Disruption of the NCS-1/frequenin-related ncsA gene in Dictyostelium discoideum accelerates development

To learn more about the function of intracellular Ca2+ in Dictyostelium discoideum, we searched databases for sequences encoding potential members of the neuronal calcium sensor (NCS) family of Ca2+-binding proteins. As a result, genes for five new putative Ca2+-binding proteins were identified. Based on amino acid sequence alignments and phylogenetic analyses, one of these genes (ncsA) was determined to be closely related to NCS-1/frequenin genes in other organisms. The protein product of ncsA (NcsA) binds 45Ca2+ and exhibits a dramatic gel mobility shift in the presence of Ca2+, suggesting that it is a Ca2+ sensor. ncsA-null cells grow normally in axenic culture. However, on bacterial lawns, the ncsA-null clones expand slowly and development begins prematurely within the plaques. In larger clones, ncsA-null cells form narrow growth zones with evenly spaced aggregates along the inner edge, and closely packed fruiting bodies. An analysis of intracellular cyclic adenosine monophosphate (cAMP) levels, developmental timing on phosphate-buffered saline (PBS) agar, and stage-specific gene expression indicate that development of ncsA-null cells is accelerated by 3-4 h. Together, these results suggest that NcsA might function in Dictyostelium to prevent cells from entering development prematurely in the presence of environmental nutrients.     

PKC-induced sensitization of Ca2+-dependent exocytosis is mediated by reducing the Ca2+ cooperativity in pituitary gonadotropes

The highly cooperative nature of Ca2+-dependent exocytosis is very important for the precise regulation of transmitter release. It is not known whether the number of binding sites on the Ca2+ sensor can be modulated or not. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca2+ sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca2+ sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca2+-triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca2+ sensor (from three to two) without significantly altering the Ca2+-binding kinetics. The reduction in the number of Ca2+-binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca2+ channels.     

Conformational properties of Ca2+-binding segments of proteins from the troponin C superfamily

The troponin C superfamily consists of about 100 Ca2+-binding proteins. Sequence variations observed in these proteins have been analyzed and lead to the following conclusions. (1) There are some strict rules defining the set of calcium ligands necessary for effective Ca2+ binding. (2) If they are fulfilled, the Ca2+ binding constant depends on tertiary interactions within a protein, as well as the free energy of secondary structures of its polypeptide chain. The former provide a constant contribution to the free energy of protein folding and the Ca2+-binding process. (3) The observed variety in Ca2+-binding constants of these proteins results from the various abilities of segments of these proteins to assume the correct secondary structure.     

Calcium-dependent hydrophobic chromatography of calmodulin, S-100 protein and troponin-C

We have demonstrated calcium-dependent hydrophobic interactions among calmodulin, S-100 protein and troponin-C and a homologous series of omega-aminoalkyl-agaroses. The three Ca2+-binding proteins were retained on the column of agarose substituted with omega- aminooctyl or even longer with alkylamine, in the presence of Ca2+ and 0.15 M NaCl. As these proteins were not retained on the column with shorter alkylamine 'arms' (N = 2, 4), they are probably successively absorbed with a higher affinity to the hydrophobic agarose column. Calmodulin and S-100 protein were eluted from the aminoocytl -agarose column with 1 mM EGTA in the presence of 0.15 M NaCl and the elution of troponin-C was Ca2+-independently carried out with 0.3 M NaCl. On the other hand, S-100 and troponin-C were eluted Ca2+-dependently from aminodecyl -agarose in the presence of 1 M NaCl and half the amount of the calmodulin applied was eluted with 1 M NaCl. As there are obvious differences among the three Ca2+-binding proteins with regard to chromatographic behavior on omega-aminoalkyl-agarose columns, our results suggest that these three proteins expose different hydrophobic regions following Ca2+-induced conformational changes and, if so, such would explain the interaction with aminoalkyl-agaroses.     

Structural independence of the two EF-hand domains of caltractin

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.     

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca²⁺-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1–101), or its short scaffolding domain (81–101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca²⁺-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca²⁺-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca²⁺-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca²⁺-signals thereby maintaining efficient phototransduction recovery and light adaptation.     

Calcium and chlorpromazine binding to the EF-hand peptides of neuronal calcium sensor-1

Neuronal calcium sensor-1, a protein of calcium sensor family, is known to have four structural EF-hands. We have synthesised peptides corresponding to all the four EF-hands and studied their conformation and calcium-binding. Our data confirm that the first putative site, a non-canonical one (EF1), does not bind calcium. We have investigated if this lack of binding is due to the presence of non-favoured residues (particularly at +x and -z co-ordinating positions) of the loop. We have mutated these residues and found that after modification the peptides bound calcium. However, these mutated peptides (EF1 and its functional mutants) do not show any Ca(2+) induced changes in far-UV CD. EF2, EF3, and EF4 peptides bind Ca(2+), EF3 being the strongest binder, followed by EF4. Our data of Ca(2+)-binding to individual EF peptides show that there are three active Ca(2+)-binding sites in NCS-1. We have also studied the binding of a neuroleptic drug, chlorpromazine, with the protein as well as with its EF-hands. CPZ binds myristoylated as well as non-myristoylated NCS-1 in Ca(2+)-dependent manner, with dynamic interaction to myristoylated protein. CPZ does not bind to EF1, but binds to functional EF-hand peptides and induces changes in far-UV CD. Our results suggest that NCS-1 could be a target of such antipsychotic and neuroleptic drugs.     

Magnesium promotes structural integrity and conformational switching action of a calcium sensor protein

Calcium binding proteins carry out various signal transduction processes upon binding to Ca2+. In general, these proteins perform their functions in a high background of Mg2+. Here, we report the role of Mg2+ on a calcium sensor protein from Entamoeba histolytica (EhCaBP), containing four Ca2+-binding sites. Mg2+-bound EhCaBP exists as a monomer with a conformation different from that of the holo- and apo-EhCaBP. NMR and biophysical data on EhCaBP demonstrate that Mg2+ stabilizes the closed conformation of the apo form. In the presence of Mg2+, the partially collapsed apo-EhCaBP gains stability and structural integrity. Mg2+ binds to only 3 out of 4 calcium binding sites in EhCaBP. The Ca2+ binding affinity and cooperativity of the conformational switching from the "closed" to the "open" state is significantly modulated by the presence of Mg2+. This fine-tuning of the Ca2+ concentration to switch its conformation is essential for CaBPs to carry out the signal transduction process efficiently.     

Identification of residues that determine the absence of a Ca(2+)/myristoyl switch in neuronal calcium sensor-1

The neuronal calcium sensor (NCS) family of Ca(2+)-binding proteins regulates a number of different processes in neurons and photoreceptor cells. The first of these proteins to be characterized, recoverin, was shown to exhibit a Ca(2+)/myristoyl switch whereby its N-terminal myristoyl group is sequestered in the Ca(2+)-free form and is exposed on Ca(2+) binding to allow the protein to become membrane-associated. It has subsequently been shown that certain other family members also exhibit this mechanism in living cells. In contrast, NCS-1 does not show the Ca(2+)/myristoyl switch and is membrane-associated even at low Ca(2+) concentrations. We have used sequence comparison combined with information from structural analyses to attempt to identify candidate residues within the NCS proteins that determine whether or not the Ca(2+)/myristoyl switch operates in cells and have tested their functional significance by mutagenesis. The results show that NCS-1 possesses residues within its N terminus that lock the myristoyl group in an exposed conformation. In addition, other structural aspects within the C-terminal domains are required to allow the switch to operate. We have determined a key role for residues within the motif EELTRK in NCS-1 in keeping the myristoyl group exposed and allowing the protein to be constitutively membrane-associated.     

ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.     

Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes

Mutant versions of the calmodulin of Drosophila melanogaster have been prepared for use in the study of Ca2+ binding and Ca2(+)-induced conformational changes. In each mutant, a conserved glutamic acid residue indicated to play a critical role in Ca2+ binding has been mutated to glutamine in one of the Ca2(+)-binding sites. Thus a series of four proteins, each with an analogous mutation in one of the four binding sites, has been generated. Here the Ca2(+)-induced conformational changes in these proteins have been examined by use of the fluorescent hydrophobic reporter molecule, 9-anthroyl choline. These studies confirm earlier work which indicates that the carboxyl-terminal pair of Ca2(+)-binding sites shows cooperative Ca2+ binding to produce a major conformational change in the protein. However, these studies provide evidence that the sites of the amino-terminal pair are more independent in their Ca2+ binding properties and contribute individually to the conformational changes associated with Ca2+ binding in the amino-terminal half of the protein. This work also indicates that mutation of either of the amino-terminal Ca2(+)-binding sites can influence the conformational change produced by Ca2+ binding to the carboxyl-terminal sites.     

Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.     

Solution structures of Ca2+-CIB1 and Mg2+-CIB1 and their interactions with the platelet integrin alphaIIb cytoplasmic domain

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.     

Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation.

Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N., Olshevskaya, E.V. & Dizhoor, A.M. (2001) J. Biol. Chem.276, 48143-48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type. CaM-GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EF-hand 1 of GCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+-induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-type GCAP-1 is critical for providing the correct conformation for target activation.     

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.     

Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca2+-dependent elution from phenyl-sepharose

Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two "intrinsic glycoproteins," while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.     

One of the Ca2+ binding sites of recoverin exclusively controls interaction with rhodopsin kinase

Recoverin is a neuronal calcium sensor protein that controls the activity of rhodopsin kinase in a Ca(2+)-dependent manner. Mutations in the EF-hand Ca2+ binding sites are valuable tools for investigating the functional properties of recoverin. In the recoverin mutant E121Q (Rec E121Q ) the high-affinity Ca2+ binding site is disabled. The non-myristoylated form of Rec E121Q binds one Ca2+ via its second Ca(2+)-binding site (EF-hand 2), whereas the myristoylated variant does not bind Ca2+ at all. Binding of Ca2+ to non-myristoylated Rec E121Q apparently triggers exposure of apolar side chains, allowing for association with hydrophobic matrices. Likewise, an interaction surface for the recoverin target rhodopsin kinase is constituted upon Ca2+ binding to the non-acylated mutant. Structural changes resulting from Ca(2+)-occupation of EF-hand 2 in myristoylated and non-myristoylated recoverin variants are discussed in terms of critical conditions required for biological activity.     

Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus,Magnaporthe grisea

We isolated a neuronal calcium sensor 1/frequenin-like gene, Mg-NCS-1, from Magnaporthe grisea and evaluated the phenotypes of null-mutants of the gene. The putative Mg-NCS-1 protein showed high similarity to the other NCS-1 proteins. The null-mutants had normal growth and pathogenicity similar to the parental strain, but their growth was suppressed in high concentrations of Ca2+ or acidic conditions.     

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.