Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains

Ca²⁺ and calmodulin (CaM), a key Ca²⁺ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca²⁺ and CaM in defense signaling, we used ³⁵S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound ³⁵S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca²⁺ whereas EGTA, a Ca²⁺ chelator, abolished binding, confirming that PICBP binds CaM in a Ca²⁺-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsis accelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca²⁺-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca²⁺-dependent CaM-binding domains.     

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery

EF-hand Ca²⁺-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca²⁺. Upon binding Ca²⁺, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca²⁺-binding affinity of CaM and most S100s in the absence of target is weak (^CaK_D > 1 μM). However, upon effector protein binding, the Ca²⁺ affinity of these proteins increases via heterotropic allostery (^CaK_D < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca²⁺ homeostasis and (ii) strict maintenance of Ca²⁺-signaling within a narrow dynamic range of free Ca²⁺ ion concentrations, [Ca²⁺]^free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca²⁺ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.     

Genes encoding calmodulin-binding proteins in the Arabidopsis genome.

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.     

The emerging function of IQD proteins as scaffolds in cellular signaling and trafficking

Calcium (Ca²⁺) signaling modules are essential for adjusting plant growth and performance to environmental constraints. Differential interactions between sensors of Ca²⁺ dynamics and their molecular targets are at the center of the transduction process. Calmodulin (CaM) and CaM-like (CML) proteins are principal Ca²⁺-sensors in plants that govern the activities of numerous downstream proteins with regulatory properties. The families of IQ67-Domain (IQD) proteins are a large class of plant-specific CaM/CML-targets (e.g., 33 members in A. thaliana) which share a unique domain of multiple varied CaM retention motifs in tandem orientation. Genetic studies in Arabidopsis and tomato revealed first roles for IQD proteins related to basal defense response and plant development. Molecular, biochemical and histochemical analysis of Arabidopsis IQD1 demonstrated association with microtubules as well as targeting to the cell nucleus and nucleolus. In vivo binding to CaM and kinesin light chain-related protein-1 (KLCR1) suggests a Ca²⁺-regulated scaffolding function of IQD1 in kinesin motor-dependent transport of multiprotein complexes. Furthermore, because IQD1 interacts in vitro with single-stranded nucleic acids, the prospect arises that IQD1 and other IQD family members facilitate cellular RNA localization as one mechanism to control and fine-tune gene expression and protein sorting.     

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca²⁺ signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca²⁺-dependent electrophoretic mobility shift and Ca²⁺ binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.     

CML9, an Arabidopsis calmodulin-like protein, contributes to plant innate immunity through a flagellin-dependent signalling pathway

Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca²⁺ signals are perceived by various Ca²⁺ sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin‐like (CML) proteins extend the Ca²⁺ toolkit in plants; they share sequence similarity with the ubiquitous and highly conserved CaM but their roles at physiological and molecular levels are largely unknown. Knowledge of the contribution of Ca²⁺ decoding proteins to plant immunity is emerging, and we report here data on Arabidopsis thaliana CML9, whose expression is rapidly induced by phytopathogenic bacteria, flagellin and salicylic acid. Using a reverse genetic approach, we present evidence that CML9 is involved in plant defence by modulating responses to bacterial strains of Pseudomonas syringae. Compared to wild‐type plants, the later responses normally observed upon flagellin application are altered in knockout mutants and over‐expressing transgenic lines. Collectively, using PAMP treatment and P. syringae strains, we have established that CML9 participates in plant innate immunity.     

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca²⁺ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca²⁺ binding to an unidentified secondary Ca²⁺ sensor. To test whether CaM could be the enigmatic Ca²⁺ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca²⁺ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca²⁺/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Calmodulin effects on steroids‐regulated plasma membrane calcium pump activity

It is now generally accepted that non‐genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca²⁺ is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca²⁺ increase is ATP‐consuming plasma membrane calcium pump (PMCA). The enzyme is coded by four genes, but isoforms diversity was detected in excitable and non‐excitable cells. It is the only ion pump stimulated directly by calmodulin (CaM). We examined the role of PMCA isoforms composition and CaM effect in regulation of Ca²⁺ uptake by estradiol, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and their sulfates in a concentration range from 10^?9 to 10^?6 M, using the membranes from rat cortical synaptosomes, differentiated PC12 cells, and human erythrocytes. In excitable membranes with full set of PMCAs steroids apparently increased Ca²⁺ uptake, although to a variable extent. In most of the cases, CaM decreased transport by 30–40% below controls. Erythrocyte PMCA was regulated by the steroids somewhat differently than excitable cells. CaM strongly increased the potency for Ca²⁺ extrusion in membranes incubated with 17‐β‐estradiol and PREG. Our results indicated that steroids may sufficiently control cytoplasmic calcium concentration within physiological and therapeutic range. The response depended on the cell type, PMCA isoforms expression profile, CaM presence, and the steroids structure. Copyright © 2009 John Wiley & Sons, Ltd.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Mammary gland Ca2+-binding (-dependent) proteins: Identification as calelectrins and calpactin I/p36

Calcium-binding (-dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross-reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with ³⁵S-methionine-labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca²⁺-dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca²⁺, we conclude that the mammary gland CBPs are also Ca²⁺-binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.     

Cloning,Recombinant Expression and Characterization of Wild Type-105-Trp-Calmodulin of the Green Alga Mougeotia scalaris

The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX-2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S-transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca²⁺-dependent shift in SDS-PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca²⁺ buffers in the PDE test, affinity to Ca²⁺ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20-fold increase of half maximal activation (K_act) in the PDE test for Mougeotia CaM relative to maize CaM, while the K_act of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp (Mougeotia) → Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling.     

Recognition of β-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca²⁺‐calmodulin‐dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T‐cell activation and cardiac hypertrophy in humans. Calcium binding to CaN_B (the regulatory subunit) triggers conformational change in CaN_A (the catalytic subunit). Two isoforms of CaN_A (α, β) are both abundant in brain and heart and activated by calcium‐saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca²⁺)₄‐CaM_1–148 bound to βCaNp, a peptide representing its CaM‐binding domain, indicated a 1:1 stoichiometry. βCaNp binding to CaM increased the affinity of calcium for the N‐ and C‐domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium‐saturated CaM domains dissociating from βCaNp were ～1 μM. A limiting K_d ≤ 1 nM was measured directly for full‐length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. βCaNp binding to ¹⁵N‐(Ca²⁺)₄‐CaM_1–148 monitored by ¹⁵N/¹HN HSQC NMR showed that association perturbed the N‐domain of CaM more than its C‐domain. NMR resonance assignments of CaM and βCaNp, and interpretation of intermolecular NOEs observed in the ¹³C‐edited and ¹²C‐¹⁴N‐filtered 3D NOESY spectrum indicated anti‐parallel binding. The sole aromatic residue (Phe) located near the βCaNp C‐terminus was in close contact with several residues of the N‐domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of βCaN. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Experimental and computational approaches for the study of calmodulin interactions

Ca²⁺, a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca²⁺ in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca²⁺ sensors that have no enzymatic activity of their own but upon binding Ca²⁺ interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca²⁺/CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca²⁺ sensor target proteins have uncovered their precise role in Ca²⁺-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network - “calcium sensor interactome” - will help us in understanding how Ca²⁺ regulates a myriad of cellular and physiological processes.     

Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca²⁺–dependent manner with a K_d of 7.6 nM and a Ca²⁺ binding K_d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca²⁺. Increasing cytosolic Ca²⁺ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca²⁺/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca²⁺ can act in an integrated fashion to gate currents through these plant ion channels.     

Pull-down of Calmodulin-binding Proteins

Calcium (Ca²⁺) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca²⁺ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)^1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca²⁺ (Ca²⁺-CaM) as opposed to its Ca²⁺-free state (ApoCaM)³.While most target proteins bind Ca²⁺-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin⁴, neurogranin (Ng)⁵, and certain myosins⁶. These proteins have been shown to play important roles in presynaptic function⁷, postsynaptic function⁸, and muscle contraction⁹, respectively. Their ability to bind and release CaM in the absence or presence of Ca²⁺ is pivotal in their function. In contrast, many proteins only bind Ca²⁺-CaM and require this binding for their activation. Examples include myosin light chain kinase¹⁰, Ca²⁺/CaM-dependent kinases (CaMKs)¹¹ and phosphatases (e.g. calcineurin)¹², and spectrin kinase¹³, which have a variety of direct and downstream effects¹⁴.The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca²⁺-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca²⁺-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function^8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca²⁺, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca²⁺ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca²⁺/CaM signaling and proteins that interact with CaM.