Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand.

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.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

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 Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ 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.

Probing Lanmodulin's Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage.

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 (koff = 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 (kon ≈ 109 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.

Ca2+-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors.

Voltage-gated sodium channels (Navs) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Nav EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the NaV IQ motif. The EFL domain and IQ motif are contiguous regions of NaV 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 (Ca2+)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 (Ca2+)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 NaV1.2 CTD increased the Ca2+-binding affinity of both CaM domains, consistent with (Ca2+)4-CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with NaV IQ motifs for (Ca2+)4-CaM. During spikes in the cytosolic Ca2+ concentration that accompany an action potential, CaM may translocate from the NaV IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel.

EF hand-like motif mutations of Nav1.4 C-terminus cause myotonic syndrome by impairing fast inactivation.

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.

Coordination of a Single Calcium Ion in the EF-hand Maintains the Off State of the Stromal Interaction Molecule Luminal Domain.

Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ 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 Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca2+ 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 Ca2+. Remarkably, we also detected an α-helix (+Ca2+) to ß-strand (-Ca2+) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.

A luminal EF-hand mutation in STIM1 in mice causes the clinical hallmarks of tubular aggregate myopathy.

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 Ca2+ 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-STIM1I115F) 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 Ca2+ 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.

Analyzing the structural and functional roles of residues from the 'black' and 'gray' clusters of human S100P protein.

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 (K1 = 4 × 106 M-1) and two cooperative low affinity (K2 = 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.

Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism.

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle's inner membrane. In mammalian cells the uniporter's activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-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 Ca2+-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 Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-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.

On the relationship between the conserved 'black' and 'gray' structural clusters and intrinsic disorder in parvalbumins.

Recently we found two highly conserved structural motifs in the members of the EF-hand protein family, which provide a supporting scaffold for their Ca2+ 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.

In silico analysis of the EF-hand proteins in the genome of Giardia intestinalis assembly A.

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 Protein IBA2 Promotes Cell Proliferation in Breast Cancers via Transcriptional Control of Cyclin D1.

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.

GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja, Plays Differential Roles in Plant Responses to Bicarbonate, Salt and Osmotic Stresses.

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.

A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter.

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.

Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity.

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.

Molecular dynamics of the neuronal EF-hand Ca2+-sensor Caldendrin.

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.

A canonical EF-loop directs Ca(2+) -sensitivity in phospholipase C-η2.

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.

S100A14, a member of the EF-hand calcium-binding proteins, is overexpressed in breast cancer and acts as a modulator of HER2 signaling.

HER2 is overexpressed in 20­25% 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 956­1154 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.

Reprogramming EF-hands for design of catalytically amplified lanthanide sensors.

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.

Extra EF hand unit (DX) mediated stabilization and calcium independency of α-amylase.

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.