Sarcoplasmic Calcium-binding Protein Research Articles

Amphioxus CaVPT and creatine kinase are crucial immune-related molecules in response to bacterial infection and immunization

Although a great progress has been made, our understanding of innate immunity is incomplete. Here, we hypothesize that the innate immune response to pathogens is attributed into a group of functional proteins. The group contains information on host status post bacterial entry (infection or immunity) and bacterial species (Gram-positive or Gram-negative bacteria). Investigation of the group of proteins may result in disclosing of biomarkers identifying the status and species. For this regard, differential proteomics approach coupled with the pattern recognition methods are used to identify biomarkers from the proteins that being specifically regulated during the innate immune response of amphioxus to Gram-positive and Gram-negative bacteria with live or dead status. Four proteins, Calcium vector protein (CaVP), sarcoplasmic calcium-binding protein (SCP), CaVP-target protein (CaVPT) and creatine kinase (CK), are selected as the key biomarkers. Since immunoprotection of CaVP and SCP has been reported, the role of CaVPT and CK are further investigated. Gut CaVPT appears in dying amphioxus, whereas humoral fluid CK downregulates and gut CK keep no change in animals with immunity. The responses are stronger in Gram-negative than Gram-positive bacteria. These results indicate that CaVPT, CK, CaVP and SCP are the most important biomarkers to uncover amphioxus innate immunity to bacteria, and the approach is an efficient way to identify key biomarkers.

Characterization of sarcoplasmic calcium binding protein (SCP) variants from freshwater crayfish Procambarus clarkii

Sarcoplasmic calcium binding protein (SCP) is an invertebrate EF-hand calcium buffering protein that has been proposed to fulfill a similar function in muscle relaxation as vertebrate parvalbumin. We have identified three SCP variants in the freshwater crayfish Procambarus clarkii. The variants (pcSCP1a, pcSCP1b, and pcSCP1c) differ across a 37 amino acid region that lies mainly between the second and third EF-hand calcium binding domains. We evaluated tissue distribution and response of the variants to cold exposure, a stress known to affect expression of parvalbumin. Expression patterns of the variants were not different and therefore do not provide a functional rationale for the polymorphism of pcSCP1. Compared to hepatopancreas, expression of pcSCP1 variants was 100,000-fold greater in axial abdominal muscle and 10-fold greater in cardiac muscle. Expression was 10-100 greater in fast-twitch deep flexor and extensor muscles compared to slow-twitch superficial flexor and extensors. In axial muscle, no significant changes of pcSCP1, calmodulin (CaM), or sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) expression were measured after one week of 4°C exposure. In contrast, large decreases of pcSCP1 were measured in cardiac muscle, with no changes in CaM or SERCA. Knockdown of pcSCP1 by dsRNA led to reduced muscle activity and decreased expression of SERCA. In summary, the pattern of pcSCP1 tissue expression is similar to parvalbumin, supporting a role in muscle contraction. However, the response of pcSCP1 to cold exposure differs from parvalbumin, suggesting possible functional divergence between the two proteins.

Gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri

To understand the role of calcium-binding proteins of invertebrates in immunological response, amphioxus sarcoplasmic calcium-binding protein (SCP) was investigated in the present study. Following gene cloning, recombinant protein expression and purification and antibody preparation, the expression and alteration of SCP in the response to bacterial challenge were detected using Western blotting. SCP was not detected in the branchia, humoral fluid, gonad or in the gut of wounded animals, but it was abundant in muscle and appeared in the gut of healthy animals using Vibrio parahaemolyticus immunization and challenge. Furthermore, whether gut SCP possessed anamnestic response was investigated using cross-immune challenge between Gram-positive and –negative bacteria. Gut SCP showed stronger anamnestic activity or pattern-recognition in response to Gram-negative bacterium V. parahaemolyticus than Gram-positive bacterium Staphylococcus aureus. The response was faster and more species-specific to V. parahaemolyticus, whereas it was slower and longer to S. aureus. The reason why the response showed significant difference between Gram-positive and -negative bacteria awaits investigation. These results indicate that gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri.

Structural basis for prokaryotic calcium-mediated regulation by a Streptomyces coelicolor calcium binding protein.

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.

A differential proteomic approach to assess the effects of chemotherapeutics and production management strategy on giant tiger shrimp Penaeus monodon

The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg − 1) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.

Greater epitope recognition of shrimp allergens by children than by adults suggests that shrimp sensitization decreases with age

Shellfish allergy is a long-lasting disorder typically affecting adults. Despite its high prevalence, there is limited information about allergenic shrimp proteins and the epitopes implicated in such allergic reactions. We sought to identify the IgE-binding epitopes of the 4 shrimp allergens and to characterize epitope recognition profiles of children and adults with shrimp allergy. Fifty-three subjects, 34 children and 19 adults, were selected with immediate allergic reactions to shrimp, increased shrimp-specific serum IgE levels, and positive immunoblot binding to shrimp. Study subjects and 7 nonatopic control subjects were tested by means of peptide microarray for IgE binding with synthetic overlapping peptides spanning the sequences of Litopenaeus vannamei shrimp tropomyosin, arginine kinase (AK), myosin light chain (MLC), and sarcoplasmic calcium-binding protein (SCP). The Wilcoxon test was used to determine significant differences in z scores between patients and control subjects. The median shrimp IgE level was 4-fold higher in children than in adults (47 vs 12.5 kU(A)/L). The frequency of allergen recognition was higher in children (tropomyosin, 81% [94% for children and 61% for adults]; MLC, 57% [70% for children and 31% for adults]; AK, 51% [67% for children and 21% for adults]; and SCP, 45% [59% for children and 21% for adults]), whereas control subjects showed negligible binding. Seven IgE-binding regions were identified in tropomyosin by means of peptide microarray, confirming previously identified shrimp epitopes. In addition, 3 new epitopes were identified in tropomyosin (epitopes 1, 3, and 5b-c), 5 epitopes were identified in MLC, 3 epitopes were identified in SCP, and 7 epitopes were identified in AK. Interestingly, frequency of individual epitope recognition, as well as intensity of IgE binding, was significantly greater in children than in adults for all 4 proteins. Children with shrimp allergy have greater shrimp-specific IgE antibody levels and show more intense binding to shrimp peptides and greater epitope diversity than adults.

Epitope Mapping of the Main Four Shrimp Allergens and Comparison of IgE Recognition Between Children and Adults

RATIONALE: Shellfish allergy is a long-lasting disorder typically persisting throughout life. We aimed to characterize the IgE-binding profiles of four shrimp allergens recognized by children and adults.METHODS: Sera from 34 children (P) and 19 adults (A), with immediate allergic reactions and elevated serum IgE to shrimp were tested by microarray analysis for IgE binding to overlapping synthetic peptides spanning the sequences of the shrimp allergens tropomyosin, myosin light chain (MLC), sarcoplasmic calcium-binding protein (SCP) and arginine kinase (AK).RESULTS: Median shrimp-specific IgE was 4 fold higher in children (47 versus 12.5 kUA/L). Frequency of allergen recognition was: tropomyosin 81% (94%P, 61%A), MLC 57% (70%P, 31%A), AK 51% (67%P, 21%A) and SCP 45% (59%P, 21%A).Identified epitopes in tropomyosin are: Epitope 1 (amino acids 1-39), Epitope 2 (40-63), Epitope 3 (61-81), Epitope 4 (82-105), Epitope 5a (115-153), 5b (142-165), 5c (156-186), Epitope 6 (190-210), Epitope 7 (246-284). In MLC: Epitope 1 (13-30), Epitope 2 (22-48), Epitope 3 (46-66), Epitope 4a (58-90), Epitope 4b (79-99), and Epitope 5 (118-141). In SCP: Epitope 1 (10-33), Epitope 2 (49-72) and Epitope 3 (130-147). And, in AK: Epitope 1 (1-18), Epitope 2a (61-87), 2b (79-96), Epitope 3 (121-141), Epitope 4 (160-192), Epitope 5 (232-249), Epitope 6 (319-342).CONCLUSIONS: Shrimp-allergic children have higher IgE levels, show more intense recognition of shrimp allergens and epitope diversity than adults, who almost only recognize tropomyosin. Our work is the first to show that sensitization to shrimp proteins is greater in children and that appears to decrease in adulthood. RATIONALE: Shellfish allergy is a long-lasting disorder typically persisting throughout life. We aimed to characterize the IgE-binding profiles of four shrimp allergens recognized by children and adults. METHODS: Sera from 34 children (P) and 19 adults (A), with immediate allergic reactions and elevated serum IgE to shrimp were tested by microarray analysis for IgE binding to overlapping synthetic peptides spanning the sequences of the shrimp allergens tropomyosin, myosin light chain (MLC), sarcoplasmic calcium-binding protein (SCP) and arginine kinase (AK). RESULTS: Median shrimp-specific IgE was 4 fold higher in children (47 versus 12.5 kUA/L). Frequency of allergen recognition was: tropomyosin 81% (94%P, 61%A), MLC 57% (70%P, 31%A), AK 51% (67%P, 21%A) and SCP 45% (59%P, 21%A). Identified epitopes in tropomyosin are: Epitope 1 (amino acids 1-39), Epitope 2 (40-63), Epitope 3 (61-81), Epitope 4 (82-105), Epitope 5a (115-153), 5b (142-165), 5c (156-186), Epitope 6 (190-210), Epitope 7 (246-284). In MLC: Epitope 1 (13-30), Epitope 2 (22-48), Epitope 3 (46-66), Epitope 4a (58-90), Epitope 4b (79-99), and Epitope 5 (118-141). In SCP: Epitope 1 (10-33), Epitope 2 (49-72) and Epitope 3 (130-147). And, in AK: Epitope 1 (1-18), Epitope 2a (61-87), 2b (79-96), Epitope 3 (121-141), Epitope 4 (160-192), Epitope 5 (232-249), Epitope 6 (319-342). CONCLUSIONS: Shrimp-allergic children have higher IgE levels, show more intense recognition of shrimp allergens and epitope diversity than adults, who almost only recognize tropomyosin. Our work is the first to show that sensitization to shrimp proteins is greater in children and that appears to decrease in adulthood.

Identification of commercial prawn and shrimp species of food interest by native isoelectric focusing

Native isoelectric focusing (IEF) of water-soluble sarcoplasmic proteins was applied to the identification of 14 shrimp species of food interest belonging to the order Decapoda. These species have different commercial values, but due to their phenotypic similarities and carapace removal in their industrial processing, incorrect food labelling and deliberate or inadvertent adulteration can occur. Each of the 14 tested species showed species-specific protein band profiles and intra-specific polymorphism was low, not preventing the correct identification of the species. Therefore, IEF of water-soluble sarcoplasmic proteins allowed the differentiation of the 14 species considered. In addition, sarcoplasmic calcium-binding proteins (SCPs) were identified by tandem mass spectrometry (MS/MS) as the major species-specific proteins in these species, opening the way to further studies focusing on their potential use as specific biomarkers.

Sarcoplasmic calcium-binding protein is an EF-hand–type protein identified as a new shrimp allergen

Shellfish allergy is a long-lasting disorder usually persisting throughout life. Despite its high prevalence, there is limited information about allergenic shrimp proteins. Characterization of shrimp allergens. Fifty-two adults and children with a history of immediate allergic reactions to shrimp and elevated serum IgE to shrimp were selected for this study. Tryptic digests from a 20-kd IgE-binding protein were analyzed by LC-MS/MS, identifying the protein as a sarcoplasmic-calcium-binding protein. cDNA encoding sarcoplasmic calcium-binding protein (SCP) from a shrimp cDNA library (Litopenaeus vannamei) was amplified by PCR, cloned into an expression vector, and sequenced. Recombinant SCP was tested with patients' sera. ELISA inhibition experiments determined the fraction of total shrimp IgE recognizing SCP. A functional assay with a rat basophilic leukemia cell line was used to determine the capacity for mediator release induced by SCP. Immunoblotting demonstrated IgE binding by 31 of 52 (59.6%) of the sera to a 20-kd shrimp protein. The protein was identified as a SCP. Amplified cDNA encoding SCP was isolated and sequenced. Open reading frame translation provided the complete amino acid sequence of shrimp SCP. Recombinant SCP was recognized by serum IgE from 20 of 52 (38.4%) subjects, of whom 17 of 20 (85%) were children. ELISA inhibition of pooled sera IgE reactivity to BS extract using recombinant SCP was significant (as high as 79%). For some subjects, mediator release induced by recombinant SCP was higher than that induced by recombinant tropomyosin. We have identified and cloned a new shrimp allergen, Lit v 4.0101, an SCP, which appears to be of particular importance in the pediatric population.

Allergènes de poissons et crustacés

Concerning fish allergy, the major allergens are the β isoforms of muscle parvalbumins. Fish parvalbumins share among themselves important homologies in amino acid composition, which gives rise to extensive cross-sensitizations. Allergic cross-reactions with frog parvalbumin have been described. Due to the frequent sensitization to parvalbumins, the role of other allergens is masked. Allergy to fish gelatin has been reported. Additional muscle allergens have been incompletely described, but their exact role remains to be defined. With respect to shellfish allergy, muscle tropomyosin is the major allergen. Tropomyosins give rise to important cross-sensitization between shellfish, but also with gastropods, mites, cockroaches and other arthropods. The myosin light chain has been shown to be an allergen in 60% of white shrimp-allergic patients. A muscle arginine kinase has been recognized as an allergen in a third of patients sensitized to black tiger shrimp. A sarcoplasmic calcium-binding protein of the same tiger shrimp is recognized by IgE antibodies of 60% of shrimp-allergic patients.

Identification and Cloning of a Sarcoplasmic Calcium-binding Protein, a new Shrimp Allergen

RATIONALE: Shellfish allergy is a long-lasting disorder usually persisting throughout life. Despite its high prevalence, there is limited knowledge about allergenic shrimp proteins. Recently a sarcoplasmic calcium-binding protein (SCP) was described as a shrimp allergen, but its allergenicity needs confirmation by recombinant methods.

Sarcoplasmic Calcium-Binding Protein: Identification as a New Allergen of the Black Tiger Shrimp Penaeus monodon

Background: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. Methods: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. Results: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. Conclusions: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

Characterization of a novel EF-hand homologue, CnidEF, in the sea anemone Anthopleura elegantissima

The superfamily of EF-hand proteins is comprised of a large and diverse group of proteins that contain one or more characteristic EF-hand calcium-binding domains. This study describes and characterizes a novel EF-hand cDNA, CnidEF, from the sea anemone Anthopleura elegantissima (Phylum Cnidaria, Class Anthozoa). CnidEF was found to contain two EF-hand motifs near the C-terminus of the deduced amino acid sequence and two regions near the N-terminus that could represent degenerate EF-hand motifs. CnidEF homologues were also identified from two other sea anemone species. A combination of bioinformatic and molecular phylogenetic analyses was used to compare CnidEF to EF-hand proteins in other organisms. The closest homologues identified from these analyses were a luciferin binding protein (LBP) involved in the bioluminescence of the anthozoan Renilla reniformis, and a sarcoplasmic calcium-binding protein (SARC) involved in fluorescence of the annelid worm Nereis diversicolor. Predicted structure and folding analysis revealed a close association with bioluminescent aequorin (AEQ) proteins from the hydrozoan cnidarian Aequorea aequorea. Neighbor-joining analyses grouped CnidEF within the SARC lineage along with AEQ and other cnidarian bioluminescent proteins rather than in the lineage containing calmodulin (CAM) and troponin-C (TNC).

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca 2+. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca 2+ is linked.

Structure of the Neuronal Protein Calexcitin Suggests a Mode of Interaction in Signalling Pathways of Learning and Memory

The three-dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus, calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium-binding protein from Amphioxus although the sequence identity is very low at 31%. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues, which have been implicated by mutagenesis in its GTPase activity, are in a short but highly conserved region of 3(10) helix close to the C terminus. This helix resides in a large loop that is partly sandwiched between the N and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice in the vicinity of the 3(10) helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.

Solution structure and internal dynamics of NSCP, a compact calcium‐binding protein

The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing. Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a rmsd of 0.87 A from the mean structure. The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces. The internal dynamics of the protein backbone were determined by studying amide hydrogen/deuterium exchange rates and 15N nuclear relaxation. The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini. For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein. Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices. Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of N(H)-H vectors. We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence.

A calmodulin-like protein in the bacterial genus Streptomyces

The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix–loop–helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its α-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant Δ cabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera.

Mechanism of Fluorescence and Conformational Changes of the Sarcoplasmic Calcium Binding Protein of the Sand Worm Nereis diversicolor upon Ca2+ or Mg2+ Binding

The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca2+ or Mg2+. Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca2+ or Mg2+. Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of these mutants show a limited decrease of the affinity for calcium, but no alterations of the cooperativity. Upon adding calcium, Trp170 shows a strong fluorescence increase, Trp57 an extensive fluorescence decrease, and Trp4 shows no fluorescence change. Therefore mutant W4F/W170F is ideally suited to analyze the fluorescence titrations and to study the binding mechanism. Mutations of the calcium ligands at the z-position in the three binding sites show no effect at site I and a total loss of cooperativity at sites III and IV. The quenching of Trp57 upon calcium binding is dependent on the presence of arginine R25, but this residue is not just a simple dynamic quencher. The role of the salt bridge R25-D58 is also investigated.

Calcium-regulated GTPase activity in the calcium-binding protein calexcitin

Calexcitin (CE) is a calcium-binding protein, closely related to sarcoplasmic calcium-binding proteins, that is involved in invertebrate learning and memory. Early reports indicated that both Hermissenda and squid CE also could bind GTP; however, the biochemical significance of GTP-binding and its relationship to calcium binding have remained unclear. Here, we report that the GTPase activity of CE is strongly regulated by calcium. CE possessed a P-loop-like structure near the C-terminal similar to the phosphate-binding regions in other GTP-binding proteins. Site-directed mutagenesis of this region showed that Gly 182, Phe 186 and Gly 187 are required for maximum affinity, suggesting that the GTP-binding motif is G-N-x-x-[FM]-G. CE cloned from Drosophila CNS possessed a similar C-terminal sequence and also bound and hydrolyzed GTP. GTPase activity in Drosophila CE was also strongly regulated by Ca 2+, exhibiting over 23-fold higher activity in the presence of 0.3 μM calcium. Analysis of the conserved protein motifs defines a new family of Ca 2+-binding proteins representing the first example of proteins endowed with both EF-hand calcium binding domains and a C-terminal, P-loop-like GTP-binding motif. These results establish that, in the absence of calcium, both squid and Drosophila CE bind GTP at near-physiological concentrations and hydrolyze GTP at rates comparable to unactivated ras. Calcium functions to increase GTP-binding and GTPase activity in CE, similar to the effect of GTPase activating proteins in other low-MW GTP-binding proteins. CE may, therefore, act as a molecular interface between Ca 2+ cytosolic oscillations and the G protein-coupled signal transduction.

NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.