Ca2+ regulation of interactions between endoplasmic reticulum chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.     

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.     

Expression and purification of recombinant and native calreticulin.

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.     

Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.     

Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Ca2+ binding and 1H NMR

Ca2+ binding to calmodulin was measured in the presence of mastoparan or caldesmon fragment. Mastoparan and caldesmon fragment were used as model compounds of enzymes and cytoskeleton proteins, respectively, working as the target of calmodulin. Although the Ca2+ bindings of the two globular domains of calmodulin occur independently in the absence of the target peptide (or proteins), mastoparan and caldesmon fragment increased the affinity of Ca2+ and, at the same time, produced the positive cooperative Ca2+ bindings between the two domains. The result of Ca2+ binding was compared with 1H NMR spectra of calmodulin in the presence of equimolar concentration of mastoparan. It is known that a conformation change of the C-terminal half-region (C-domain) occurs by the Ca2+ binding to C-domain. A further change in conformation of C-domain was demonstrated by the Ca2+ binding to the N-terminal half-region (N-domain) in the presence of mastoparan. It indicates that the two domains of calmodulin get into communication with each other in the associated state with the target, and we concluded that the Ca2+ binding to the N-domain is responsive to the development of calmodulin function.     

Characterization of Haemonchus contortus calreticulin suggests its role in feeding and immune evasion by the parasite

Haemonchus contortus, a gastrointestinal parasite of sheep and goat feeds on the blood of its host and causes bleeding at the biting site. In this report, we demonstrate that the Ca2+ binding protein, calreticulin (CalR), is present in excretory/secretory products of adult worms. The secreted CalR enhanced plasma coagulation time. Using recombinant fragments, this property has been mapped to C-terminal part of the molecule which has binding sites for Ca2+ as well as clotting factors. Complement protein C1q bound to immobilized CalR and C1q dependent lysis of sensitized sheep erythrocytes was inhibited by CalR, a function mapped to N-domain of the protein. Factor X and a 24 kDa polypeptide derived from prothrombin but not prothrombin bound to immobilized CalR. The binding site for 24 kDa polypeptide in the CalR molecule has been localized in the P-domain. Our results suggest at least two functions for secreted CalR: first, to prevent blood clotting by binding to Ca2+ and clotting factors thus enabling parasite to feed on the host blood and second to modulate the host immune response by binding to complement C1q thereby facilitating parasite's survival within the host.     

Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for "calciosomes"

In the present study, we have shown that calreticulin is a major Ca(2+)-sequestering protein in pancreatic microsomes. This protein is a peripheral membrane protein and could be extracted from the microsomal membrane with carbonate buffer at pH 11.4. Calreticulin was identified in the membrane fractions by immunoblotting with a specific antibody, by a 45Ca2+ overlay technique, and by NH2-terminal amino acid analysis of the purified protein. Immunocytochemical localization of calreticulin in pancreatic acinar cells and pancreatic fibroblasts showed that the protein is localized to the ER membranes in these cells. We were unable to detect calsequestrin or any calsequestrin-like proteins in the pancreas and found no evidence for the existence of large numbers of specialized, calreticulin-containing vesicles which could be an equivalent of the calsequestrin-containing calciosomes previously reported in this tissue. Purified pancreatic calreticulin binds Ca2+ with both a low and a high capacity (approximately 1 mol of Ca2+/mol of protein and approximately 20-23 mol of Ca2+/mol of protein). The concentrations of Ca2+ required for half-maximal saturation of the low and high capacity sites were approximately 4-6 microM and approximately 1.5 mM, respectively. We conclude that calreticulin, which is confined to the lumen of the ER, plays a major role in Ca2+ storage in pancreatic cells.     

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.     

The solution structure of apocalmodulin from Saccharomyces cerevisiae implies a mechanism for its unique Ca2+ binding property

We have determined the solution structure of calmodulin (CaM) from yeast (Saccharomyces cerevisiae) (yCaM) in the apo state by using NMR spectroscopy. yCaM is 60% identical in its amino acid sequence with other CaMs, and exhibits its unique biological features. yCaM consists of two similar globular domains (N- and C-domain) containing three Ca(2+)-binding motifs, EF-hands, in accordance with the observed 3 mol of Ca(2+) binding. In the solution structure of yCaM, the conformation of the N-domain conforms well to the one of the expressed N-terminal half-domains of yCaM [Ishida, H., et al. (2000) Biochemistry 39, 13660-13668]. The conformation of the C-domain basically consists of a pair of helix-loop-helix motifs, though a segment corresponding to the forth Ca(2+)-binding site of CaM deviates in its primary structure from a typical EF-hand motif and loses the ability to bind Ca(2+). Thus, the resulting conformation of each domain is essentially identical to the corresponding domain of CaM in the apo state. A flexible linker connects the two domains as observed for CaM. Any evidence for the previously reported interdomain interaction in yCaM was not observed in the solution structure of the apo state. Hence, the interdomain interaction possibly occurs in the course of Ca(2+) binding and generates a cooperative Ca(2+) binding among all three sites. Preliminary studies on a mutant protein of yCaM, E104Q, revealed that the Ca(2+)-bound N-domain interacts with the apo C-domain and induces a large conformational change in the C-domain.     

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.     

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.     

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.     

The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.     

Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.     

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.     

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+.

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 A from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15N-1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H-15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.     

The role of gamma-carboxyglutamyl residues in the positive cooperative binding of Ca2+ to blood coagulation factor X

1. The calcium binding properties of factor X and its analogous decarboxyprotein have been compared with the aid of flow rate dialysis and ultraviolet difference spectroscopy. 2. Factor X binds approx. 20 mol of calcium per mol of protein. The first four sites exhibit positive cooperativity. 3. Changes in the ultraviolet difference spectrum when Ca2+ is bound suggest a conformational change. 4. In decarboxyfactor X low affinity of Ca2+ and no ligand-induced conformational change was observed. It is concluded that the presence of gamma-carboxyglutamate residues is a prerequisite for positive cooperative Ca2+ binding.     

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.