Calcium-binding by p26olf, an S100-like protein in the frog olfactory epithelium.

Frog p26olf is a novel S100-like Ca2+-binding protein found in olfactory cilia. It consists of two S100-like domains aligned sequentially, and has a total of four Ca2+-binding sites (known as EF-hands). In this study, to elucidate the mechanism of Ca2+-binding to each EF-hand (named EF-A, -B, -C and -D from the N-terminus of p26olf), we examined Ca2+-binding in wild-type p26olf and also in its mutants in which a glutamate at the -z coordinate position within each Ca2+-binding loop was substituted for a glutamine. Flow dialysis experiments showed that the wild-type binds nearly four Ca2+ per molecule maximally, while all the mutants bind approximately three Ca2+. Although EF-B and -D are p26olf-specific EF-hands and their role in Ca2+-binding is not known, the result unequivocally showed that they actually bind Ca2+. The overall Ca2+-binding affinity decreased in the three mutants. The decrease was very large in the mutants of EF-A and -B, which suggested that the Ca2+-affinities are high in EF-A and -B in the wild-type. Assuming the presence of four steps of Ca2+-binding, we determined the dissociation constant of each step in wild-type p26olf. To assign which step takes place at which EF-hand, we measured the antagonistic effect of K+ on each step, as the effect of K+ is thought to be a function of the number of the carboxyl groups in an EF-hand. Although the actual Ca2+-binding mechanism may not be so simple, this study together with the mutation study suggested a tentative Ca2+-binding model of p26olf: the order of Ca2+-binding to p26olf is EF-B, EF-A, EF-C and EF-D. Based on these results, we speculate that similar Ca2+-binding takes place in an S100 dimer.     

S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner.

S100B(betabeta) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19-31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627-11631), S100B(betabeta) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 +/- 7 microM) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(betabeta) in the presence of Ca2+ (IC50 = 17 +/- 5 microM; IC50 = 1 +/- 0.5 microM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (residues 61-72) of each S100beta subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ([Ca]IC50 = 29.3 +/- 17.6 microM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+-binding domain of S100B(betabeta).     

Solution structure of human Mts1 (S100A4) as determined by NMR spectroscopy

Mts1 is a member of the S100 family of Ca2+-binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca2+-dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1', and 4') to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca2+-binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel beta-sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF-hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family.     

The primary structure of a new Mr 18,000 calcium vector protein from amphioxus

The amino acid sequence of a new Ca2+-binding protein (CaVP) from Amphioxus muscle (Cox, J. A., J. Biol. Chem. 261, 13173-13178) has been determined. The protein contains 161 amino acid residues and has a molecular weight of 18,267. The N terminus is blocked by an acetyl group. The two functional Ca2+-binding sites have been localized based on homology with known Ca2+-binding domains, on internal homology and on secondary structure prediction, and appear to be the domains III and IV. The C-terminal half of CaVP, which contains the two Ca2+-binding sites, shows a remarkable similarity with human brain calmodulin (45%) and with rabbit skeletal troponin C (40%). Functional domain III contains 2 epsilon-N-trimethyllysine residues in the alpha-helices flanking the Ca2+-binding loop. Sequence determination revealed two abortive Ca2+-binding domains in the N-terminal half of CaVP with a similarity of 24 and 30% as compared with calmodulin and troponin C, respectively. This half is also characterized by the presence of a disulfide bridge linking the N-terminal helix of domain I to the C-terminal helix of domain II. This disulfide bond is very resistant to reduction in the native state, but not in denatured CaVP. The optically interesting aromatic chromophores (2 tryptophan and 1 tyrosine residues) are all located in the nonfunctional domain II.     

Structural basis for diversity of the EF-hand calcium-binding proteins

The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca2+-binding and target interaction properties. Here, our current understanding of the Ca2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11-14 amino acid residues in the Ca2+-binding loop are analyzed within the framework of the recently proposed two-step Ca2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca2+-binding loops in the two-EF-hand domain. This structure, named EFbeta-scaffold, defines the position of the bound Ca2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca2+-binding loop. It is proposed that the nature of the first ligand of the Ca2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca2+-induced conformational change in the EF-hand proteins.     

Three-dimensional structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor

The three-dimensional structure of a sarcoplasmic Ca2(+)-binding protein from the sandworm Nereis diversicolor has been determined at 3.0 A resolution using multiple isomorphous replacement techniques. The NH2-terminal half of the molecule contains one variant Ca2(+)-binding domain with a novel helix-loop-helix conformation and one Ca2(+)-binding domain that is no longer functional because of amino acid changes. The overall conformation of this pair of domains is different from any previously described Ca2(+)-binding protein. The COOH-terminal half of the protein contains two Ca2(+)-binding domains with the usual helix-loop-helix configuration and is similar to calmodulin and troponin C. Unlike calmodulin or troponin C, there is no exposed alpha-helix connecting the two halves of the molecule, so the overall structure is much more compact.     

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.     

Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy

Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.     

Structural studies on the Ca2+-binding domain of human nucleobindin (calnuc)

Nucleobindin, also known as calnuc, participates in Ca2+ storage in the Golgi, as well as in other biological processes that involve DNA-binding and protein-protein interactions. We have determined the three-dimensional solution structure of the Ca(2+)-binding domain of nucleobindin by NMR showing that it consists of two EF-hand motifs. The NMR structure indicates that the phi and psi angles of residues in both motifs are very similar, despite the noncanonical sequence of the C-terminal EF-hand, which contains an arginine residue instead of the typical glycine at the sixth position of the 12-residue loop. The relative orientation of the alpha-helices in the N-terminal EF-hand falls within the common arrangement found in most EF-hand structures. In contrast, the noncanonical EF-hand deviates from the average orientation. The two helix-loop-helix moieties are in the open conformation characteristic of the Ca(2+)-bound state. We find that both motifs bind Ca2+ with apparent dissociation constants of 47 and 40 microM for the noncanonical and the canonical EF-hand, respectively. The Ca(2+)-binding domain of nucleobindin is unstructured in the absence of Ca2+ and folds upon Ca2+ addition. NMR relaxation data and structural studies of the folded domain indicate that it undergoes slow dynamics, suggesting that it is floppier and less compact than a globular domain.     

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).     

Characterization of p26olf, a novel calcium-binding protein in the frog olfactory epithelium

We have previously shown that p26olf is a novel S100-like Ca(2+)-binding protein in the frog olfactory epithelium. In this paper, we characterized the Ca(2+) binding property of p26olf, examined the precise localization in the frog olfactory epithelium, and searched for the possible target proteins of p26olf. By flow dialysis experiments using (45)Ca, p26olf was suggested to bind approximately 4 Ca(2+). Circular dichroism measurement showed that binding of Ca(2+) to p26olf induces an increase in the apparent content of both alpha-helix and beta-sheet with an apparent K(d) value of 2.4 micrometer. Electron microscopic observation disclosed p26olf immunoreactivity in the cilia, dendritic knob, and dendrite of olfactory receptor cells. Blot overlay analysis and affinity purification of p26olf-binding proteins showed that p26olf binds to a frog beta-adrenergic receptor kinase-like protein in a Ca(2+)-dependent manner. These results suggested that p26olf has some roles in the olfactory transduction or adaptation.     

Molecular cloning and characterization of the human S100A14 gene encoding a novel member of the S100 family

S100 proteins form a growing subfamily of proteins related by Ca2+-binding motifs to the Efhand Ca2+-binding protein superfamily. By analyzing a human lung cancer cell line subtraction cDNA library, we have identified and characterized a new member of the human S100 family that we named S100A14 (GenBank acc. no. NM_020672). It encodes a mRNA present in several normal human tissues of epithelial origin, with the highest level of expression in colon. The full-length cDNA is 1067 nt in length, with a coding region predicting a protein of 104 amino acids that is 68% homologous to the S100A13 protein. The deduced amino acid sequence of the human S100A14 and its mouse homolog (identified as GenBank entry) contains two EF-hand Ca2+-binding domains, a myristoylation motif, a glycosylation site, and several potential protein kinase phosphorylation sites. We have mapped this gene to human chromosome 1q21, within a region where at least 15 other S100 genes are tightly clustered. A 3.2-kb genomic fragment containing the entire S100A14 was cloned and sequenced. The gene is split into four exons and three introns spanning a total of 2165 bp of genomic sequence. We examined the intracellular distribution of the epitope-tagged S100A14 protein in two human lung carcinoma cell lines and one immortalized monkey cell line. Pronounced staining was observed in the cytoplasm, suggesting an association with the plasma membrane and in the perinuclear area. We also provide evidence for heterogenic expression of S100A14 in tumors, demonstrating its overexpression in ovary, breast, and uterus tumors and underexpression in kidney, rectum, and colon tumors, a pattern suggesting distinct regulation with potentially important functions in malignant transformation.     

A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.     

Amino acid sequence of the Ca2(+)-triggered luciferin binding protein of Renilla reniformis

The complete amino acid sequence of the Ca2(+)-triggered luciferin binding protein (LBP) of Renilla reniformis has been determined. The apoprotein has an unblocked amino terminus and contains 184 residues with a calculated Mr of 20,541. LBP is a member of the EF-hand superfamily of Ca2(+)-binding proteins and bears three predicted EF-hand domains. The sequence and organization of EF-hand domains are similar to those of the Ca2(+)-dependent photoprotein, aequorin.     

The three-dimensional solution structure of Ca(2+)-bound S100A1 as determined by NMR spectroscopy

S100A1 is an EF-hand-containing Ca(2+)-binding protein that undergoes a conformational change upon binding calcium as is necessary to interact with protein targets and initiate a biological response. To better understand how calcium influences the structure and function of S100A1, the three-dimensional structure of calcium-bound S100A1 was determined by multidimensional NMR spectroscopy and compared to the previously determined structure of apo. In total, 3354 nuclear Overhauser effect-derived distance constraints, 240 dihedral constraints, 160 hydrogen bond constraints, and 362 residual dipolar coupling restraints derived from a series of two-dimensional, three-dimensional, and four-dimensional NMR experiments were used in its structure determination (>21 constraints per residue). As with other dimeric S100 proteins, S100A1 is a symmetric homodimer with helices 1, 1', 4, and 4' associating into an X-type four-helix bundle at the dimer interface. Within each subunit there are four alpha-helices and a short antiparallel beta-sheet typical of two helix-loop-helix EF-hand calcium-binding domains. The addition of calcium did not change the interhelical angle of helices 1 and 2 in the pseudo EF-hand significantly; however, there was a large reorientation of helix 3 in the typical EF-hand. The large conformational change exposes a hydrophobic cleft, defined by residues in the hinge region, the C terminus, and regions of helix 3, which are important for the interaction between S100A1 and a peptide (TRTK-12) derived from the actin-capping protein CapZ.     

Three-dimensional solution structure of the calcium-signaling protein apo-S100A1 as determined by NMR.

S100A1, a member of the S100 protein family, is an EF-hand containing Ca(2+)-binding protein (93 residues per subunit) with noncovalent interactions at its dimer interface. Each subunit of S100A1 has four alpha-helices and a small antiparallel beta-sheet consistent with two helix-loop-helix calcium-binding domains [Baldiserri et al. (1999) J. Biomol. NMR 14, 87-88]. In this study, the three-dimensional structure of reduced apo-S100A1 was determined by NMR spectroscopy using a total of 2220 NOE distance constraints, 258 dihedral angle constraints, and 168 backbone hydrogen bond constraints derived from a series of 2D, 3D, and 4D NMR experiments. The final structure was found to be globular and compact with the four helices in each subunit aligning to form a unicornate-type four-helix bundle. Intermolecular NOE correlations were observed between residues in helices 1 and 4 from one subunit to residues in helices 1' and 4' of the other subunit, respectively, consistent with the antiparallel alignment of the two subunits to form a symmetric X-type four-helix bundle as found for other members of the S100 protein family. Because of the similarity of the S100A1 dimer interface to that found for S100B, it was possible to calculate a model of the S100A1/B heterodimer. This model is consistent with a number of NMR chemical shift changes observed when S100A1 is titrated into a sample of (15)N-labeled S100B. Helix 3 (and 3') of S100A1 was found to have an interhelical angle of -150 degrees with helix 4 (and 4') in the apo state. This crossing angle is quite different (>50 degrees ) from that typically found in other EF-hand containing proteins such as apocalmodulin and apotroponin C but more similar to apo-S100B, which has an interhelical angle of -166 degrees. As with S100B, it is likely that the second EF-hand of apo-S100A1 reorients dramatically upon the addition of Ca(2+), which can explain the Ca(2+) dependence that S100A1 has for binding several of its biological targets.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution.

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.