Low Affinity Calcium Binding Sites Research Articles

Versatility of subtilisin: A review on structure, characteristics, and applications.

Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.

Determination of the Calcium‐Dependent Activation Mechanism for Metacaspase 1 from Schizophyllum commune

Metacaspases are cysteine proteases implicated in initiating programmed cell death in plants and fungi, and have recently been shown to play a key role in the plant immune response. These proteins are distantly related to caspases, the enzymes that induce apoptosis in animals. Notably, metacaspases require calcium binding for activity, whereas caspases are instead activated by upstream signalling pathways that utilize calcium as a signal transducer. Metacaspase activity is greatest in the presence of millimolar levels of calcium. However, the exact mechanism by which calcium activates the metacaspases is still unclear. Our lab has performed an extensive characterization of metacaspase1 from the fungus Schizophyllum commune (ScMCA-Ia) to determine the likely conformational changes needed for metacaspase activation. The available metacaspase crystal structures from yeast and T. brucei demonstrate calcium-binding to a conserved site of four aspartic acid residues outside of the active site. While calcium itself binds to this allosteric site, the calcium-binding Asps are found on flexible loops and are only a few residues away from the active site. Sequence alignments demonstrate that these Asp residues are conserved in ScMCA-Ia and mutagenesis studies that exchange Asp for Ala reveal that this site is crucial for activity on small peptide substrates. We have used differential scanning fluorimetry to determine the calcium-binding constants of ScMC-Ia and associated mutants. Mutating calcium-binding Asp residues to Glu renders the enzymes inactive, however differential scanning fluorimetry indicates that the mutants still bind to calcium with Kd values similar to wildtype, in the µM range. This suggests that calcium-binding to this high-affinity site changes the position of calcium binding residues, altering the positions of the loops bearing these residues, and ultimately changing the orientation of the active site. Even slight changes to the calcium binding site (Asp to Glu) cannot be tolerated. In addition to the micromolar binding site, a second site that binds calcium in the mM range has been inferred from the low millimolar calcium levels needed for optimal metacaspase activity. Kinetics experiments suggest that the low-affinity calcium binding site is important for substrate binding. Together, these findings comprise a complete model for metacaspase activation in which high-affinity calcium binding reorients the active site for catalysis and low-affinity binding alters the substrate binding pocket to enhance substrate binding and catalysis. This mechanistic understanding of metacaspase activation and conformational changes is needed to design metacaspase-specific inhibitors to treat fungal diseases.

Type III metacaspases: calcium-dependent activity proposes new function for the p10 domain.

Metacaspases are a subgroup of caspase homologues represented in bacteria, algae and plants. Although type I and type II metacaspases are present in plants, recently discovered and uncharacterized type III metacaspases can only be found in algae which have undergone secondary endosymbiosis. We analysed the expression levels of all 13 caspase homologues in the cryptophyte Guillardia theta invivo and biochemically characterized its only type III metacaspase, GtMC2, invitro. Type III metacaspase GtMC2 was shown to be an endopeptidase with a preference for basic amino acids in the P1 position, which exhibited specific N-terminal proteolytic cleavage for full catalytic efficiency. Autolytic processing, as well as the activity of the mature enzyme, required the presence of calcium ions in low millimolar concentrations. In GtMC2, two calcium-binding sites were identified, one with a dissociation constant at low and the other at high micromolar concentrations. We show high functional relatedness of type III metacaspases to type I metacaspases. Moreover, our data suggest that the low-affinity calcium-binding site is located in the p10 domain, which contains a well-conserved N-terminal region. This region can only be found in type I/II/III metacaspases, but is absent in calcium-independent caspase homologues.

The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis.

Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum. Calreticulin is detectable on the surface of apoptotic cells under some apoptosis-inducing conditions, where it promotes the phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains: a globular domain, an extended arm-like P-domain, and a C-terminal acidic region containing multiple low-affinity calcium binding sites. We show that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared with other phospholipids and that this interaction is calcium dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher-affinity calcium-dependent mode that is acidic region dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower-affinity P-domain and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that cooperates with other PS-binding factors to promote the phagocytosis of apoptotic cells.

Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion

Human MMP-26 (matrix metalloproteinase-26) (also known as endometase or matrilysin-2) is a putative biomarker for human carcinomas of breast, prostate and other cancers of epithelial origin. Calcium modulates protein structure and function and may act as a molecular signal or switch in cells. The relationship between MMPs and calcium has barely been studied and is absent for MMP-26. We have investigated the calcium-binding sites and the role of calcium in MMP-26. MMP-26 has one high-affinity and one low-affinity calcium binding site. High-affinity calcium binding was restored at physiologically low calcium conditions with a calcium-dissociation constant of 63 nM without inducing secondary and tertiary structural changes. High-affinity calcium binding protects MMP-26 against thermal denaturation. Mutants of this site (D165A or E191A) lose enzymatic activity. Low-affinity calcium binding was restored at relatively high calcium concentrations and showed a K(d2) (low-affinity calcium-dissociation constant) value of 120 microM, which was accompanied with the recovery of enzymatic activity reversibly and tertiary structural changes, but without secondary structural rearrangements. Mutations at the low-affinity calcium-binding site (C3 site), K189E or D114A, induced enhanced affinity for the Ca2+ ion or an irreversible loss of enzymatic activity triggered by low-affinity calcium binding respectively. Mutation at non-calcium-binding site (V184D at C2 site) showed that C2 is not a true calcium-binding site. Observations from homology-modelled mutant structures correlated with these experimental results. A human breast cancer cell line, MDA-MB-231, transfected with wild-type MMP-26 cDNA showed a calcium-dependent invasive potential when compared with controls that were transfected with an inactive form of MMP-26 (E209A). Calcium-independent high invasiveness was observed in the K189E mutant MDA-MB-231 cell line.

A Flagellum-specific Calcium Sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.

Structural Consequences of Cysteine Substitutions C1977Y and C1977R in Calcium-binding Epidermal Growth Factor-like Domain 30 of Human Fibrillin-1

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.

Identification of a Functional Switch for Actin Severing by Cytoskeletal Proteins

Actin severing is vital for the organization of the actin cytoskeleton during cell motility. Severing of F-actin by the homologous proteins villin and gelsolin requires unphysiologically high calcium concentrations (20-200 microM). Here we demonstrate that high calcium releases an autoinhibited conformation in villin that is maintained by two low affinity calcium binding sites (aspartic acids 467 and 715) that interact with a cluster of basic residues in the S2 domain of villin. Mutation of either of these sites as well as tyrosine phosphorylation alters the conformation of villin resulting in a protein that can sever actin in nanomolar calcium. These results suggest that tyrosine phosphorylation rather than high calcium may be the mechanism by which villin and other related proteins sever actin in vivo.

Effect of His-Gly-Lys Motif Derived from Domain 5 of High Molecular Weight Kininogen on Suppression of Cancer Metastasis Both in Vitro and in Vivo

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. (2001) Biochem. Biophys. Res. Commun. 288, 975-980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg. Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.

Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states.

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 A crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as approximately 30 degrees C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.

Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca 2+ affinity of regulatory Ca 2+ sites in single channel recordings and [ 3H]ryanodine binding

The actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid ( p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20–100 μM) increased the mean open probability ( P o) of the calcium channel at subactivating (20 nM), maximally activating (20–100 μM) and inhibitory (1–4 mM) Ca 2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 μM 4-CMPS caused a transient increase in P o, followed by a low activity state within 1 min. At inhibitory Ca 2+ concentrations P o was increased to values observed with maximally activating Ca 2+ or lower, inhibitory Ca 2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [ 3H]Ryanodine binding was increased up to four-fold with 3–15 μM 4-CMPS/ p-CMB (Hill coefficient 1.7–2.0) at 4 μM Ca 2+ and reduced at high concentrations (50–200 μM). The increase in [ 3H]ryanodine binding by 10 μM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [ 3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca 2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 μM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.

The effects of pH on the generation of turbidity and elasticity associated with fibrinogen-fibrin conversion by thrombin are remarkably influenced by sialic acid in fibrinogen.

In fibrinogen-fibrin conversion by thrombin, the polymerization of a fibrin monomer is accompanied by gelation and an increase turbidity. Since sialic acids at the terminal of the carbohydrate chains bound to fibrinogen are part of the low affinity calcium binding site necessary for polymerization, they are closely involved in the network structure of fibrin clots. Fibrin clots derived from asialofibrinogen exhibited definite differences in turbidity and elasticity compared with those derived from intact fibrinogen, and were markedly dependent on the pH during the reaction. The turbidity during polymerization of fibrin, evaluated according to the absorbance at 350 nm, was maximum at pH 6.5-7.0, but it decreased in the other pH ranges, with the changes being unremarkable at higher pH levels but remarkable at lower pH ranges. The turbidity of fibrin derived from asialofibrinogen was far higher than that from intact fibrinogen near neutrality, but decreased rapidly and was lower than in intact fibrinogen at higher and lower pH ranges. Concerning the elasticity evaluated by thromboelastography, the coagulation time (k) and the maximum amplitude (ma) were lower in asialofibrinogen, indicating a deterioration of the clotting function of fibrinogen with the loss of sialic acid. These results suggest that sialic acid bound to fibrinogen is closely related to the fibrin network formation in blood coagulation, which is the most important function of fibrinogen, and plays a functional role in the stabilization of fibrin clot formation against environmental changes, including pH.

Molecular model of the extracellular lectin-like domain in CD69.

CD69 is described as a T cell activation antigen, but the ligand and physiological function of CD69 are currently unknown. The sequence of the extracellular domain of CD69 shows some similarity with that of calcium-dependent (C-type) lectins. Using comparative computer modeling and inverse folding calculations, we have generated and analyzed a detailed three-dimensional model of the extracellular domain of CD69 based on the crystal structure of the mannose binding protein. The sequence of CD69 appears to be highly compatible with the C-type lectin fold, and assessment of the model using inverse folding calculations suggests its overall correctness. Compared with mannose binding protein and the selectins, CD69 displays significant deletions in loop regions. In addition, residues that form conserved calcium binding sites found in the C-type lectin family are not conserved in CD69. This suggests the presence of structural features in CD69 that depart from some of the conserved motifs seen in two crystal structures of C-type lectins. The CD69 model shows cavity-shaped hydrophobic regions surrounded by charged residues. One of these cavities is proximal to a potential low affinity calcium binding site and may be implicated in specific interactions with ligands.

Smooth muscle calponin-caltropin interaction: effect on biological activity and stability of calponin.

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer: 1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD).(ABSTRACT TRUNCATED AT 250 WORDS)

Coexistence of high- and low-affinity calcium binding sites of the sarcoplasmic reticulum calcium pump

We have recently shown [Mészáros, L. G., & Bak, J. (1992) Biochemistry 31, 1195-1200] that, during the rapid phase of Ca2+ uptake into sarcoplasmic reticulum (SR), internalization and binding of Ca2+ to the cytoplasmic high-affinity binding sites of the Ca2+ ATPase occur simultaneously, resulting in a transient supernumerary Ca/ATP stoichiometry. Here we address the question of whether the cytoplasmic high-affinity and the luminal low-affinity Ca2+ binding sites of the SR Ca2+ ATPase also coexist. SR vesicles were loaded with Ca2+ (0-10 mM), and then the kinetics of EP formation and decomposition as well as the maximum level of EP formed from radiolabeled ATP were determined at conditions which only allow single-cycle reactions to occur: empty or Ca-loaded SR vesicles (in micromolar extravesicular Ca2+) were either mixed with ATP plus millimolar EGTA or added in amounts that set a Ca2+ ATPase/ATP ratio of 80-85 at the initiation of the reaction. The rates of EP formation and decomposition were both significantly reduced in Ca-loaded, compared to empty (ionomycin-treated), vesicles. However, the value of EPmax was unaltered by Ca-loading, suggesting the existence of the enzyme intermediate, E.Ca2(cyt).Ca2(lum), i.e., the coexistence of the cytoplasmic and the luminal Ca2+ binding sites of the Ca-pump. These results suggest that the uphill transport of Ca2+ might not be based on an alternating relocation and conversion of the Ca2+ binding sites of the Ca2+ ATPase.

Sialic acid in fibrinogen: effects of sialic acid on fibrinogen-fibrin conversion by thrombin and properties of asialofibrin clot.

The final stage in a series of blood coagulating reactions is fibrinogen-fibrin conversion by thrombin. This reaction consists of fibrinopeptide A and fibrinopeptide B release, polymerization of fibrin monomer, and stabilized fibrin formation by factor XIII. The latter two reactions require calcium. In the present study there was no difference in the rate of thrombin-induced fibrinopeptide release between fibrinogen and asialofibrinogen where sialic acid in the terminal end of carbohydrate moiety of fibrinogen was removed by neuraminidase, but turbidity associated with asialofibrin clot formation was increased more rapidly. In asialo-derivatives, the dissolution time of the clots in high concentrated urea solution tended to be shortened and rigidity as a gel tended to be decreased. In measurement by thromboelastography there was no difference in the reaction time (r) between fibrinogen and asialofibrinogen, but the maximum amplitude (ma) was obviously decreased in asialofibrinogen. Furthermore, when the rate of cross-link formation between gamma chains by F-XIII was compared, the production of gamma-dimer in the same reaction time was found to be lower and formation of stabilized fibrin tended to be retarded in asialofibrinogen. Sialic acid in fibrinogen thus may clearly influence the polymerization of fibrin-monomer and the formation of cross-linked fibrin in a series of reactions for fibrinogen-fibrin conversion. This may be consistent with the theory that fibrinogen sialic acid residues are low affinity calcium-binding sites and influence fibrin assembly.

Microdomains of high calcium concentration in a presynaptic terminal.

Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion, supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation of a low-affinity calcium-binding site at the active zone.

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.

Volume changes in high‐affinity calcium binding of the sarcoplasmic reticulum calcium‐transport enzyme

The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium-transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non-saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium binding. A reaction scheme in which two unidirectional substrate-driven reactions transfer high-affinity into low-affinity calcium-binding sites was applied to determine binding-volume increments. It has been inferred from the pressure dependence of the volume-generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding-volume increments of opposite signs (+84 to +207 ml/mol and -3 to -136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 +/- 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding-volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D.W. & Dorrier, T.E. (1986) Biochem. Biophys. Res. Commun. 38, 199-204].

Probing the relationship between .alpha.-helix formation and calcium affinity in troponin C: proton NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides

Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.