Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

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

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

High-affinity Ca2+-binding site inhibiting Ca2+ release from sarcoplasmic reticulum

Ca2+ release from sarcoplasmic reticulum membranes, activated by alkaline pH occurs only when EGTA is present in the release medium. Addition of very low concentrations of Ca2+ to the medium inhibits Ca2+ release. The concentration of free Ca2+ required for 50% inhibition ranges from between 5 and 20 nM in different experiments and/or membrane preparations, irrespective of whether the free Ca2+ concentration is controlled by EGTA or CDTA. Other divalent cations such as Mn2+, Ba2+, Cu2+, Cd2+ and Mg2+ also exert an inhibitory effect on Ca2+ release, with higher or lower potency than that of Ca2+. The inactivation of Ca2+ release by Ca2+ is reversible. We suggest the involvement of high-affinity Ca2+-binding sites in the control of Ca2+ release.     

Point amino acid substitutions in Ca2+-binding centers of recoverin. III. Mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca(2+)-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca(2+)-binding site. This site was reconstructed from the fourth potential Ca(2+)-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca(2+)-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild type recoverin.     

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

Cardiac myofibrilsisolated from trout heart have been demonstrated to have a highersensitivity for Ca²⁺ than mammalian cardiac myofibrils.Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts,we have previously demonstrated that this comparatively highCa²⁺ sensitivity is due, in part, to trout cTnC (ScTnC)having twice the Ca²⁺ affinity of mammalian cTnC (McTnC)over a broad range of temperatures. The amino acid sequence of ScTnC is92% identical to McTnC. To determine the residues responsible for thehigh Ca²⁺ affinity, the function of a number of ScTnC andMcTnC mutants was characterized by monitoring an intrinsic fluorescentreporter that monitors Ca²⁺ binding to site II (F27W). Theremoval of the COOH terminus (amino acids 90-161) from ScTnC andMcTnC maintained the difference in Ca²⁺ affinity betweenthe truncated cTnC isoforms (ScNTnC and McNTnC). The replacement ofGln²⁹ and Asp³⁰ in ScNTnC with thecorresponding residues from McNTnC, Leu and Gly, respectively, reducedCa²⁺ affinity to that of McNTnC. These results demonstratethat Gln²⁹ and Asp³⁰ in ScTnC are required forthe high Ca²⁺ affinity of site II.

     

Calelectrins are a ubiquitous family of Ca2+-binding proteins purified by Ca2+-dependent hydrophobic affinity chromatography by a mechanism distinct from that of calmodulin

The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.     

The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: III. A mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca⁺²-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca⁺²-binding site. This site was reconstructed from the fourth potential Ca⁺²-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca⁺²-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca²⁺ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin. For communication II, see [1].     

A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.     

Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX

Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bj?rk, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.     

The interaction of Ca2+ with human factor IX

The binding isotherms of Ca²⁺ and Sr²⁺ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca²⁺, a total of 16.0 ± 1.0 sites, of K_D 7.3 ± 0.2 × 10^?4m, are present on human Factor IX. Similar analysis of the Sr²⁺ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K_D of 1.9 ± 0.1 × 10^?3m. Both Sr²⁺ and Mn²⁺ effectively displace Ca²⁺ from human Factor IX; whereas Mg²⁺ is considerably less potent in this regard. Conversely, Ca²⁺ is capable of nearly complete displacement of Sr²⁺ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca²⁺ concentration. Sr²⁺ can substitute for Ca²⁺ in this activation process. Mn²⁺ cannot, in itself, substitute for Ca²⁺ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca²⁺. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca²⁺, Sr²⁺, and Mn²⁺ all allow activation to proceed equally well.     

Metal ion blockage of tritium incorporation into gamma-carboxyglutamic acid of prothrombin. Stoichiometry of gamma-carboxyglutamic acid to Gd3+ for the high affinity sites

Prothrombin possesses two high affinity and four low affinity gamma-carboxyglutamic acid (Gla)-dependent gadolinium binding sites. Earlier work (Price, P. A., Williamson, M. K., and Epstein, D. J. (1981) J. Biol. Chem. 256, 1172-1176) has shown that tritium can be specifically incorporated at the gamma-carbon of Gla in proteins at pH 5. In the present work we show that inclusion of saturating concentrations of Ca2+ in nondenaturing buffer systems ranging from pH 5.5 to 8.5 prevents the exchange of tritium into all 10 Gla residues of prothrombin. Similarly, saturating concentrations of Gd3+ prevent tritium incorporation into Gla at pH 5.5. Positive cooperativity was observed for the binding of Gd3+ to human prothrombin (at pH 5.5) for the two high affinity sites (Kd congruent to 35 nM). The four low affinity sites bind Gd3+ with a Kd congruent to 5 microM. Incubation of prothrombin ranging in concentrations from 10 to 40 microM with 2 eq of Gd3+ at pH 5.5 prevents 5.7 (average of seven determinations) Gla residues from tritium incorporation. Sedimentation velocity experiments conducted at pH 5.5 indicate that prothrombin in the presence of saturating concentrations of Gd3+ polymerizes, most likely, to a trimer. Further, in the presence of 2 eq of Gd3+, calculated percent weight average concentration of monomer prothrombin is congruent to 100% at 10 microM, approximately equal to 95% at 20 microM, and congruento to 80% at 40 microM protein concentration. Thus, it appears that under conditions in which prothrombin primarily exists as a monomer, occupancy of the initial two metal binding sites by Gd3+ involves six Gla residues.     

Bovine myeloperoxidase and lactoperoxidase each contain a high affinity site for calcium

Both bovine myeloperoxidase and lactoperoxidase contain one calcium per iron with no other metal present in significant amount. Calcium is bound with high affinity and is removed upon exposure to 6 M guanidine hydrochloride/EGTA which results in precipitation of protein. Computer amino acid sequence analyses of human myeloperoxidase reveal two plausible calcium binding sites. This is the first evidence for the presence of calcium in these peroxidases.     

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

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

Ca2+-dependent structural changes in bovine blood coagulation factor Va and its subunits

The calcium dependence of the structures of bovine blood coagulation factor Va and its subunits (Vh and Vl) has been examined spectroscopically in order to characterize the conformational changes which accompany the binding of Ca2+ to Vh and Vl to form factor Va. The far-UV CD spectra of the isolated subunits indicate that the secondary structures of both Vh and Vl are predominantly beta-sheet (greater than 45%), with little alpha-helix content (less than 15%). No change in the far-UV CD spectrum was observed when factor Va was formed by the addition of Ca2+ to an equimolar mixture of Vl and Vh. Hence, no detectable change in secondary structure occurs during the formation of factor Va. In contrast, the addition of Ca2+ to an equimolar mixture of Vh and Vl caused a small (2%) increase in the total intrinsic fluorescence intensity and a blue shift in the emission spectrum that resulted from a tertiary structural change and/or the association of nonpolar surfaces at the subunit interface. This fluorescence change correlated closely with the appearance of functional factor Va, since the rate of the spectral change was the same as the rate of recovery of cofactor activity, and since both were half-maximal near 50 microM Ca2+. This fluorescence change required both subunits, was reversed by the addition of EDTA, and was observed only with metal ions that can substitute for Ca2+ in reconstituting factor Va activity from Vh and Vl (Mn2+ and Tb3+; not Mg2+). When a sample containing ANS (8-anilino-1-naphthalenesulfonate) and an equimolar mixture of calcium-free Vh and Vl was titrated with Ca2+, the ANS emission intensity decreased by about 30%, most likely because the association of Vl and Vh caused nonpolar regions at the subunit-subunit interface to become inaccessible for ANS binding. The calcium dependence of this spectral change yielded a Kd of 51 +/- 2 microM, and the rate of the decrease in ANS fluorescence occurred at nearly the same rate as the recovery of factor Va activity. Thus, both intrinsic and extrinsic fluorescence data, as well as other data, indicate that the calcium binding site in factor Va has an apparent Kd of 50 microM under our conditions and that the calcium-mediated binding between Vl and Vh involves hydrophobic interactions between the subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro gamma-carboxylation of a 59-residue recombinant peptide including the propeptide and the gamma-carboxyglutamic acid domain of coagulation factor IX. Effect of mutations near the propeptide cleavage site

We report the expression in Escherichia coli of a fusion protein that contains the propeptide sequence and gamma-carboxyglutamic acid domain (residues -18 to 41) of human factor IX (FIXGla). CNBr was used to release FIXGla from the fusion protein. The 59-amino acid peptide is an efficient substrate for in vitro gamma-carboxylation. Its Km,app (0.55 microM) is several thousand-fold lower than that of the commonly used substrate FLEEL and about 5 times lower than proPT28 or proFIX28, (Hubbard, B. R., Jacobs, M., Ulrich, M. M. W., Walsh, C., Furie, B., and Furie, B. C. (1989) J. Biol. Chem. 264, 14145-14150). In addition, FIXGla is the first peptide substrate that is carboxylated in vitro to more than one gamma-carboxyglutamic acid/molecule (6-11 gamma-carboxyglutamic acids/molecule). We created peptides with mutations identical to FIXSan Dimas or FIXCambridge as well as a peptide with both mutations in the propeptide sequence and examined the effect of the mutations on in vitro carboxylation. Enzyme kinetic studies revealed no significant difference in Vmax/Km values between normal and mutant substrates. Maximum carbon dioxide incorporation was achieved with the double mutant. From these data we conclude the following. 1) FIXGla and its mutants are excellent substrates for studying the mechanism of gamma-carboxylase. 2) Although arginines at positions -4 and -1 are highly conserved in the propeptide sequence of all the vitamin K-dependent proteins, neither is critical for gamma-carboxylation.     

Ca 2+-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues

We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these “metallo-zipper” structures, Gla residues spaced at i, i + 4, i + 7, i + 11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca²⁺ and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH₂-(γLSγEAK)₃-CONH₂ (peptide 1) and NH₂-γLSγEAKγLSγQANγLSγKAE-CONH₂ (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca²⁺, but not Mg²⁺. Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i + 4, i + 7, i + 11, i + 14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.     

pH-Dependent structural changes at Ca(2+)-binding sites of coagulation factor IX-binding protein

Coagulation factor IX-binding protein, isolated from Trimeresurus flavoviridis (IX-bp), is a C-type lectin-like protein. It is an anticoagulant consisting of homologous subunits, A and B. Each subunit has a Ca(2+)-binding site with a unique affinity (K(d) values of 14muM and 130muM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca(2+) binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca(2+) release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca(2+)-binding sites with each other and with those of the solved structures under alkaline conditions; pH 7.8 and pH 8.0 (complexed form). At pH 6.5, Glu43 in the Ca(2+)-binding site of subunit A displayed two conformations. One (minor) is that in the alkaline state, and the other (major) is that at pH 4.6. However, the corresponding Gln43 residue of subunit B is in only a single conformation, which is almost identical with that in the alkaline state. At pH 4.6, Glu43 of subunit A adopts a conformation similar to that of the major conformer observed at pH 6.5, while Gln43 of subunit B assumes a new conformation, and both Ca(2+) positions are occupied by water molecules. These results showed that Glu43 of subunit A is much more sensitive to protonation than Gln43 of subunit B, and the conformational change of Glu43 occurs around pH6.5, which may correspond to the step of Ca(2+) release.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.