Cloning and sequencing of the gene for Tetrahymena calcium-binding 25-kDa protein (TCBP-25)

With the intention of studying calcium-dependent ciliary reversal in Tetrahymena, we isolated a Tetrahymena calcium-binding protein of 10 kDa (TCBP-10) which was not calmodulin and reported its properties (Ohnishi, K., and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978-13985). However, immunoblotting with an antiserum against TCBP-10 and sequencing of the cDNAs and partial genomic DNAs for this calcium-binding protein prove that this previously reported TCBP-10 is the degraded product of a 25-kDa calcium-binding protein. Thus, we correct the name of the protein from TCBP-10 to TCBP-25. From the analysis of the cDNA for TCBP-25, it is shown to be composed of 218 amino acid residues and its molecular weight is estimated to be 24,702. This protein is predicted to contain four EF-hand-type calcium binding domains and to be a member of the calmodulin family. Little sequence homology with other proteins was shown by a computer search, except in the EF-hand regions. The special feature of TCBP-25 is that the distance between calcium-binding domains II and III is extraordinarily long for a calmodulin family protein having four calcium-binding domains. The genomic DNA for TCBP-25 contains two introns situated at short distances before calcium-binding domains I and III, implying gene duplication in genealogy.     

Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2

Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.     

The third calmodulin family protein in Tetrahymena. Cloning of the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23)

Recently, we proved the existence of the second calmodulin family protein in Tetrahymena (Tetrahymena calcium-binding protein of 25 kDa, TCBP-25) by analyzing its cDNA (Takemasa, T., Ohnishi, K., Kobayashi, T., Takagi, T., Konishi, K., and Watanabe, Y. (1989) J. Biol. Chem. 264, 19293-19301). During the amino acid sequence determination of TCBP-25, we became aware of the fact that another polypeptide carrying calcium-binding domains of EF-hand type existed in addition to Tetrahymena calmodulin and TCBP-25. This third calmodulin family protein from Tetrahymena was confirmed by isolating its cDNA clones. One of the cloned cDNAs contains 763 nucleotides and encodes a protein that is composed of 207 amino acid residues and has a molecular mass of 23,413 daltons. This predicted protein possesses four EF-hand type calcium-binding domains, so we have designated it as Tetrahymena calcium-binding protein of 23 kDa (TCBP-23). TCBP-23 is similar (35% homology) but clearly different from TCBP-25. The TCBP-23 gene is actively transcribed in vivo as a 0.84-kilobase RNA. Thus, it follows that Tetrahymena cells have three different calmodulin family proteins: calmodulin, TCBP-25 and TCBP-23. These proteins are expected to provide important clues for solving the mechanisms of calcium-dependent phenomena, such as ciliary reversal.     

Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.     

Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein

ALG-2 is a EF hand calcium binding protein with sequence homologies to calmodulin. Vito et al have shown that ALG-2 expression is required for apoptosis following a number of death stimuli,1 although nothing is known about the effectors which underlie ALG-2 function. Here we have used ALG-2 as bait in a yeast two hybrid screen of a mouse brain cDNA library. We found that ALG-2 binds to itself and to a novel protein that we call ALG-2 interacting protein X, Alix. Using co-immunoprecipitation experiments, we confirmed ALG-2/ALG-2 binding and demonstrated that this interaction is calcium independent. ALG-2/Alix interaction was also validated by co-immunoprecipitation, but in this case, the binding was found to be strictly calcium dependent. Alix seems highly conserved throughout evolution since it shows significant homologies to a putative C. elegans protein (YNK-1) and to proteins of A. nidulans (PalA) and S. cerevisiae (BRO1). Alix is a potential regulator or downstream effector of ALG-2 action.     

Purification and some properties of a new Ca2+-binding protein (TCBP-10) present in tetrahymena cilium

A new Ca2+-binding protein, different from calmodulin, has been detected in the cilium and cell body of Tetrahymena. This protein, designated as TCBP-10, has been purified from the cells to homogeneity. TCBP-10 is an acidic protein (pI = 4.5) which shows a Ca2+-dependent mobility shift in alkali-glycerol-polyacrylamide gel electrophoresis. The protein is resistant to heat and trichloroacetic acid. The molecular weight of the protein is 10,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 22,000 by Sephadex G-50 gel filtration, suggesting that the native form of the protein is a dimer. The protein has a molar extinction coefficient of 6,500 at 282 nm. Equilibrium dialysis experiments revealed that the protein binds 1 mol of Ca2+/mol of protein with a dissociation constant of 27 microM. The protein contains a relatively large quantity of acidic amino acids, single residues of cysteine, histidine, and tryptophan, and no methionine. These properties are similar to those of some low molecular weight Ca2+-binding proteins belonging to the calmodulin family. Thus, the cilium of Tetrahymena contains a second Ca2+-binding protein in addition to calmodulin. We consider that TCBP-10 and calmodulin may play important cooperative roles in the Ca2+-regulation of ciliary movement in Tetrahymena.     

Calmodulin Target Database

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for a -helix formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

The (1)H NMR structure of bovine Pb(2+)-osteocalcin and implications for lead toxicity

Structural information on the effect of Pb(2+) on proteins under physiologically relevant conditions is largely unknown. We have previously shown that low levels of lead increased the amount of osteocalcin bound to hydroxyapatite (BBA 1535:153). This suggested that lead induced a more compact structure in the protein. We have determined the 3D structure of Pb(2+)-osteocalcin (49 amino acids), a bone protein from a target tissue, using (1)H 2D NMR techniques. Lead, at a stoichiometry of only 1:1, induced a similar fold in the protein as that induced by Ca(2+) at a stoichiometry of 3:1. The structure consisted of an unstructured N-terminus and an ordered C-terminal consisting of a hydrophobic core (residues 16-49). The genetic algorithm-molecular dynamics simulation predicted the lead ion was coordinated by the Gla 24 and Gla 21 residues. It is proposed that mineral binding occurs via uncoordinated Gla oxygen ions binding to calcium in hydroxyapatite. A comparison of Pb(2+)- and Ca(2+)-osteocalcin suggests Pb(2+), at a lower stoichiometry, may induce similar conformational changes in proteins and subsequent molecular processes normally controlled by calcium alone. This may contribute to a molecular mechanism of lead toxicity for calcium binding proteins. Lead exposure may alter the amount of mineral bound osteocalcin and contribute to abnormal bone remodeling.     

Calcium-binding crystallins from Yersinia pestis. Characterization of two single betagamma-crystallin domains of a putative exported protein

Betagamma-crystallin is a superfamily with diverse members from vertebrate lens to microbes. However, not many members have been identified and studied. Here, we report the identification of a putative exported protein from Yersinia pestis as a member of the betagamma-crystallin superfamily. Even though calcium has been known to play an important role in the physiology and virulence of the Yersinia genus, calcium-binding proteins have not yet been identified. We have studied the calcium-binding properties of two of the three crystallin domains present in this putative exported protein designated "Yersinia crystallin." These two domains (D1 and D2) have unique AA and BB types of arrangement of their Greek key motifs unlike the domains of other members of the betagamma-crystallin superfamily, which are either AB or BA types. These domains bind two calcium ions with low and high affinity-binding sites. We showed their calcium-binding properties using various probes for calcium and the effect of calcium on their secondary and tertiary structures. Although both domains bind calcium, D1 underwent drastic changes in secondary and tertiary structure and hydrodynamic volume upon calcium binding. Domain D1, which is intrinsically unstructured in the apo form, requires calcium for the typical betagamma-crystallin fold. Calcium exerted an extrinsic stabilization effect on domain D1 but not on D2, which is also largely unstructured. We suggest that this protein might be involved in calcium-dependent processes, such as stress response or physiology in the Yersinia genus, similar to its microbial relatives and mammalian lens crystallins.     

Involvement of a 25 kDa Tetrahymena Ca2+-binding protein in pronuclear exchange

The Tetrahymena Ca2+-binding protein of 25 kDa (TCBP-25) is a calmodulin family protein containing four EF-hand type calcium-binding domains. TCBP-25 is localized in the whole cell cortex and around both the migratory and stationary pronuclei at the pronuclear exchange stage during conjugation. TCBP-25 is expected to play an important role in conjugation, though its function during sexual reproduction has not been elucidated. According to the localization of this protein and its timing, three possible roles of TCBP-25 are proposed. TCBP-25 may play a role in 1) differentiating the two functional pronuclei from the degenerative post-meiotic nuclei, 2) the process of pronuclear exchange and 3) pronuclear fusion. To test these hypotheses, the localization of TCBP-25 in conjugation mutants (cnj10, cnj7 and bcd2) was examined. The results ruled out the first and the third hypotheses and suggested that TCBP-25 may play a role in pronuclear exchange. In the next step we succeeded in reducing expression of the TCBP-25 gene using the antisense ribosome system, and we analyzed the phenotype of the transformants. The knock down of TCBP-25 function also suggests that TCBP-25 plays a role in the pronuclear exchange and in the maintenance of cell shape.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.     

Identification of ubiquitinated proteins in Arabidopsis

Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.     

How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X.

Domains homologous to the epidermal growth factor (EGF) are important building blocks for extracellular proteins. Proteins containing these domains have been shown to function in such diverse biological processes as blood coagulation, complement activation, and the developmental determination of embryonic cell fates. Many of these proteins require calcium for their biological function. In the case of coagulation factors IX and X and anticoagulants proteins C and S, calcium has been found to bind to the EGF-like domains. We have now determined the three-dimensional structure of the calcium-bound form of the NH2-terminal EGF-like domain in coagulation factor X by two-dimensional NMR and simulated folding. Ligands to the calcium ion are the two backbone carbonyls in Gly-47 and Gly-64, as well as the side chains in Gln-49, erythro-beta-hydroxyaspartic acid (Hya) 63, and possibly Asp-46. The conserved Asp-48 is not a ligand in our present structures. The remaining ligands are assumed to be solvent molecules or, in the intact protein, ligands from neighboring domains. Other proteins interacting in a calcium-dependent manner may also contribute ligands. A comparison with the calcium-free form shows that calcium binding induces strictly local structural changes in the domain. Residues corresponding to the side chain ligands in factor X are conserved in many other proteins, such as the integral membrane protein TAN-1 of human lymphocytes and its developmentally important homolog, Notch, in Drosophila. Calcium binding to EGF-like domains may be crucial for numerous protein-protein interactions involving EGF-like domains in coagulation factors, plasma proteins, and membrane proteins. Therefore, there is reason to believe that this novel calcium site plays an important role in the biochemistry of extracellular proteins.     

Solution structure of a novel calcium binding protein, MTH1880, from Methanobacterium thermoautotrophicum

MTH1880 is a hypothetical protein from Methanobacterium thermoautotrophicum, a target organism of structural genomics. The solution structure determined by NMR spectroscopy demonstrates a typical alpha + beta-fold found in many proteins with different functions. The molecular surface of the protein reveals a small, highly acidic pocket comprising loop B (Asp36, Asp37, Asp38), the end of beta2 (Glu39), and loop D (Ser57, Ser58, Ser61), indicating that the protein would have a possible cation binding site. The NMR resonances of several amino acids within the acidic binding pocket in MTH1880, shifted upon addition of calcium ion. This calcium binding motif and overall topology of MTH1880 differ from those of other calcium binding proteins. MTH1880 did not show a calcium-induced conformational change typical of calcium sensor proteins. Therefore, we propose that the MTH1880 protein contains a novel motif for calcium-specific binding, and may function as a calcium buffering protein.     

The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells

The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca(2+)-regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation.     

The structure of the Arabidopsis thaliana SOS3: molecular mechanism of sensing calcium for salt stress response

The Arabidopsis thaliana SOS3 gene encodes a calcium sensor that is required for plant salt tolerance. The SOS3 protein binds to and activates the self-inhibited SOS2 protein kinase, which mediates the expression and activities of various transporters important for ion homeostasis under salt stress. SOS3 belongs to a unique family of calcium-binding proteins that contain two pairs of EF hand motifs with four putative metal-binding sites. We report the crystal structure of a dimeric SOS3 protein in complex with calcium, and with calcium and manganese. Analytical ultracentrifugation experiments and circular dichroism measurements show that calcium binding is responsible for the dimerization of SOS3. This leads to a change in the global shape and surface properties of the protein that may be sufficient to transmit the Ca(2+) signal elicited during salt stress.     

Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants.

Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth. The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q. This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif. We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site. By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene. We have isolated three independent phage mutants that are able to grow on an IHF- host. They carry different mutations scattered in the roi gene and specifying single amino-acid changes. The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type. Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA. The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed.     

Biochemical characterization of a truncated penta-EF-hand Ca2+ binding protein from maize

Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.     

SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor

SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.