Calcium (Ca 2+ ) signaling is involved in the regulation of diverse biological functions through association with several proteins that enable them to respond to abiotic and biotic stresses. Though Ca 2+ -dependent signaling has been implicated in the regulation of several physiological processes in Chlamydomonas reinhardtii , Ca 2+ sensor proteins are not characterized completely . C. reinhardtii has diverged from land plants lineage, but shares many common genes with animals, particularly those encoding proteins of the eukaryotic flagellum (or cilium) along with the basal body. Calcineurin, a Ca 2+ /calmodulin-dependent protein phosphatase, is an important effector of Ca 2+ signaling in animals, while calcineurin B-like proteins (CBLs) play an important role in Ca 2+ sensing and signaling in plants. The present study led to the identification of 13 novel CBL-like Ca 2+ sensors in C. reinhardtii genome . One of the archetypical genes of the newly identified candidate, CrCBL-like1 was characterized. The ability of CrCBL-like1 protein to sense as well as bind Ca 2+ were validated using two-step Ca 2+ -binding kinetics. The CrCBL-like1 protein localized around the plasma membrane, basal bodies and in flagella, and interacted with voltage-gated Ca 2+ channel protein present abundantly in the flagella, indicating its involvement in the regulation of the Ca 2+ concentration for flagellar movement. The CrCBL-like1 transcript and protein expression were also found to respond to abiotic stresses, suggesting its involvement in diverse physiological processes. Thus, the present study identifies novel Ca 2+ sensors and sheds light on key players involved in Ca 2+ signaling in C. reinhardtii , which could further be extrapolated to understand the evolution of Ca 2+ mediated signaling in other eukaryotes.

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca 2+ , adenine nucleotides and NADH, with NADH exerting a major influence on the K 0.5 value for Ca 2+ . We investigated two potential Ca 2+ -binding sites on E1, which we term site 1 (D 114 ADLD) and site 2 (E 139 SDLD). Comparison of sequences from vertebrates with those from Ca 2+ -insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca 2+ , whereas variant ∆site1 (in which the sequence of site 1 is replaced by A 114 AALA) exhibits an almost complete loss of Ca 2+ activation. Variant ∆site2 (in which the sequence is replaced with A 139 SALA) shows no measurable change in Ca 2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca 2+ -binding site, which is distinct from other previously described Ca 2+ -binding sites.

CDPKs (calcium-dependent protein kinases), which contain both calmodulin-like calcium binding and serine/threonine protein kinase domains, are only present in plants and some protozoans. Upon activation by a stimulus, they transduce the signal through phosphorylation cascades to induce downstream responses, including transcriptional regulation. To understand the functional specificities of CDPKs, 14 Arabidopsis CPKs (CDPKs in plants) representative of the three main subgroups were characterized at the biochemical level, using HA (haemagglutinin)-tagged CPKs expressed in planta . Most of them were partially or mainly associated with membranes, in agreement with acylation predictions. Importantly, CPKs displayed highly variable calcium-dependences for their kinase activities: seven CPKs from subgroups 1 and 2 were clearly sensitive to calcium with different intensities, whereas six CPKs from subgroup 3 exhibited low or no calcium sensitivity to two generic substrates. Interestingly, this apparent calcium-independence correlated with significant alterations in the predicted EF-hands of these kinases, although they all bound calcium. The noticeable exception, CPK25, was calcium-independent owing to the absence of functional EF-hands. Taken together, the results of the present study suggest that calcium binding differentially affects CDPK isoforms that may be activated by distinct molecular mechanisms.

Two missense mutations, R33Q and L167H, of hCASQ2 (human cardiac calsequestrin), a protein segregated to the lumen of the sarcoplasmic reticulum, are linked to the autosomal recessive form of CPVT (catecholaminergic polymorphic ventricular tachycardia). The effects of these mutations on the conformational, stability and Ca 2+ sensitivity properties of hCASQ2, were investigated. Recombinant WT (wild-type) and mutant CASQ2s were purified to homogeneity and characterized by spectroscopic (CD and fluorescence) and biochemical (size-exclusion chromatography and limited proteolysis) methods at 500 and 100 mM KCl, with or without Ca 2+ at a physiological intraluminal concentration of 1 mM; Ca 2+ -induced polymerization properties were studied by turbidimetry. In the absence of Ca 2+ , mutations did not alter the conformation of monomeric CASQ2. For L167H only, at 100 mM KCl, emission fluorescence changes suggested tertiary structure alterations. Limited proteolysis showed that amino acid substitutions enhanced the conformational flexibility of CASQ2 mutants, which became more susceptible to tryptic cleavage, in the order L167H>R33Q>WT. Ca 2+ at a concentration of 1 mM amplified such differences: Ca 2+ stabilized WT CASQ2 against urea denaturation and tryptic cleavage, whereas this effect was reduced in R33Q and absent in L167H. Increasing [Ca 2+ ] induced polymerization and precipitation of R33Q, but not that of L167H, which was insensitive to Ca 2+ . Based on CASQ2 models, we propose that the Arg 33 →Gln exchange made the Ca 2+ -dependent formation of front-to-front dimers more difficult, whereas the Leu 167 →His replacement almost completely inhibited back-to-back dimer interactions. Initial molecular events of CPVT pathogenesis begin to unveil and appear to be different depending upon the specific CASQ2 mutation.

AFPs (antifreeze proteins) are produced by many organisms that inhabit ice-laden environments. They facilitate survival at sub-zero temperatures by binding to, and inhibiting, the growth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large (>1 MDa) hyperactive Ca 2+ -dependent AFP. We have cloned, expressed and characterized a 322-amino-acid region of the protein where the antifreeze activity is localized that shows similarity to the RTX (repeats-in-toxin) family of proteins. The recombinant protein requires Ca 2+ for structure and activity, and it is capable of depressing the freezing point of a solution in excess of 2 °C at a concentration of 0.5 mg/ml, therefore classifying it as a hyperactive AFP. We have developed a homology-guided model of the antifreeze region based partly on the Ca 2+ -bound β-roll from alkaline protease. The model has identified both a novel β-helical fold and an ice-binding site. The interior of the β-helix contains a single row of bound Ca 2+ ions down one side of the structure and a hydrophobic core down the opposite side. The ice-binding surface consists of parallel repetitive arrays of threonine and aspartic acid/asparagine residues located down the Ca 2+ -bound side of the structure. The model was tested and validated by site-directed mutagenesis. It explains the Ca 2+ -dependency of the region, as well its hyperactive antifreeze activity. This is the first bacterial AFP to be structurally characterized and is one of only five hyperactive AFPs identified to date.

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca 2+ ions (log K 1 =7.1±0.4, log K 2 =4.7±0.6, log K 3 =3.6±0.7 and log K 4 =4.6±0.6 in physiological salt buffers) with a [Ca 2+ ] 0.5 of approx. 25 μM. The tertiary structure of secretagogin changes significantly upon Ca 2+ binding, but not upon Mg 2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca 2+ binding, but not upon Mg 2+ binding. These properties suggest that secretagogin belongs to the ‘sensor’ family of Ca 2+ -binding proteins. However, in contrast with the prototypical Ca 2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca 2+ -induced exocytosis in neurons and in neuroendocrine cells. K d was determined to be 1.2×10 −7 M in the presence of Ca 2+ and 1.5×10 −6 M in the absence of Ca 2+ . The comparatively low Ca 2+ affinity for secretagogin and the fact that it undergoes Ca 2+ -induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca 2+ signalling to exocytotic processes.

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca 2+ binding for MMP-12 activity and the properties of the different Ca 2+ sites, the activity as a function of [Ca 2+ ] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca 2+ ]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the K D was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The p K a values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca 2+ ] and pH ranges studied suggest that Ca 2+ may be involved in controlling the activity of MMP-12.

Annexin A13 is considered the original progenitor of the 11 other members of vertebrate annexins, a superfamily of calcium/phospholipid-binding proteins. It is highly tissue-specific, being expressed only in intestinal and kidney epithelial cells. Alternative splicing generates two isoforms, both of which bind to rafts. In view of the lack of structural information supporting the physiological role of this annexin subfamily, we have cloned, expressed and purified human annexin A13b to investigate its structural and functional properties. The N-terminus of annexin A13b: (i) destabilizes the conserved protein core, as deduced from the low melting temperature in the absence (44 °C) or presence of calcium (55 °C), and (ii) impairs calcium-dependent binding to acidic phospholipids, requiring calcium concentrations >400 μM. Truncation of the N-terminus restores thermal stability and decreases the calcium requirement for phospholipid binding, confirming its essential role in the structure–function relationship of this annexin. Non-myristoylated annexin A13b only binds to acidic phospholipids at high calcium concentrations. We show for the first time that myristoylation of annexin A13b enables the direct binding to phosphatidylcholine, raft-like liposomes and acidic phospholipids in a calcium-independent manner. The conformational switch induced by calcium binding, from a ‘closed’ to an ‘open’ conformation with exposure of Trp 227 , can be mimicked by a decrease in pH, a process that may be relevant for membrane interactions. Our studies confirm that the common structural and functional characteristics that are dependent on the protein core of vertebrate annexins are likely to be common conserved features, whereas their variable N-termini confer distinct functional properties on annexins, as we report for myristoylation of annexin A13b.

Neutral trehalases mobilize trehalose accumulated by fungal cells as a protective and storage carbohydrate. A structural feature of these enzymes is the presence of an EF-like motif similar to that shown by many Ca 2+ -binding proteins. In this study we provide direct evidence for physical binding of Ca 2+ to neutral trehalase (Ntp1p) of the fission yeast Schizosaccharomyces pombe , and show that aspartic residues at positions 97 and 108 in the conserved putative Ca 2+ -binding motif of Ntp1p appear to be responsible for this interaction. Mutations in these residues do not interfere with the ability of Ntp1p to associate in vivo with trehalose-6-phosphate synthase, but prevent activation of neutral trehalase triggered by the addition of glucose or by subjecting cells to stressing conditions. Strains expressing Ntp1p variants that are unable to bind Ca 2+ partially resemble those devoid of the ntp1 + gene in terms of trehalose hyperaccumulation. Gel filtration of cell extracts from wild-type cells after EDTA treatment or from cells containing Ntp1p with mutations in aspartic acid residues within the Ca 2+ -binding site revealed that Ntp1p eluted mainly in an inactive conformation instead of the dimeric or trimeric active form of the enzyme. These results suggest that activation of S. pombe Ntp1p under different conditions depends upon Ca 2+ binding through the Ca 2+ -binding motif as a prerequisite for correct enzyme oligomerization to its active form. Given the high degree of conservation of the Ca 2+ accommodation site, this might be a general mechanism regulating neutral trehalase activity in other yeasts and filamentous fungi.

Annexin A11 is one of the 12 vertebrate subfamilies in the annexin superfamily of calcium/phospholipid-binding proteins, distinguishable by long, non-homologous N-termini rich in proline, glycine and tyrosine residues. As there is negligible structural information concerning this annexin subfamily apart from primary sequence data, we have cloned, expressed and purified recombinant mouse annexin A11 to investigate its structural and functional properties. CD spectroscopy reveals two main secondary-structure contributions, α-helix and random coil (approx. 30% each), corresponding mainly to the annexin C-terminal tetrad and the N-terminus respectively. On calcium binding, an increase in α-helix and a decrease in random coil are detected. Fluorescence spectroscopy reveals that its only tryptophan residue, located at the N-terminus, is completely exposed to the solvent; calcium binding promotes a change in tertiary structure, which does not affect this tryptophan residue but involves the movement of approximately four tyrosine residues to a more hydrophobic environment. These calcium-induced structural changes produce a significant thermal stabilization, with an increase of approx. 14 °C in the melting temperature. Annexin A11 binds to acidic phospholipids and to phosphatidylethanolamine in the presence of calcium; weaker calcium-independent binding to phosphatidylserine, phosphatidic acid and phosphatidylethanolamine was also observed. The calcium-dependent binding to phosphatidylserine is accompanied by an increase in α-helix and a decrease in random-coil contents, with translocation of the tryptophan residue towards a more hydrophobic environment. This protein induces vesicle aggregation but requires non-physiological calcium concentrations in vitro . A three-dimensional model, consistent with these data, was generated to conceptualize annexin A11 structure–function relationships.

The effect of temperature and calcium ions on the denaturation of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature ( T d ) of 83 °C in the presence of excess calcium ions was observed, whereas T d decreased to 48 °C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy E A of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent E A can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.

Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro , cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met 49 . Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc—HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca 2+ , via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P 1 site, such as valine, alanine or phenylalanine. The K m and k cat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca 2+ ions. At 70°C, a 10-fold increase in Ca 2+ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca 2+ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca 2+ -binding site in Ak.1 protease. Other metal ions, such as Sr 2+ , increase the thermostability of the enzyme, but to a significantly lower degree than does Ca 2+ . The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca 2+ -binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca 2+ -binding EF-hands in each subunit, but their relative affinities for Ca 2+ are not known. In the present study mutations were made in both subunits to reduce the Ca 2+ -binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca 2+ did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca 2+ were otherwise identical to that of the Ca 2+ -bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca 2+ requirement ( K d ) was approx. 350 μ M. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K d , which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K d values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca 2+ and provides the major driving force for conformational change.