EF-hand Research Articles

Cysteine oxidation to the sulfinic acid induces oxoform-specific lanthanide binding and fluorescence in a designed peptide

Cysteine sulfinic acid (Cys-SO2–) is a protein post-translational modification that is formed reversibly under oxidative conditions. A short, encodable peptide was developed whose metal binding and terbium luminescence are dependent on cysteine (Cys) oxidation to the sulfinic acid. The protein design is based on the modification of a key metal-binding aspartate (Asp) in a canonical EF-Hand motif (DKDADGWISPAEAK) to Cys. In this design, Cys in the thiol oxidation state does not mimic the native Asp, and thus the peptide binds terbium(III) (Tb3+) poorly and exhibits weak terbium luminescence (fluorescence). In contrast, when Cys is oxidized to the Cys sulfinic acid oxoform, the Cys sulfinate effectively mimics Asp, resulting in a significant increase in terbium affinity and luminescence. Asp residues at positions 1, 3, and 5 of the EF-Hand motif were examined as potential sites for Cys oxidation-responsive metal binding. The peptide with Cys at residue 1 exhibited the highest Tb3+ affinity in both oxidation states. The peptide with Cys at residue 3 exhibited a 4.2-fold distinction in affinity between the oxidation states. Most significantly, the peptide with Cys at residue 5 had only modest Tb3+ affinity as the Cys thiol, but exhibited a 30-fold increase in Tb3+ affinity and an 18-fold increase in Tb3+ luminescence on Cys oxidation to the sulfinic acid. This peptide (Ac-DKDACGWISPAEAK-NH2) exhibited selective Tb3+ binding via Cys-SO2– over the thiol, S-glutathionyl, S-nitrosyl, and sulfonic acid oxoforms, indicating substantially greater Lewis basicity of the sulfinate than the sulfonate. NMR spectroscopy and quantum homology modeling indicated that the designed peptide binds metal with an overall geometry similar to that of an EF-Hand motif, with the Cys sulfinate effectively replacing Asp as a metal-binding ligand. This peptide was applied to detect Cys oxidation to the sulfinic acid by fluorescence spectroscopy, suggesting its broader application in understanding Cys sulfinic acid biology.

Mechanism of Ca2+-mediated NOX modulated in ROS metabolism induced by T-2 toxin in potato tuber

T-2 toxin at low concentrations can induce ROS accumulation and modulate host resistance in plants. NOX plays crucial roles in ROS production and is regulated by Ca2+via direct binding to EF-hand motifs. In this study, the effect of EGTA (Ca2+ chelating agent) on the expression and enzymatic activity of NOX, as well as the activities and corresponding gene expressions involved in ROS metabolism and cell membrane integrity, were investigated in treated slices. Results indicated that EGTA treatment significantly affected gene expression and activity of NOX, and reduced ROS accumulation and cell membrane integrity and the enzymatic activities and gene expression involved in ROS metabolism when exposed to treatment. The addition of exogenous Ca2+ restored the initial relative transcript abundance, ROS accumulation and their activities. Results suggest that Ca2+ affected by EGTA plays a crucial role in NOX activity regulation, ultimately affecting ROS metabolism in slices induced by T-2 toxin.

Calmodulin and Calmodulin Binding Proteins in Dictyostelium: A Primer.

Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational changes effected by calcium ion binding to its four EF hands are compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in calmodulin binding to target proteins are discussed with examples of the diversity of calmodulin binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize calmodulin binding proteins is covered followed by the ways Dictyostelium is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, Dictyostelium is in a position to become a leader in the field of calmodulin research.

Molecular cloning and biochemical characterization of the phospholipid scramblase SCRM-1 from Caenorhabditis elegans

In this study, the SCRM-1 gene from Caenorhabditis elegans was cloned and overexpressed in E. coli to study the biochemical properties of scramblase. This is the first report showing that this scramblase from C. elegans possesses a Ca2+-dependent and head group-independent scramblase activity. The SCRM-1 of C.elegans possesses functional domains including a single EF-hand-like Ca2+ binding domain, as human scramblases do. A point mutation in the EF-hand-like Ca2+ binding motif results in loss of scramblase activity. Other biochemical assays like carbocyanine staining, Tb3+ luminescence, Tryptophan fluorescence, and CD spectroscopy strongly proved the role of the EF-hand motif for functional activity. The increase in protein size in solution upon incubating with Ca2+ shows ligand-dependent oligomerization and conformational changes.

Swiprosin1/EFhd2 is involved in the monoaminergic and locomotor responses of psychostimulant drugs.

Psychostimulants are widely abused drugs that may cause addiction in vulnerable individuals. While the reward circuitry of the brain is involved in addiction establishment, various pathways in the brain may provide protection at the molecular level that limits the acute and chronic effects of drugs. These targets may be used for strategies designed to prevent and treat addiction. Swiprosin-1/EF hand domain 2 (EFhd2) is a Ca2+ -binding cytoskeletal adaptor protein involved in sensation-seeking behaviour, anxiety and alcohol addiction. Here, we tested how EFhd2 contributes to the physiological and behavioural effects of the psychostimulant drugs methamphetamine (METH) and cocaine. An in vivo microdialysis study in EFhd2 knockout mice revealed that EFhd2 controls METH- and cocaine-induced changes in extracellular dopamine, serotonin and noradrenaline levels through different mechanisms in the nucleus accumbens and prefrontal cortex. Electrophysiological recordings in a slice preparation showed that a lack of EFhd2 increases dopaminergic neuronal activity in the ventral tegmental area and increases the sensitivity of neurons to stimulation. We report a role of EFhd2 in METH-induced locomotor activation and in the conditioned locomotor effects. No role, however, was observed in the establishment of METH- or cocaine-induced conditioned place preference. These findings may suggest that EFhd2 modulates the activity of the dopaminergic system and the neurochemical effects of METH and cocaine, which translate into a modulation of the behavioural effects of these drugs at the level of the acute and conditioned locomotor activity.

LQTS-Associated Mutants of Calmodulin Show Disrupted Interaction with L-Type Calcium Channels

Long QT syndrome (LQTS) is a major cause of sudden cardiac death, and is characterised by dysfunctional ion channel activity. Genetic screening of clinical populations has identified mutations in the calcium sensor calmodulin (CaM). However, little is currently known about the molecular mechanism of CaM-associated LQTS. A small but ubiquitous protein, CaM is involved in the calcium-dependent regulation of multiple targets such as Cav1.2, where it mediates calcium-dependent inactivation by interaction with CaM binding domains present on the channel, including the N-terminal spatial calcium transforming element (NSCaTE) and the C-terminal IQ domain. Ergo, CaM mutations that affect binding to Cav1.2 may affect channel modulation. We investigated the interaction of clinically relevant CaM mutants D132E, D134H, Q136P and E141G with Cav1.2. Here, we show that these mutations in the fourth EF hand of CaM differentially alter CaM's stability and structure, in addition to its interaction with Cav1.2 CaM binding domains. Circular dichroism experiments reveal alterations to thermal stability and secondary structure content. This is corroborated by HSQC NMR spectra showing differences in conformation which are exacerbated by interaction with NSCaTE or IQ domains. Furthermore, isothermal titration calorimetry demonstrates that in calcium-saturating conditions, Q136P and E141G isoforms have a significantly decreased affinity for NSCaTE (up to 6-fold), whilst D132E, D134H and E141G have an increased affinity for IQ (up to 3-fold). Therefore, mutations have clear but distinct effects on the interaction of CaM with Cav1.2, suggesting differences in the mechanisms underlying the pathophysiology of the disease. This is likely to disrupt channel function, leading to prolonged calcium influx and manifestation of the LQTS phenotype. This work is funded by the Wellcome Trust PhD Studentship (to NG) and the British Heart Foundation Intermediate Basic Science Research Fellowship FS/17/56/32925 (to NH).

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

Analysis of Arabidopsis TPK2 and KCO3 reveals structural properties required for K+ channel function

ABSTRACT Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.

EF-hand like Region in the N-terminus of Anoctamin 1 Modulates Channel Activity by Ca2+ and Voltage.

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca2+ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca2+ binding. Because the Ca2+ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca2+ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca2+ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca2+-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca2+ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca2+. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca2+ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca2+ and modulate the activation by Ca2+ and voltage.

Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase

The aim of this experiment was to study the effects of calcium ion on the hydrolysis of cationic and anionic substrate by human butyrylcholinesterase (HuBChE). The hydrolysis of aspirin, an anionic substrate, by HuBChE was markedly increased in the presence of increasing concentrations of calcium ion (∼20 mM), as shown by the increasing kcat (∼18-fold). Butyrylthiocholine (BTC), a cationic substrate, was biphasically hydrolyzed with substrate activation; a second BTC molecule caused a 3-fold increase in kcat. At both lower and higher concentrations of BTC, its hydrolysis by HuBChE was slightly slowed down by the addition of calcium ion. Other cationic substrates, propranolol derivatives with butyryl and valeryl groups, were R-preferentially hydrolyzed by HuBChE; the rate of hydrolysis of these compounds was nearly the same in the absence and presence of calcium ion. These data indicate differential effects of calcium ion on HuBChE activity with anionic and cationic substrates. Furthermore, during the hydrolysis of aspirin in the presence of calcium ions, we demonstrated the existence of 2 additional binding sites for calcium, with Km values of 1.8 and 5.9 mM. These binding sites exhibited much lower affinities than the EF-hand motif, previously identified as a high-affinity calcium-binding site.

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

ABSTRACTSTIM 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.

Role of calcium in the defense response induced by brassinosteroids in strawberry plants

Brassinosteroids (BRs) are steroidal hormones essential for the normal growth and development of plants. The aim of this work was to study the role of calcium, in the defense response induced by two BRs e.g. BB16 and EP24, and to investigate the expression levels of defense-related genes in strawberry plants treated with these BRs. An increase in the calcium influx in response to treatment with BB16 or EP24 was observed. By comparing Fragaria vesca and Fragaria ananassa genome sequences 28 nucleotide sequences coding for calcium-sensing proteins called CMLs were predicted. Bioinformatic analyses of the sequences revealed that all of them contained the characteristic EF-hand motif of Ca2+ binding, and regulatory cis-elements related to stress. qPCR studies showed that both BB16 and EP24 cause the upregulation of selected CMLs genes e.g. FaCML1a, FaCML36, FaCML42 and FaCML45, and defense marker genes, such as PR1, ERF1 and GLS5. Results also showed that whereas BB16 induced the upregulation of MYB30, EP24 induced the upregulation of PRX27. These results provide valuable information of the response mechanisms induced by BRs and demonstrate the role of calcium in the signaling pathways induced by BRs in strawberry plants.

Sequential activation of STIM1 links Ca2+ with luminal domain unfolding.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

Effects of Membrane and Biological Target on the Structural and Allosteric Properties of Recoverin: A Computational Approach.

Recoverin (Rec) is a prototypical calcium sensor protein primarily expressed in the vertebrate retina. The binding of two Ca2+ ions to the functional EF-hand motifs induces the extrusion of a myristoyl group that increases the affinity of Rec for the membrane and leads to the formation of a complex with rhodopsin kinase (GRK1). Here, unbiased all-atom molecular dynamics simulations were performed to monitor the spontaneous insertion of the myristoyl group into a model multicomponent biological membrane for both isolated Rec and for its complex with a peptide from the GRK1 target. It was found that the functional membrane anchoring of the myristoyl group is triggered by persistent electrostatic protein-membrane interactions. In particular, salt bridges between Arg43, Arg46 and polar heads of phosphatidylserine lipids are necessary to enhance the myristoyl hydrophobic packing in the Rec-GRK1 assembly. The long-distance communication between Ca2+-binding EF-hands and residues at the interface with GRK1 is significantly influenced by the presence of the membrane, which leads to dramatic changes in the connectivity of amino acids mediating the highest number of persistent interactions (hubs). In conclusion, specific membrane composition and allosteric interactions are both necessary for the correct assembly and dynamics of functional Rec-GRK1 complex.

Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress.

VIP1 is a bZIP transcription factor in Arabidopsis thaliana. When cells are exposed to mechanical stress, VIP1 transiently accumulates in the nucleus, where it regulates the expression of its target genes and suppresses mechanical stress-induced root waving. The nuclear-cytoplasmic shuttling of VIP1 is regulated by phosphorylation and calcium-dependent signaling, but specific regulators of these processes remain to be identified. Here, inhibitors of protein phosphatase 2A (PP2A) are shown to inhibit both the mechanical stress-induced dephosphorylation and nuclear accumulation of VIP1. The PP2A B subunit, which recruits substrates of PP2A holoenzyme, is classified into B, B', B'', and B''' families. Using bimolecular fluorescence complementation, in vitro pull-down, and yeast two-hybrid assays, we show that VIP1 interacts with at least two of the six members of the Arabidopsis PP2A B''-family subunit, which have calcium-binding EF-hand motifs. VIP1AAA, a constitutively nuclear-localized VIP1 variant with substitutions in putative phosphorylation sites of VIP1, suppressed the root waving induced by VIP1-SRDX (a repression domain-fused variant of VIP1). These results support the idea that VIP1 is dephosphorylated by PP2A and that the dephosphorylation suppresses the root waving. The phosphorylation sites of VIP1 and its homologs were narrowed down by in vitro phosphorylation, yeast two-hybrid, and protein subcellular localization assays.

Experimental Insight into the Structural and Functional Roles of the 'Black' and 'Gray' Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs.

Recently, we have found that calcium binding proteins of the EF-hand superfamily (i.e., a large family of proteins containing helix-loop-helix calcium binding motif or EF-hand) contain two types of conserved clusters called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster), which provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domains. Cluster I is more conservative and mostly incorporates aromatic amino acids, whereas cluster II includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. Recoverin is EF-hand Ca2+-binding protein containing two ‘black’ clusters comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172 (C-terminal domain) as well as two ‘gray’ clusters comprised of F70, Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain). To understand a role of these residues in structure and function of human recoverin, we sequentially substituted them for alanine and studied the resulting mutants by a set of biophysical methods. Under metal-free conditions, the ‘black’ clusters mutants (except for F35A and E169A) were characterized by an increase in the α-helical content, whereas the ‘gray’ cluster mutants (except for K119A) exhibited the opposite behavior. By contrast, in Ca2+-loaded mutants the α-helical content was always elevated. In the absence of calcium, the substitutions only slightly affected multimerization of recoverin regardless of their localization (except for K119A). Meanwhile, in the presence of calcium mutations in N-terminal domain of the protein significantly suppressed this process, indicating that surface properties of Ca2+-bound recoverin are highly affected by N-terminal cluster residues. The substitutions in C-terminal clusters generally reduced thermal stability of recoverin with F172A (‘black’ cluster) as well as W156A and K119A (‘gray’ cluster) being the most efficacious in this respect. In contrast, the mutations in the N-terminal clusters caused less pronounced differently directed changes in thermal stability of the protein. The substitutions of F172, W156, and K119 in C-terminal domain of recoverin together with substitution of Q46 in its N-terminal domain provoked significant but diverse changes in free energy associated with Ca2+ binding to the protein: the mutant K119A demonstrated significantly improved calcium binding, whereas F172A and W156A showed decrease in the calcium affinity and Q46A exhibited no ion coordination in one of the Ca2+-binding sites. The most of the N-terminal clusters mutations suppressed membrane binding of recoverin and its inhibitory activity towards rhodopsin kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated rhodopsin phosphorylation regardless of the presence of calcium. Taken together, these data confirm the scaffolding function of several cluster-forming residues and point to their critical role in supporting physiological activity of recoverin.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma

Design of a Protein Motif Responsive to Tyrosine Nitration and an Encoded Turn-Off Sensor of Tyrosine Nitration.

Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-hand metal-binding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. Nuclear magnetic resonance spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. To develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF-hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with a tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity ( Kd = 1.6 μM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched upon nitration. The greater terbium affinity upon tyrosine nitration resulted in a large dynamic range and sensitivity to substoichiometric nitration. An improved approach for the synthesis of peptides containing nitrotyrosine was also developed, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.

Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in apple (Malus × domestica)

Changes in intracellular calcium (Ca2+) levels in response to developmental processes or external stimuli serve as signals in eukaryotic cells. These Ca2+ signals are likely perceived through sensor proteins that bind Ca2+ by EF-hand (a helix-loop-helix structure) motif. Calmodulins (CaMs), a group of well-characterized Ca2+ sensors, and calmodulin-like (CMLs) are implicated in a large number of diverse cellular processes, including plant development and stress responses. In this study, apple (Malus × domestica) genes encoding CaM and CML proteins that only possess EF-hand motifs with no other functional domains were analyzed. A total of 4 MdCaM and 58 MdCML genes were identified, which are spread among 16 out of the 17 apple chromosomes. Bioinformatics analyses, including protein characteristics, conserved domain, evolutionary relationships and chromosomal locations, demonstrated the conservation and divergence of MdCaMs/CMLs. In addition, expression analysis showed that MdCaMs/CMLs are expressed in more than one tissue, including shoot tips, roots, mature leaves, flowers and fruit. Furthermore, the expression of some MdCaM/CML members responded to plant hormones (abscisic acid, jasmonic acid) and salt stress, suggesting a potential role of these genes in responses to biotic and abiotic stress. Overexpression of stress-induced MdCML3 gene significantly improved the tolerance of apple calli to salinity and ABA. The identification and characterization of MdCaMs/CMLs in apple lays a foundation for future functional studies of these genes.

14-3-3 proteins mediate the localization of Centrin2 to centrosome

14-3-3ε and 14-3-3γ localize to the centrosome and regulate centrosome duplication, by inhibiting cdc25C function. As 14-3-3γ and 14-3-3ε form a complex with centrosomal proteins, we asked if this ability was required to regulate centrosome duplication. The results in this report demonstrate that 14-3-3ε and 14-3-3γ form a complex with Centrin2 and that the binding site is located in the N-terminal EF hand in Centrin2, EF1. A Centrin2 mutant that does not form a complex with 14-3-3 proteins displays a punctate cytoplasmic localization and does not localize to the centrosome. These results suggest that in addition to negatively regulating centrosome duplication as previously reported, 14-3-3 proteins might also be required for centriole biogenesis by regulating the localization of Centrin2 at the centrosome.