Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin

Flow microcalorimetric titrations of calmodulin with melittin at 25 degrees C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca2+ (Comte, M., Maulet, Y., and Cox, J. A. (1983) Biochem, J. 209, 269-272) is entirely entropy driven (delta H0 = 30.3 kJ X mol-1; delta S0 = 275 J X K-1 X mol-1). Neither the proton nor the Mg2+ concentrations have any significant effect on the strength of the complex. In the absence of Ca2+, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg2+. Enthalpy titrations of metal-free calmodulin with Ca2+ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: protons, absorbed by the nonspecific calmodulin-melittin complex, are released upon binding of the first Ca2+; Ca2+ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant greater than or equal to 10(7) M-1, i.e. 2 orders of magnitude higher than that of free calmodulin; the latter is even more entropy driven (delta H0 = 7.2 kJ X site-1; delta S0 = 158 J X K-1 X site-1) than binding to free calmodulin (delta H0 = 4.7 kJ X site-1; delta S0 = 112 J X K-1 X site-1), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.     

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.     

The interaction of calmodulin with melittin

Studies utilizing the interaction of melittin with the 1-106 fragment of calmodulin, the protection of calmodulin from tryptic digestion by melittin, and the interaction of the carbocyanine dye Stains-all with the calmodulin-melittin complex have indicated that complex formation of calmodulin with melittin involves the alpha-helical connecting bridge joining the N- and C-terminal lobes of calmodulin.     

High-affinity formation of a 2:1 complex between gramicidin S and calmodulin.

Two molecules of gramicidin S, a very rigid cyclic decapeptide rich in beta-sheet structure, can bind in a Ca2+-dependent way to a calmodulin molecule in the presence as well as in the absence of 4 M-urea. The flow-microcalorimetric titration of 25 microM-calmodulin with gramicidin S at 25 degrees C is endothermic for 21.3 kJ.mol-1; the enthalpy change is strictly linear up to a ratio of 2, indicating that the affinity constant for binding of the second gramicidin S is at least 10(7) M-1. In 4 M-urea the peptide quantitatively displaces seminalplasmin from calmodulin, as monitored by tryptophan fluorescence. An iterative data treatment of these competition experiments revealed strong positive co-operativity with K1 less than 5 X 10(5) M-1 and K1.K2 = 2.8 X 10(12) M-2. A competition assay with the use of immobilized melittin enabled us to monitor separately the binding of the second gramicidin S molecule: the K2 value is 1.9 X 10(7) M-1. By complementarity, the K1 value is 1.5 X 10(5) M-1. In the absence of urea the seminalplasmin displacement is incomplete: the data analysis shows optimal fitting with K1 less than 2 X 10(4) M-1 and K1.K2 = 3.2 X 10(11) M-2 and reveals that the mixed complex (calmodulin-seminalplasmin-gramicidin S) is quite stable and is even not fully displaced from calmodulin at high concentrations of gramicidin S. The activation of bovine brain phosphodiesterase by calmodulin is not impaired up to 0.2 microM-gramicidin S. According to our model the ternary complex enzyme-calmodulin-gramicidin is relatively important and displays the same activity as the binary complex enzyme-calmodulin. Gramicidin S also displaces melittin from calmodulin synergistically, as monitored by c.d. Our studies with gramicidin S reveal the importance of multipoint attachments in interactions involving calmodulin and confirm the heterotropic co-operativity in the binding of calmodulin antagonists first demonstrated by Johnson [(1983) Biochem. Biophys. Res. Commun. 112, 787-793].     

Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.     

Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme

A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the L-citrulline formation of nNOS from L-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.     

Comparison of S100b protein with calmodulin: interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein kinase C

To gauge similarities between S100b protein and calmodulin, interactions were observed between S100b and melittin and between S100b and tau, the microtubule-associated proteins. The interaction of melittin with S100b protein in the presence and absence of calcium was studied by fluorescence polarization, UV difference spectroscopy, and sulfhydryl derivatization. Whether calcium was present or not in the solution, melittin and S100b form a complex of molar ratios up to 2:1. Further binding of melittin occurred, but it resulted in precipitation of S100b, as is true of the corresponding case of melittin binding to calmodulin. In the absence of calcium, the interaction of melittin and S100b shielded the tryptophan (Trp) of the former protein and exposed cysteine-84 beta (Cys-84 beta) of the latter protein, leaving the tyrosine-16 beta (Tyr-16 beta) of S100b unaffected. Calcium addition to the complex partially restored the exposure of Trp of melittin and caused changes in the environment of Tyr-16 beta (unlike the environmental changes induced for Tyr-16 beta by calcium in the absence of melittin). The conformational changes induced in S100b by interaction with melittin increased its affinity for calcium and offset the inhibition of calcium binding otherwise observed in the presence of potassium ions. This corroborated the previous finding that S100b affinity for calcium greatly depends on the protein conformation. The phenomena described above are similar to the interactions of melittin with calmodulin and thus suggest that S100b and calmodulin have a common structural domain not only that binds melittin but also that may interact with common target proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide binding by calmodulin and its proteolytic fragments and by troponin C

Calmodulin and troponin C exhibit calcium-dependent binding of 1 mol/mol of dynorphin. The dissociation constants of the complexes, determined in 0.20 N KC1-1.0 mM CaCI2, pH 7.3, are 0.6 microM for calmodulin, 2.4 microM for rabbit fast skeletal muscle troponin C, and 9 microM for bovine heart troponin C. Experiments with deletion peptides of dynorphin show that peptide chain length and especially charge affect the binding of the peptides by calmodulin. Dynorphin, but not mastoparan or melittin, inhibits adenosinetriphosphatase activity in a reconstituted rabbit skeletal muscle actomyosin assay. The inhibition is partially reversed by the addition of calmodulin or troponin C in the presence of calcium. Calmodulin also exhibits calcium-dependent binding of a synthetic peptide corresponding to positions 104-115 of rabbit fast skeletal muscle troponin I. Mastoparan is a tetradecapeptide from the vespid wasp having exceptional affinity for calmodulin, with Kd approximately 0.3 nM [Malencik, D.A., & Anderson, S.R. (1983) Biochem. Biophys. Res. Commun. 114, 50]. The addition of 1 mol/mol of mastoparan to the complex of calmodulin with dynorphin results in complete dissociation of dynorphin. Similar titrations of the skeletal muscle troponin C-dynorphin complex produce a gradual dissociation consistent with a dissociation constant of 0.2 microM for the troponin C-mastoparan complex. Fluorescence anisotropy measurements using the intrinsic tryptophan fluorescence of mastoparan X show strongly calcium-dependent binding by proteolytic fragments of calmodulin. binding by proteolytic fragments of calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of calmodulin with amphiphilic peptides

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.     

Association of melittin with the isolated myosin light chains

Melittin is a 26-residue peptide which undergoes high-affinity calcium-dependent binding by calmodulin [Barnette, M.S., Daly, R., & Weiss, B. (1983) Biochem. Pharmacol. 32, 2929; Comte, M., Maulet, Y., & Cox, J.A. (1983) Biochem. J. 209, 269; Anderson, S.R., & Malencik, D.A. (1986) Calcium Cell Funct. 6, 1]. The results in this paper show that three different types of myosin light chain--the smooth muscle regulatory light chain, the smooth muscle essential light chain, and the skeletal muscle regulatory 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chain--also associate with melittin. The resulting complexes have dissociation constants ranging from 1.1 to 2.5 microM in the presence of 0.10 M NaCl and from approximately 50 to approximately 130 nM in solutions of 20 mM 3-(N-morpholino)propanesulfonic acid alone. The regulatory smooth muscle myosin light chain exhibits two equivalent melittin binding sites while each of the others displays only one. The myosin light chains evidently contain elements of structure related to the macromolecular interaction sites present in calmodulin and troponin C but not in parvalbumin. The association of melittin and other peptides with the light chains requires consideration whenever assays of the calmodulin-dependent activity of myosin light chain kinase are used to determine peptide binding by calmodulin. The binding measurements performed on the DTNB light chain and melittin necessitated derivation of the equation relating complex formation to the observed fluorescence anisotropy of a solution containing three fluorescent components. This analysis is generally applicable to equilibria involving the association of two fluorescent molecules emitting in the same wavelength range.     

Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 A. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 A, or with POPC vesicles, 4.9 A. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 A, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp-19 to dansyl distance was near 20 A. This value is somewhat smaller than expected for the free alpha-helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.     

Crystallization of a complex between ribonuclease T1 and 2'-guanylic acid

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.     

Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium.

The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.     

蜂毒肽片段的合成及其与钙调蛋白的相互作用

采用固相法设计合成了４个蜂毒肽片段：Ｍｅｌ１２、Ｍｅ１１３、Ｍｅｌ１４、Ｍｅｌ１５。应用电泳技术,抑制钙依赖性的磷酸二酯酶酶活方法和荧光技术研究了这些多肽与钙调蛋白的相互作用。结果表明这些多肽与钙调蛋白均形成１：１复合物,抑制钙依赖性的磷酸二酯酶的活性,其中Ｍｅｌ１４和Ｍｅｌ１５对钙调蛋白的结合活性与完整的蜂毒肽比较接近。     

Energetics of target peptide binding by calmodulin reveals different modes of binding

Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.     

Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

Effect of melittin on prostaglandin production by guinea-pig uterus.

Melittin, an activator of phospholipase (PL) A-2, increased the outputs of prostaglandin (PG) F-2 alpha and 6-keto-PGF-1 alpha, but not of PGE-2, from Day-7 guinea-pig uterus superfused in vitro. Reducing the extracellular calcium concentration (by omitting calcium chloride from the superfusing fluid) partially inhibited the stimulatory effect of melittin on uterine PG production. TMB-8 (an intracellular calcium antagonist) completely prevented the stimulation of PGF-2 alpha and 6-keto-PGF-1 alpha output by melittin, although the production of both PGs tended to increase after stopping the melittin and TMB-8 treatments. TMB-8 also inhibited the increases in outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 and prevented contraction of the uterus induced by exogenous PLA-2. Trifluoperazine (a calmodulin antagonist) had no inhibitory effect on the increases in outputs of PGF-2 alpha and 6-keto-PGF-1 alpha produced by melittin; it potentiated the stimulatory effect of melittin on 6-keto-PGF-1 alpha output and allowed melittin to increase PGE-2 output. When melittin was applied twice to the superfused uterus with an interval of 1 h between each treatment, partial refractoriness of the responses to melittin was seen: the magnitudes of the increases in PGF-2 alpha and 6-keto-PGF-1 alpha outputs were 40-50% less after the second treatment than after the first treatment. These results show that melittin stimulates the synthesis of PGF-2 alpha and PGI-2 (measured as 6-keto-PGF-1 alpha) in guinea-pig uterus by mechanisms which are calcium dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of melittin with troponin C

Melittin has been found to interact with troponin C with high affinity in the presence of Ca²⁺. The association constant approaches in magnitude that for melittin and calmodulin. The interaction results in a shift to lower wavelengths of the emission band of Trp-19 of melittin and in an increased shielding of Trp-19 from quenching. A major increase occurs in the α-helical content of combined melittin. Formation of the complex inhibits tryptic hydrolysis of the connecting strand. The properties of fluorescent labels attached to Met-25 and to AEDANS-98 are altered as a result of the interaction. It is concluded that the combined melittin makes extensive contact with the connecting strand and adjacent portions of the N- and C-terminal lobes.     

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: divalent cation interactions monitored by direct binding and microcalorimetry

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.     

Isolation and characterization of a new Mr 18,000 protein with calcium vector properties in amphioxus muscle and identification of its endogenous target protein

A new Ca2+-binding protein, called CaVP, has been detected in muscle of the cephalochordate amphioxus and purified to electrophoretic homogeneity. The Mr 18,000 protein (pI = 4.9) binds 2 Ca2+ atoms in a noncooperative way with an intrinsic binding constant of 8.2 X 10(6) M-1. Ca2+, but not Mg2+, induces a 10% increase in alpha-helical content in the metal-free protein. CaVP does not interact with chlorpromazine, but forms a Ca2+-dependent complex with melittin. In situ, CaVP forms a high affinity Ca2+-dependent complex with an Mr 36,000 protein present in muscle extracts of amphioxus. This complex has been purified by gel filtration and ion exchange chromatography, and the target protein further purified after dissociation of the complex in the presence of Ca2+-chelating agents and 6 M urea. The nearly pure Mr 36,000 protein also forms a Ca2+-dependent complex with calmodulin which, however, is less stable during electrophoresis than the CaVP-Mr 36,000 protein complex. Amphioxus CaVP does not substitute for calmodulin in a specific enzyme assay nor for troponin C in restoring Ca2+ sensitivity to skinned muscle fibers. Its polyclonal antibody does not cross-react with the latter two activators. No immunological cross-reacting counterpart of CaVP was found in organs of fish and rat. Its relative abundance in amphioxus muscle indicates that CaVP must underlie an important new limb of Ca2+ regulation in this particular muscle.