Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Crystal structure of the Ca(2+)-form and Ca(2+)-binding kinetics of metastasis-associated protein, S100A4

S100A4 takes part in control of tumour cell migration and contributes to metastatic spread in in vivo models. In the active dimeric Ca(2+)-bound state it interacts with multiple intracellular targets. Conversely, oligomeric forms of S100A4 are linked with the extracellular function of this protein. We report the 1.5A X-ray crystal structure of Ca(2+)-bound S100A4 and use it to identify the residues involved in target recognition and to derive a model of the oligomeric state. We applied stopped-flow analysis of tyrosine fluorescence to derive kinetics of S100A4 activation by Ca(2+) (k(on)=3.5 microM(-1)s(-1), k(off)=20s(-1)).     

C-terminal domain of Kv4.2 and associated KChIP2 interactions regulate functional expression and gating of Kv4.2

The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.     

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

Localization of the Ca(2+)-binding protein, Bra r 1, in anthers and pollen tubes

Calcium plays an essential role during pollen development and pollen tube growth, and several Ca(2+)-binding proteins are expressed in anthers. We have previously reported that Brassica pollen allergens encoded by Bra r 1 and Bra r 2 show sequence similarities to Ca(2+)-binding proteins [Toriyama et al. (1995) Plant Mol. Biol. 29: 1157]. Herein, we report that both genes are expressed in the diploid tapetum and haploid microspores, as detected by in situ RNA hybridization. Immunoblot analysis revealed that Bra r 1 and Bra r 2 were accumulated in anthers during pollen development. When pollen grains were suspended in an aqueous solution, both proteins were mainly detected in the pollen extracellular fraction, indicating that Bra r 1 and Bra r 2 are released from the pollen upon hydration. Localization of Bra r 1 was further investigated in sections of anthers and pollen tubes. Bra r 1 was detected in the tapetum, microspores and pollen grains. In longitudinal sections of cross-pollinated pistils. Bra r 1 was detected throughout pollen tubes elongating in transmitting-tissue. These findings suggest that Bra r 1 may be involved in pollen-pistil interaction and pollen tube growth.     

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.     

Cab45, a novel (Ca2+)-binding protein localized to the Golgi lumen

We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca2+ due to the presence of six EF-hand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol Chem. 269:19142- 19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi. Cab45 is the first calcium- binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment.     

Identification and characterization of two penta-EF-hand Ca(2+)-binding proteins in Dictyostelium discoideum

Penta-EF-hand (PEF) proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit are a newly classified family of Ca(2+)-binding proteins that possess five EF-hand-like motifs. We identified two mutually homologous PEF proteins, designated DdPEF-1 and DdPEF-2 (64% amino acid residue identities), in the cellular slime mold Dictyostelium discoideum. Both PEF proteins showed a higher similarity to mammalian ALG-2 and peflin (Group I PEF proteins) than to calpain and sorcin subfamily (Group II PEF proteins) in the first EF-hand (EF-1) regions. Northern blot analyses revealed that DdPEF-1 and DdPEF-2 were constitutively expressed throughout development of Dictyostelium, but their levels of expression were developmentally regulated. In situ hybridization analyses demonstrated that DdPEF-1 was expressed in both the anterior prestalk and the posterior prespore regions of the tipped aggregate, slugs and early culminants. On the other hand, DdPEF-2 was dominantly expressed in the anterior tip region of these multicellular structures. Both PEF proteins were detected as 22-23-kDa proteins in soluble fractions in the presence of EGTA but in particulate fractions in the presence of Ca(2+) by Western blotting using specific monoclonal antibodies. Together with the finding of PEF-like sequences in DNA databases of plants, fungi and protists, our results strongly suggest that Group I PEF proteins are ubiquitously present in all eukaryotes and play important roles in basic cellular functions.     

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6

S100A11 is a member of the S100 family of EF-hand Ca²⁺-binding proteins, which is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca²⁺-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca²⁺-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH₂ terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH₂- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca²⁺] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma. smooth muscle; protein-protein interaction     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Annexins--a new family of Ca(2+)-binding proteins

Annexins, the Ca(2+)- and phospholipid-binding proteins, are able to induce Ca(2+)-dependent aggregation of biomembranes. All the representatives of this family contain four or eight tandem repeats, 60-80 amino acids each. All these repeats include a highly conservative 17-member amino acid consensus sequence (an endonexin fold). The central domain comprises all these repeats and contains, in addition, the site(s) with a binding affinity for Ca2+ and phospholipids. Annexins are devoid of the classical "EF-hand" Ca(2+)-binding domain and can therefore be assigned to a new family of Ca(2+)-binding proteins.     

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.     

Purification, properties, and molecular cloning of a novel Ca(2+)-binding protein in radish vacuoles

To understand the roles of plant vacuoles, we have purified and characterized a major soluble protein from vacuoles of radish (Raphanus sativus cv Tokinashi-daikon) taproots. The results showed that it is a novel radish vacuole Ca(2+)-binding protein (RVCaB). RVCaB was released from the vacuolar membrane fraction by sonication, and purified by ion exchange and gel filtration column chromatography. RVCaB is an acidic protein and migrated on sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 43 kD. The Ca(2+)-binding activity was confirmed by the (45)Ca(2+)-overlay assay. RVCaB was localized in the lumen, as the protein was recovered in intact vacuoles prepared from protoplasts and was resistant to trypsin digestion. Plant vacuoles store Ca(2+) using two active Ca(2+) uptake systems, namely Ca(2+)-ATPase and Ca(2+)/H(+) antiporter. Vacuolar membrane vesicles containing RVCaB accumulated more Ca(2+) than sonicated vesicles depleted of the protein at a wide range of Ca(2+) concentrations. A cDNA (RVCaB) encoding a 248-amino acid polypeptide was cloned. Its deduced sequence was identical to amino acid sequences obtained from several peptide fragments of the purified RVCaB. The deduced sequence is not homologous to that of other Ca(2+)-binding proteins such as calreticulin. RVCaB has a repetitive unique acidic motif, but not the EF-hand motif. The recombinant RVCaB expressed in Escherichia coli-bound Ca(2+) as evidenced by staining with Stains-all and migrated with an apparent molecular mass of 44 kD. These results suggest that RVCaB is a new type Ca(2+)-binding protein with high capacity and low affinity for Ca(2+) and that the protein could function as a Ca(2+)-buffer and/or Ca(2+)-sequestering protein in the vacuole.     

Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain.

Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa. Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium-binding sites. To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed. The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901. The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments. The results demonstrate that the four binding sites can be occupied by Ca2+. Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns. Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb. Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions. To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay. The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb.     

Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin.

Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied. In each mutant, a conserved glutamic acid residue at position 12 in all of the Ca(2+)-binding loops has been mutated in one site. In one series the residue is changed to glutamine; in the second series the change is to lysine. The Ca(2+)-binding properties of these mutants and the wild-type protein under pseudo-physiological conditions are presented. In addition, Ca(2+)-induced changes to the environment of the single tyrosine residue (Tyr-138) have been studied for some of the mutants. Ca2+ binding to the wild-type protein is best modeled as two pairs of sites with a higher affinity pair that shows strong cooperativity. For all but one of these eight mutant proteins, only three Ca(2+)-binding events can be detected. In three of the amino-terminal mutants, the three residual sites are (i) a pair of relatively high affinity sites and (ii) a weakened low affinity site. For all four carboxyl-terminal mutations, the residual sites are three relatively low affinity sites. In general, mutations to sites 2 and 4 prove more deleterious than mutations to sites 1 and 3. The Ca(2+)-induced conformational changes in the vicinity of Tyr-138 are relatively undisturbed by mutations of site 1. However, the changes to Tyr-138 in the carboxyl-terminal site mutants indicate that upon disruption of the cooperative binding at the high affinity sites, conformational change in the carboxyl terminus occurs in two phases. It appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site. The final conformations of site 3 and 4 mutants are thus significantly different.