse functions.

Single site-directed mutations at each of the four EF-hand loops of potassium channel-interacting protein 2.2 (KChIP2.2) were carried out to explore the functional roles of EF-hands in KChIP2.2. In contrast to those on EF-hands 1 and 2, mutations on EF-hands 3 or 4 distorted the high affinity Ca(2+)-binding site of KChIP2.2. However, the Mg(2+)-binding ability of KChIP2.2 was marginally affected by the mutations. The gross conformation of mutated KChIP2.2 was indistinguishable from wild-type KChIP2.2 as revealed by CD spectra. The results of size exclusion chromatography showed that, with exception of EF-hand 4 mutant, mutations on EF-hands 1, 2 or 3 caused KChIP2.2 to form oligomer. Pull-down assay revealed that, unlike wild-type KChIP2.2, the interaction between mutated KChIP2.2 and Kv4.2 was not notably enhanced by Ca(2+) and Mg(2+). Coexpression of Kv4.2 and KChIP2.2 in HeLa cells revealed that mutations on EF-hands did not alter the intracellular co-localization of KChIP2.2 and Kv4.2. Together with previous findings that EF-hand mutants of KChIP proteins are unable to regulate the kinetics of Kv4.2, our data show that the intact EF-hands should be crucial for the formation of active conformation of KChIP2.2 when the protein is loaded with Ca(2+) and Mg(2+).

Premature stop codons in CACNA1A, which encodes the alpha(1A) subunit of neuronal P/Q-type (Ca(V)2.1) Ca(2+) channels, cause episodic ataxia type 2 (EA2). CACNA1A undergoes extensive alternative splicing, which contributes to the pharmacological and kinetic heterogeneity of Ca(V)2.1-mediated Ca(2+) currents. We identified three novel heterozygous stop codon mutations associated with EA2 in an alternately spliced exon (37A), which encodes part of an EF-hand motif required for Ca(2+)-dependent facilitation. One family had a C to G transversion (Y1854X). A dinucleotide deletion results in the same premature stop codon in a second family, and a further single nucleotide change leads to a different truncation (R1858X) in a de novo case of EA2. Expression studies of the Y1854X mutation revealed loss of Ca(V)2.1-mediated current. Because these mutations do not affect the alternate exon 37B, these findings reveal unexpected dependence of cerebellar function on intact exon 37A-containing Ca(V)2.1 channels.

Background: Among the EF-Hand calcium-binding proteins the subgroup of S100 proteins constitute a large family with numerous and diverse functions in calcium-mediated signaling. The evolutionary origin of this family is still uncertain and most studies have examined mammalian family members., Results: We have performed an extensive search in several teleost genomes to establish the s100 gene family in fish. We report that the teleost S100 repertoire comprises fourteen different subfamilies which show remarkable similarity across six divergent teleost species. Individual species feature distinctive subsets of thirteen to fourteen genes that result from local gene duplications and gene losses. Eight of the fourteen S100 subfamilies are unique for teleosts, while six are shared with mammalian species and three of those even with cartilaginous fish. Several S100 family members are found in jawless fish already, but none of them are clear orthologs of cartilaginous or bony fish s100 genes. All teleost s100 genes show the expected structural features and are subject to strong negative selection. Many aspects of the genomic arrangement and location of mammalian s100 genes are retained in the teleost s100 gene family, including a completely conserved intron/exon border between the two EF hands. Zebrafish s100 genes exhibit highly specific and characteristic expression patterns, showing both redundancy and divergence in their cellular expression. In larval tissue expression is often restricted to specific cell types like keratinocytes, hair cells, ionocytes and olfactory receptor neurons as demonstrated by in situ hybridization., Conclusion: The origin of the S100 family predates at least the segregation of jawed from jawless fish and some extant family members predate the divergence of bony from cartilaginous fish. Despite a complex pattern of gene gains and losses the total repertoire size is remarkably constant between species. On the expression level the teleost S100 proteins can serve as precise markers for several different cell types. At least some of their functions may be related to those of their counterparts in mammals. Accordingly, our findings provide an excellent basis for future studies of the functions and interaction partners of s100 genes and finally their role in diseases, using the zebrafish as a model organism.

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

To contribute to the study of the calcium-signaling mechanism of egg, we cloned and characterized a 26 kDa Ca(2+)-binding protein from Xenopus laevis eggs, a homologue of Rana catesbeiana dicalcin (renamed from p26olf) that was isolated from the olfactory epithelium. The primary structure of Xenopus dicalcin shows approximately 61% identity to that of Rana dicalcin and consists of two S100-like regions aligned in tandem, as seen in Rana dicalcin. Genomic Southern blot analysis indicated that Xenopus dicalcin is a unique orthologue of Rana dicalcin. Northern blot analysis showed that Xenopus dicalcin mRNA is expressed in Xenopus eggs and also in other tissues. These results indicated that Xenopus dicalcin is a novel S100-like Ca(2+)-binding protein in Xenopus eggs.

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

In this study, we were concerned with the structural role of the surface-exposed extracellular loops of the N-terminal transmembrane (TM) domain of OmpA. A variant of the TM domain of outer membrane protein A (OmpA) with all four such loops shortened, which we call the beta-barrel platform (BBP), was successfully refolded. This indicates that the removed parts of the surface-exposed loops indeed do not contain amino acid sequences critical for this membrane protein's refolding in vitro. BBP has the potential to be used as a template beta-barrel membrane protein structure for the development of novel functions, although our results also highlight the potential difficulties that can arise when functionality is being engineered into the loop regions of membrane proteins. We have used solution nuclear magnetic resonance spectroscopy to determine the global fold of BBP+EF, BBP with a metal ion-binding EF-hand inserted in one of the shortened loops. BBP and BBP+EF in dihexanoylphosphatidylcholine micelles are eight-stranded antiparallel beta-barrels, and BBP represents the smallest beta-structured integral membrane protein known to date.

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics)., ((c) 2006 Wiley-Liss, Inc.)