EF-hand Research Articles

Ca(II) and Zn(II) Cooperate To Modulate the Structure and Self-Assembly of S100A12.

S100A12 is a member of the Ca2+ binding S100 family of proteins that functions within the human innate immune system. Zinc sequestration by S100A12 confers antimicrobial activity when the protein is secreted by neutrophils. Here, we demonstrate that Ca2+ binding to S100A12's EF-hand motifs and Zn2+ binding to its dimeric interface cooperate to induce reversible self-assembly of the protein. Solution and magic angle spinning nuclear magnetic resonance spectroscopy on apo-, Ca2+-, Zn2+-, and Ca2+,Zn2+-S100A12 shows that significant metal binding-induced chemical shift perturbations, indicative of conformational changes, occur throughout the polypeptide chain. These perturbations do not originate from changes in the secondary structure of the protein, which remains largely preserved. While the overall structure of S100A12 is dominated by Ca2+ binding, Zn2+ binding to Ca2+-S100A12 introduces additional structural changes to helix II and the hinge domain (residues 38-53). The hinge domain of S100A12 is involved in the molecular interactions that promote chemotaxis for human monocyte, acute inflammatory responses and generates edema. In Ca2+-S100A12, helix II and the hinge domain participate in binding with the C-type immunoglobulin domain of the receptor for advanced glycation products (RAGE). We discuss how the additional conformational changes introduced to these domains upon Zn2+ binding may also impact the interaction of S100A12 and target proteins such as RAGE.

EFCAB2 is a novel calcium-binding protein in mouse testis and sperm.

Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.

Regulation of the Ca2+‐sensing capacity of STIM1 by the G protein‐coupled estrogen receptor

The stromal interaction molecule 1 (STIM1) is essential for activation of store‐operated Ca2+entry (SOCE). STIM1 contains in its intra‐endoplasmic reticular (ER) domain a canonical EF hand (cEF) that houses a Ca2+‐binding loop. This loop serves as a Ca2+sensor; Ca2+leaving its binding loop upon ER Ca2+depletion initiates conformational changes leading to STIM1 oligomerization and SOCE activation. We recently reported that activation of the G protein‐coupled estrogen receptor (GPER) clamps intracellular Ca2+signals via multifaceted inhibitory actions on both Ca2+efflux and SOCE; the latter results partly from alteration in phosphorylation at serines 575, 608 and 621 of STIM1. Here, we identified a dynamic interaction between GPER and STIM1 that directly controls the Ca2+‐sensing capacity of STIM1. GPER coimmunoprecipitates with STIM1 in resting primary endothelial cells, an association enhanced by treatment with either GPER agonist G‐1 or activation of SOCE using thapsigargin. This effect is not altered by combined S575/608/621A substitutions. Coimmunoprecipitation analysis using truncated versions of STIM1 showed that GPER associates with the cEF domain. Confocal microscopy identified a GPER's submembrane domain that localizes GPER to the ER. To assess the role of GPER‐STIM1 interaction in controlling STIM1's Ca2+sensing capacity, we generated a series of biosensors that incorporated STIM1's Ca2+‐binding loop in singlet, doublet or triplet. These biosensors faithfully report Ca2+binding to the loop, with tripled dynamic range by the triple‐loop biosensor and loss of Ca2+binding by charge substitutions targeting strategic elements for Ca2+binding. Using peptides corresponding to submembrane domains of GPER and these STIM1 biosensors, we identified a robust Ca2+‐independent interaction between a submembrane domain and the Ca2+‐binding loop. The charge substitutions that abolish Ca2+binding only reduce but do not negate the interaction, indicating that Ca2+and the GPER domain bind to different regions of the loop. Consistently, we have observed that GPER overexpression decreases SOCE and GPER knockdown substantially increases SOCE. These data reveal a new interaction between GPER and STIM1 that directly controls STIM1's Ca2+‐sensing capacity and SOCE activation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteins with calmodulin-like domains: structures and functional roles.

The appearance of modular proteins is a widespread phenomenon during the evolution of proteins. The combinatorial arrangement of different functional and/or structural domains within a single polypeptide chain yields a wide variety of activities and regulatory properties to the modular proteins. In this review, we will discuss proteins, that in addition to their catalytic, transport, structure, localization or adaptor functions, also have segments resembling the helix-loop-helix EF-hand motifs found in Ca2+-binding proteins, such as calmodulin (CaM). These segments are denoted CaM-like domains (CaM-LDs) and play a regulatory role, making these CaM-like proteins sensitive to Ca2+ transients within the cell, and hence are able to transduce the Ca2+ signal leading to specific cellular responses. Importantly, this arrangement allows to this group of proteins direct regulation independent of other Ca2+-sensitive sensor/transducer proteins, such as CaM. In addition, this review also covers CaM-binding proteins, in which their CaM-binding site (CBS), in the absence of CaM, is proposed to interact with other segments of the same protein denoted CaM-like binding site (CLBS). CLBS are important regulatory motifs, acting either by keeping these CaM-binding proteins inactive in the absence of CaM, enhancing the stability of protein complexes and/or facilitating their dimerization via CBS/CLBS interaction. The existence of proteins containing CaM-LDs or CLBSs substantially adds to the enormous versatility and complexity of Ca2+/CaM signaling.

Characterization of the Ca2+-coordination structures of L- and T-plastins in combination with their synthetic peptide analogs by FTIR spectroscopy

FTIR spectroscopy was employed to characterize the coordination structures of divalent cations (M2+ = Ca2+ or Mg2+) bound by L- and T-plastins, which contain two EF-hand motifs. We focused on the N-terminal headpieces in the L- and T-plastins to analyze the regions of COO− stretching and amide-I in solution. The spectral profiles indicated that these headpieces have EF-hand calcium-binding sites because bands at 1551 cm−1 and 1555 cm−1 were observed for the bidentate coordination mode of Glu at the 12th position of the Ca2+-binding site of Ca2+-loaded L-plastin and T-plastin, respectively. The amide-I profile of the Mg2+-loaded L-plastin headpiece was identical with that of the apo L-plastin headpiece, meaning that L-plastin has a lower affinity for Mg2+. The amide-I profiles for apo, Mg2+-loaded and Ca2+-loaded T-plastin suggested that aggregation was generated in protein solution at a concentration of 1 mM. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of data obtained for synthetic peptide analogs corresponding to the Ca2+-binding site.

Evolutionary-Conserved Allosteric Properties of Three Neuronal Calcium Sensor Proteins.

Neuronal Calcium Sensors (NCS) are highly conserved proteins specifically expressed in neurons. Calcium (Ca2+)-binding to their EF-hand motifs results in a conformational change, which is crucial for the recognition of a specific target and the downstream biological process. Here we present a comprehensive analysis of the allosteric communication between Ca2+-binding sites and the target interfaces of three NCS, namely NCS1, recoverin (Rec), and GCAP1. In particular, Rec was investigated in different Ca2+-loading states and in complex with a peptide from the Rhodopsin Kinase (GRK1) while NCS1 was studied in a Ca2+-loaded state in complex with either the same GRK1 target or a peptide from the D2 Dopamine receptor. A Protein Structure Network (PSN) accounting for persistent non-covalent interactions between amino acids was built for each protein state based on exhaustive Molecular Dynamics simulations. Structural network analysis helped unveiling the role of key amino acids in allosteric mechanisms and their evolutionary conservation among homologous proteins. Results for NCS1 highlighted allosteric inter-domain interactions between Ca2+-binding motifs and residues involved in target recognition. Robust long range, allosteric protein-target interactions were found also in Rec, in particular originating from the EF3 motif. Interestingly, Tyr 86, involved the hydrophobic packing of the N-terminal domain, was found to be a key residue for both intra- and inter-molecular communication with EF3, regardless of the presence of target or Ca2+ ions. Finally, based on a comprehensive topological PSN analysis for Rec, NCS1, and GCAP1 and multiple sequence alignments with homolog proteins, we propose that an evolution-driven correlation may exist between the amino acids mediating the highest number of persistent interactions (high-degree hubs) and their conservation. Such conservation is apparently fundamental for the specific structural dynamics required in signaling events.

Structural Modulation of RyR1 by MgATP and Free Mg2+ in Lipid Membrane using CryoEM

Mg2+ is the most abundant divalent cation in living cells. In the skeletal muscle cytosol, out of the total Mg2+ concentration of 8 mM, around 1 mM is free Mg2+ and around 5 mM exists in the form of MgATP, the biological active form of cellular ATP. Mg2+ is constitutively bound to RyR1 in its free and complexed forms. In fact, preferential binding of Mg2+ to the acidic side chains with similar coordination geometry as Ca2+, allows it to be a potential antagonistic cation of Ca2+, which has much lower cytoplasmic concentrations (<1 μM - 100 μM). Free Mg2+ is thought to inhibit RyRs through its association with Ca2+ binding sites at cytoplasmic or luminal regions, while the effect of MgATP per se is novel in the context of excitation-contraction coupling. We studied the effect of free Mg2+ and MgATP on RyR1 bound to a POPC bilayer enclosed by membrane scaffold protein (nanodisc). CryoEM reconstructions of RyR1 nanodiscs complexed with MgAMPPCP at ∼3.8 Å resolution show that in the absence of any detergents or FKBP, the RyR1 assumes a closed conformation with a pore radius that makes impermeant to Ca2+ ions. Discernible densities for nanodisc and MgATP were observed. Binding of MgATP involves global motion of the central and cytoplasmic domains towards the T-tubule and the fourfold axis with reorganization of the P2 domain. Local motion of the TM helices, U-motif, VSC and EF hands were also observed with concomitant closure of the ATP cavity at the intersubunit interface. With the cryoEM structure of RyR1-MgATP, we seek to explain the mechanistic basis of RyR1 inhibition during the physiological resting state of skeletal muscle. Supported by grants AHA 14GRNT19660003, MDA352845, NIH R01 AR068431, and NIH U24GM116789.

Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum.

Apoptosis-linked gene 2 (ALG-2) is a Ca2+-binding protein with five repetitive EF-hand motifs, named penta-EF-hand (PEF) domain. It interacts with various target proteins and functions as a Ca2+-dependent adaptor in diverse cellular activities. In the cytoplasm, ALG-2 is predominantly localized to a specialized region of the endoplasmic reticulum (ER), called the ER exit site (ERES), through its interaction with Sec31A. Sec31A is an outer coat protein of coat protein complex II (COPII) and is recruited from the cytosol to the ERES to form COPII-coated transport vesicles. I will overview current knowledge of the physiological significance of ALG-2 in regulating ERES localization of Sec31A and the following adaptor functions of ALG-2, including bridging Sec31A and annexin A11 to stabilize Sec31A at the ERES, polymerizing the Trk-fused gene (TFG) product, and linking MAPK1-interacting and spindle stabilizing (MISS)-like (MISSL) and microtubule-associated protein 1B (MAP1B) to promote anterograde transport from the ER.

Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain.

Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain. The encodable protein motif comprises a 7- or 8-residue sequence (DKDADXW or DKDADXXW), derived from EF-Hand calcium-binding domains, that is necessary but not sufficient for binding terbium, combined with a protein phosphorylation site (Ser or Thr at residue 9) that, upon phosphorylation, completes the metal-binding motif. Thus, the motif binds metal poorly and exhibits weak terbium luminescence when not phosphorylated. Upon phosphorylation, the peptide binds metal with significantly higher affinity and exhibits robust terbium luminescence. Phosphorylation results in up to a 23× increase in terbium luminescence. Minimal phosphorylation-dependent motifs as small as 9 residues (DKDADGWIS) were developed. NMR spectroscopy on this lanthanum(iii)·phosphopeptide complex confirmed that binding occurs in a manner similar to that in an EF-Hand, despite the absence of the conserved Glu12 typically present in an EF-Hand. By combining molecular design with known protein kinase recognition sequences, minimal protein kinase-inducible domains were developed that were responsive to phosphorylation by Protein Kinase A (PKA: DKDADRRW(S/pS)IIAK), Protein Kinase C (PKC: DKDADGWI(T/pT)FRRKA), and Casein Kinase 1 (CK1: DKDADDWA(S/pS)I). Phosphorylation by PKA was quantified in HeLa cell extracts, with a 4.4× increase in fluorescence (terbium luminescence) observed at 544 nm. The optimized minimal motif includes alternating aspartate residues at positions 1, 3, and 5, plus binding through the main-chain carbonyl at position 7; a lysine at position 2 to provide electrostatic balance and reduce binding in the absence of phosphorylation; an alanine at residue 4 to promote the αL conformation observed at that position of the EF Hand; a tryptophan at residue 7 or 8 to sensitize terbium luminescence; and a phosphorylation site with serine or threonine at residue 9. Residues at positions 6; 7 or 8; and 10 or later may be changed to provide kinase specificity. In the CK1-responsive peptide, the acidic residues in the proto-terbium-binding motif are employed as part of the kinase recognition sequence. This work thus presents fundamental rules for the design of compact phosphorylation-responsive terbium-binding motifs, with potential further application to motifs responsive to other protein post-translational modifications.

Quantification of Human Swiprosin-1/EFhd2 Expression on Protein and RNA Level.

Many Ca2+-binding proteins are differentially regulated under pro-inflammatory conditions in different organs. Specific quantification of RNA and protein expression of those proteins demands validated protocols. Peripheral blood mononuclear cells (PBMC) can mirror an inflammatory status originating from several organs and can therefore be an important diagnostic tool. Swiprosin-1/EFhd2 (EFhd2) is a ~30kDa Ca2+ and F-actin binding, cytoskeletal protein with two central EF hands and a C-terminal coiled-coil domain. Unbiased gene expression analyses and proteomics revealed that EFhd2 is regulated under pro-inflammatory conditions in several cell types and tissues. Here we describe validated protocols to quantify the expression of the human orthologue of Swiprosin-1/EFhd2 on RNA and protein level in PBMC. Both methods reveal that EFhd2 is stronger expressed in monocytes than in B cells of healthy donors. Thus, initial experiments relying on qPCR are likely to provide results with functional relevance. The higher expression of EFhd2 in monocytes could be related to monocyte migration under inflammatory conditions.

Solution structure of TbCentrin4 from Trypanosoma brucei and its interactions with Ca2+ and other centrins.

Centrin is a conserved calcium-binding protein that plays an important role in diverse cellular biological processes such as ciliogenesis, gene expression, DNA repair and signal transduction. In Trypanosoma brucei, TbCentrin4 is mainly localized in basal bodies and bi-lobe structure, and is involved in the processes coordinating karyokinesis and cytokinesis. In the present study, we solved the solution structure of TbCentrin4 using NMR (nuclear magnetic resonance) spectroscopy. TbCentrin4 contains four EF-hand motifs consisting of eight α-helices. Isothermal titration calorimetry experiment showed that TbCentrin4 has a strong Ca2+ binding ability. NMR chemical shift perturbation indicated that TbCentrin4 binds to Ca2+ through its C-terminal domain composed of EF-hand 3 and 4. Meanwhile, we revealed that TbCentrin4 undergoes a conformational change and self-assembly induced by high concentration of Ca2+ Intriguingly, localization of TbCentrin4 was dispersed or disappeared from basal bodies and the bi-lobe structure when the cells were treated with Ca2+in vivo, implying the influence of Ca2+ on the cellular functions of TbCentrin4. Besides, we observed the interactions between TbCentrin4 and other Tbcentrins and revealed that the interactions are Ca2+ dependent. Our findings provide a structural basis for better understanding the biological functions of TbCentrin4 in the relevant cellular processes.

Redox-Responsive Protein Design: Design of a Small Protein Motif Dependent on Glutathionylation.

Cysteine S-glutathionylation is a protein post-translational modification that promotes cellular responses to changes in oxidative conditions. The design of protein motifs that directly depend on defined changes to protein side chains provides new methods for probing diverse protein post-translational modifications. A canonical, 12-residue EF-hand motif was redesigned to be responsive to cysteine glutathionylation. The key design principle was the replacement of the metal-binding Glu12 carboxylate of an EF-hand with a motif capable of metal binding via a free carboxylate in the glutathione-conjugated peptide. In the optimized peptide (DKDADGWCG), metal binding and terbium luminescence were dependent on glutathionylation, with weaker metal binding in the presence of reduced cysteine but increased metal affinity and a 3.5-fold increase in terbium luminescence at 544 nm when cysteine was glutathionylated. Nuclear magnetic resonance spectroscopy indicated that the structure at all residues of the glutathionylated peptide changed in the presence of metal, with chemical shift changes consistent with the adoption of an EF-hand-like structure in the metal-bound glutathionylated peptide. This small protein motif consists of canonical amino acids and is thus genetically encodable, for its potential use as a localized tag to probe protein glutathionylation.

Protein-Metal-Ion Interactions Studied by Mass Spectrometry-Based Footprinting with Isotope-Encoded Benzhydrazide.

Metal ions, usually bound by various amino-acid side chains in proteins, play multiple roles in protein folding, conformational change, cellular communication, and catalysis. Ca(II) and Mg(II), abundant among biologically relevant cations, execute their cellular functions associated with the conformational change of bound proteins. They bind with proteins where carboxylic acid residues are dominant ligands. To develop mass spectrometry for mapping protein-binding sites, we implemented a new carboxyl group footprinter, benzhydrazide, and refined it with isotope encoding. The method uses carbodiimide chemistry to footprint carboxylic residues, whereby 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide activates a carboxyl group followed by nucleophilic attack by benzhydrazide forming a stable labeled product. We tested the effectiveness of isotope-encoded benzhydrazide by studying Ca2+ and Mg2+ binding of calmodulin, an EF-hand protein. The footprinting results indicate that the four active sites for metal-ion binding (EF hands I, II, III, and IV) and the linker region (peptide 78-86) undergo conformational changes upon Ca(II) and Mg(II) binding, respectively. The outcome is consistent with previously reported results and 3-D structures, thereby validating a new reagent that is more reactive and discriminating for specific amino-acid protein footprinting. This reagent should be important for locating metal-binding sites of other metalloproteins.

Characterization of neurocalcin delta membrane binding by biophysical methods.

Neurocalcin delta (NCALD) is a member of the neuronal calcium sensors protein family. In the retina, NCALD is expressed by ganglion and amacrine cells. NCALD is composed of 4 EF-hand motifs but only 3 of them may bind calcium. The binding of calcium induces a conformational change of the protein which leads to the extrusion of its N-terminal myristoyl group as well as some hydrophilic residues. This mechanism, named calcium-myristoyl switch, is presumably involved in its membrane binding. The parameters responsible for the interaction of NCALD with membranes are only partially known. The purpose of this study was thus to gather more information on the membrane binding behavior of NCALD using lipid monolayers, including the influence of the lipid composition, the calcium and the myristoyl group. NCALD was injected underneath different lipid monolayers and this model membrane allowed the determination of the binding parameters as maximum insertion pressure (MIP) and synergy. The values of MIP are larger when monolayers were composed of a saturated phospholipid with phosphoethanolamine polar head. This trend is confirmed by polarization modulation infrared reflection absorption spectroscopy measurements. Moreover, the observations by fluorescence microscopy show that NCALD preferentially interacts with phospholipids which are in the liquid-condensed physical state, as found in membrane microdomains. This observation could explain the changes of NCALD expression level in the brains of patients suffering from Alzheimer's disease because of the alteration of lipid composition in microdomains structures.

EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity.

Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell-mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important regulator of T cell cytotoxicity and further studies should evaluate its role in T cell-mediated inflammation.

A role for Saccharomyces cerevisiae Centrin (Cdc31) in mitochondrial function and biogenesis.

Centrins belong to a family of proteins containing calcium-binding EF-hand motifs that perform well-established roles in centrosome and spindle pole body (SPB) duplication. Yeast encodes a single Centrin protein (Cdc31) that binds components in the SPB. However, further studies revealed a role for Centrins in mRNA export, and interactions with contractile filaments and photoreceptors. In addition, human Centrin-2 can bind the DNA-lesion recognition factor XPC, and improve the efficiency of nucleotide excision repair. Similarly, we reported that yeast Cdc31 binds Rad4, a functional counterpart of the XPC DNA repair protein. We also found that Cdc31 is involved in the ubiquitin/proteasome system, and mutations interfere with intracellular protein turnover. In this report, we describe new findings that indicate a role for Cdc31 in the energy metabolism pathway. Cdc31 and cdc31 mutant proteins showed distinct interactions with proteins in energy metabolism, and mutants showed sensitivity to oxidative stress and poor growth on non-fermentable carbon. Significant alteration in mitochondrial morphology was also detected. Although it is unclear how Cdc31 contributes to so many unrelated mechanisms, we propose that by controlling SPB duplication Centrin proteins might link the cellular responses to DNA damage, oxidative load and proteotoxic stresses to growth control.

Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the CALM genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+ by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.

EF-hand domain containing 2 (Efhc2) is crucial for distal segmentation of pronephros in zebrafish

BackgroundThe blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function.Resultsefhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development.ConclusionIn this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development.

Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium.

Lanthanides (Lns) have been shown recently to be essential cofactors in certain enzymes in methylotrophic bacteria. Here we identify in the model methylotroph, Methylobacterium extorquens, a highly selective LnIII-binding protein, which we name lanmodulin (LanM). LanM possesses four metal-binding EF hand motifs, commonly associated with CaII-binding proteins. In contrast to other EF hand-containing proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concentrations of all LnIII (Ln = La-Lu, Y), whereas it only responds to CaII at near-millimolar concentrations. Mutagenesis of conserved proline residues present in LanM's EF hands, not encountered in CaII-binding EF hands, to alanine pushes CaII responsiveness into the micromolar concentration range while retaining picomolar LnIII affinity, suggesting that these unique proline residues play a key role in ensuring metal selectivity in vivo. Identification and characterization of LanM provides insights into how biology selectively recognizes low-abundance LnIII over higher-abundance CaII, pointing toward biotechnologies for detecting, sequestering, and separating these technologically important elements.

Transgelin-1 (SM22α) interacts with actin stress fibers and podosomes in smooth muscle cells without using its actin binding site

Transgelin-1 (SM22α) has been recognized as a smooth muscle marker and a tumor suppressor, but many details of the working mechanisms remain unclear. Transgelin-1 belongs to the calponin family of actin-binding proteins with an N-terminal calponin homology domain (CH-domain) and a C-terminal calponin-like module (CLIK23). Here, we demonstrate that transgelin-1 interacts with actin stress fibers and podosomes in smooth muscle cells via its type-3 CH-domain, while CLIK23 is dispensable for the binding to the actin structures. We further suggest that the EF-hand motif in transgelin-1 contributes to proper folding of the CH-domain and in turn to the interaction with the actin structures. These results are in contrast to the ones reported in in vitro studies that demonstrated CLIK23 was necessary for the transgelin-1–actin binding, while the CH-domain was not. Besides, within cells, transgelin-1 phosphorylation at Ser181 in CLIK23 did not affect its colocalization with the actin structures, while the same phosphorylation was reported in in vitro studies to negatively regulate actin binding. Thus, our results suggest the molecular basis of intracellular interaction between transgelin-1 and actin, distinct from that in vitro. The actin binding capability intrinsic to CLIK23 may not appear within cells probably because of the weaker competition for actin binding compared to other actin binding molecules.