Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Alix and ALG-2 are involved in tumor necrosis factor receptor 1-induced cell death

Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFalpha receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixDeltaALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixDeltaALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.     

A novel flagellar Ca2+-binding protein in trypanosomes

A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.     

Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca2+-binding EF-hand sequence

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM-40) is a metal-binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid-G₁, and that a synthetic peptide (amino acids 54–73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide-bonded region was in part responsible for the growth-suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648–2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C-terminal EF-hand sequence comprising a high-affinity Ca²⁺-binding site of SPARC and represented by a synthetic peptide (amino acids 254–273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790–25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [³H]-thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019–0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose-responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti-SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell-surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration-dependent: peptide 4.2 K, containing an Asp₂₅₈ → Lys substitution, and peptide 4.2 AA, in which the two disulfide-bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF-hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild-type 4.2 sequence, that was indicative of a compromised affinity for Ca²⁺. Moreover, ablation of the disulfide bond in peptide 4.2 AA potentially destabilized the Ca²⁺-binding loop structure, as assessed by fluorescence spectroscopy, such that the peptide competed poorly for the binding of [¹²⁵I]-peptide 4.2 to BAE cells. We conclude both that Ca²⁺-coordinating Asp at position 258 and the conformation of peptide 4.2 are necessary for the inhibition of DNA synthesis by SPARC in cultured endothelial cells.     

67 kDa calcimedin, a new Ca2+-binding protein.

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.     

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.     

The EF-hand Ca2+-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca2+-binding requirements

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.     

Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins.

ALG-2 is a newly discovered Ca2+-binding protein which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca2+-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca2+-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca2+-binding motif is likely to participate in the formation of the dimer complex. Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca2+-binding integrity when individually expressed in bacteria. In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands. Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca2+-binding sites. Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca2+ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein. Furthermore, our studies demonstrated that Ca2+ binding to both strong Ca2+-binding sites of ALG-2 is required for ion-induced aggregation of the protein. We also report here the expression, purification, and partial characterization of a Ca2+-binding-deficient ALG-2 mutant (Glu47Ala/Glu114Ala). In light of its much decreased affinity for Ca2+, this mutant could prove to be instrumental in elucidating the Ca2+-mediated function of ALG-2 within the context of its cellular environment.     

Membrane-bound forms of Ca2+-dependent protein modulator: Ca2+-dependent and independent binding of modulator protein to the particulate fraction from brain.

Ca2+-dependent binding of modulator protein to the particulate fraction was studied. The particulate fraction from one gram of rat brain bound in a Ca2+-dependent fashion 144 microgram of modulator protein, representing more than one third of the total soluble modulator protein in this tissue. The binding site was present in both the mitochondrial and microsomal fractions, the specific activity of the microsomes being the higher. The binding was reversible with a physiological concentration of Ca2+, and was temperature-dependent, and the site can be saturated with modulator protein (4.5 microgram modulator protein per mg of microsomal protein). Tryptic digestion of the membranes caused complete disappearance of the binding activity, but heat-treatment for 5 min at 70 degrees C caused only 40% loss of activity. The binding site may be a known or unknown enzyme(s), the activity of which is regulated by Ca2+ and modulator. Alternatively, this binding site may be a nonenzymic protein that regulates the concentration of free modulator protein in the cell.     

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.     

Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity.     

Localization of a K+ -binding site involved in dephosphorylation of the sarcoplasmic reticulum Ca2+ -ATPase

K+ plays an important role for the function of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA), but its binding site within the molecule has remained unidentified. We have located the binding site for a K+ ion in the P-domain by means of x-ray crystallography using crystals prepared in the presence of the K+ congener Rb+. Backbone carbonyls from the loop containing residues 711-715 together with the side chain of Glu732 define the K+/Rb+ site in the Ca2+ -ATPase conformation with bound Ca2+, ADP, and AlF4-. Functional analysis of Ca2+ -ATPase mutants with alterations to Glu732 shows that this site is indeed important for the stimulatory effect of K+ on the dephosphorylation rate. Comparison with the Ca2+ -ATPase in a dephosphorylated E2 conformation suggests that the K+ site is involved in the correct movement and positioning of the A-domain during translocation and dephosphorylation.     

Ca2+-binding proteins in nuclei

Nuclei isolated from skeletal muscle of 15-day-old chick embryos, adult chickens, rabbits and from rat liver contain on the average 8-18 nmol Ca2+/mg protein. Digestion of nuclei with DNAase I and RNAase at 37 degrees C for 8--12 h reduced the Ca2+ binding by more than 90%. After nuclease treatment, Ca2+-binding proteins were identified in the nonhistone chromosomal protein fractions and in the insoluble residue by equilibrium dialysis and centrifuge transport, in media of 0.1 M KCl and 1 mM MgCl2. The interaction of Ca2+-binding proteins with chromatin may be of importance in the regulation of the gene expression in response to changes in cytoplasmic and nucleoplasmic free-Ca2+ concentration.     

Novel gene encoding a Ca2+-binding protein and under hexokinase-dependent sugar regulation

A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.     

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.     

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.