Calcium release from intact calmodulin and calmodulin fragment 78-148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum

Calcium release from high and low-affinity calcium-binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78-148, purified by high-pressure liquid chromatography, containing only the high-affinity calcium-binding sites, was determined by fluorescence stopped-flow with 2-p-toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1-77 and 78-148 each contain a calcium-dependent TNS-binding site, as shown by the calcium-dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25 degrees C and 10 degrees C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kobsslow 10.6 s-1 and 3.0 s-1 at 25 degrees C and 10 degrees C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-1 and 3.5 s-1 at 25 degrees C and 10 degrees C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites on the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (kobsfast 280 s-1 at 10 degrees C) but not with fragment 78-148, is most probably due to the conformational change associated with calcium release from low-affinity sites on the N-terminal half. The calmodulin fragments 1-77 and 78-148 neither activated calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin-dependent activation at a concentration approximately 1000-fold greater (5 microM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.     

Cooperativity among calmodulin's drug binding sites

The binding of felodipine, a dihydropyridine Ca2+ antagonist, to calmodulin has been studied by equilibrium dialysis and fluorescence techniques. Analysis using the Hill equation gives a Hill coefficient of 2. A plot of bound [felodipine] vs. free [felodipine]2 gives a Bmax of 1.9 mol/mol and a K0.5 of 22 microM. Two calmodulin antagonists, prenylamine and R24571, which have previously been shown to potentiate the fluorescent enhancement observed when felodipine binds to calmodulin [Johnson, J. D. (1983) Biochem. Biophys. Res. Commun. 112, 787], produce a reduction in Hill coefficient to 0.7 and 1.0, respectively, and account for the observed potentiation of felodipine binding. Titrations of felodipine with calmodulin in the absence and presence of prenylamine and R24571 suggest that these drugs decrease the K0.5 of calmodulin for felodipine by 25-fold. Thus, potentiating drugs (prenylamine and R24571) bind to either of the two felodipine binding sites and, through an allosteric mechanism, result in felodipine binding to the remaining site with greatly enhanced affinity. Two types of potentiating drugs are observed. Prenylamine exhibits a Hill coefficient of 0.8 whereas felodipine, R24571, and diltiazem exhibit Hill coefficients of 2 in their potentiation of felodipine binding. Titrations of felodipine and calmodulin with Ca2+ exhibit cooperativity with a Hill coefficient of 4. Half-maximal binding occurs near pCa 6.0. In the presence of R24571, the calcium dependence of felodipine binding is biphasic, now exhibiting a much higher affinity (pCa 7.6) component. A model is presented to explain the relationship of these various allosterically regulated conformers of calmodulin and their interactions and activation with its target proteins.     

Static and kinetic studies of complex formations between calmodulin and mastoparanX

Ca(2+)-induced complex formation between calmodulin (CaM) and mastoparanX (MasX) was studied by a fluorescence spectroscopy and by a stopped-flow method. The measurements of the fluorescence anisotropy in the presence of calcium and the fluorescence titration with Ca(2+) revealed that the N- and C-domains of CaM bound cooperatively MasX, while the tryptic fragments of CaM (TR(1)C, 1-77 and TR(2)C, 78-148) bound independently MasX. The Trp-fluorescence stopped-flow experiments revealed that the Ca(2+)-induced binding of CaM and MasX was composed of two processes: one was a rapid binding of the N-domain of CaM to MasX, which was induced by the rapid Ca(2+) binding to the N-sites of CaM. The other was a slow biphasic process. Its fast phase was the binding of the C-domain of CaM to MasX, which was induced by the slow Ca(2+) binding to the C-sites. Interestingly, the kinetics of the slow process varied with the Ca(2+) concentrations. At the low Ca(2+) concentrations, its rate constant increased to around 20 s(-1) as the Ca(2+) concentration increased. At the high Ca(2+) concentrations, the Ca(2+)-induced binding of the C-domain of CaM to MasX proceeded at a constant rate around 20 s(-1). This suggested an existence of a rate-limiting step for the Ca(2+)-induced binding of the C-domain of CaM to MasX at the high Ca(2+) concentrations. The slow phase of the slow process may be a rearrangement of the CaM-MasX complex. These results led to our model of a molecular kinetic mechanism of the Ca(2+)-induced complex formation between CaM and MasX.     

Binding of amphiphilic peptides to a carboxy-terminal tryptic fragment of calmodulin

Calmodulin (CaM) fragments 1-77 (CaM 1-77) and 78-148 (CaM 78-148) were prepared by tryptic cleavage of CaM. CaM 78-148 exhibited Ca2+-dependent binding to mastoparan X, Polistes mastoparan, and melittin with apparent dissociation constants less than 0.2 microM as judged from changes in the fluorescence spectrum and anisotropy of the single tryptophan residue of each of these cationic, amphiphilic peptides. This interaction was accompanied by a large spectral blue shift of the peptide fluorescence spectrum. These findings are consistent with earlier results [Malencik, D.A., & Anderson, S.R. (1984) Biochemistry 23, 2420-2428] on the binding of mastoparan X to CaM fragment 72-148. The binding of the peptide to CaM 78-148 also caused a significant loss of the accessibility of the peptide tryptophan to the fluorescence quencher acrylamide. The CaM 78-148 induced effects on the fluorescence spectra and tryptophan accessibility of the peptides were most pronounced for mastoparan X, a peptide with tryptophan on the apolar face of the putative amphiphilic helix. The data were comparable with results from parallel experiments on the Ca2+-dependent interaction of these peptides with intact CaM. Difference circular dichroic spectra suggested that binding to CaM 78-148 was associated with the induction of considerable degrees of helicity in the amphiphilic peptides, which by themselves have predominantly random coil structures in aqueous solution. This finding is also reminiscent of the interaction of these peptides with intact CaM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective affinity chromatography with calmodulin fragments coupled to sepharose

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.     

Limited digestion of calmodulin with trypsin in the presence or absence of various metal ions

The limited proteolysis of bovine brain calmodulin with trypsin in the presence or absence of various metal ions was reinvestigated in detail by HPLC. With metal ion-free calmodulin, limited proteolysis occurred at Arg 37 and Arg 106 with a cleavage ratio of 1 to 5, resulting in fragments consisting of residues 1-37, 38-148, 1-106 and 107-148. Fragments 1-37 and 107-148 accumulated under metal ion-free conditions. In the presence of calcium ions, the susceptibility of these sites to trypsin decreased and limited proteolysis occurred at Lys 77 as already reported by other workers. Fragment 78-148 accumulated, whereas fragment 1-77 was unstable under calcium-bound conditions, giving smaller peptides. Upon binding of manganese ions, calmodulin underwent a change of susceptibility to trypsin, resulting in cleavage at Lys 77, as observed for calcium-bound calmodulin. In the presence of zinc or magnesium ions, calmodulin was cleaved at the same sites as metal ion-free calmodulin under conditions where calmodulin would be expected to bind the respective ions.     

A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.     

Agonist and antagonist properties of calmodulin fragments

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.     

Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase.     

Is the binding of magnesium (II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance

Previous reports on the interaction between calmodulin (CaM) and Mg2+ range from no binding to a binding constant of 10(4) M-1 [for a summary, see Cox, J. A., Comte, M., Malnoe, A., Berger, D., & Stein, E. A. (1984) Met. Ions Biol. Syst. 17, 215-273]. In order to resolve the controversy, we used 25Mg NMR to study the binding of Mg2+ to apo-CaM, CaM.Ca2(2)+ (in which sites III and IV are occupied by Ca2+), CaM.La2(3)+ (in which sites I and II are occupied by La3+), and the two tryptic fragments of calmodulin, TR1C (containing sites I and II of CaM) and TR2C (containing sites III and IV of CaM). In each system, a "titration set" and a "temperature set" were obtained, and the spectral data were analyzed by total band-shape analysis to calculate the association constant (Ka) and off-rate (koff). As in the case of Ca2+ binding, sites I and II and sites III and IV were treated as two sets of equivalent sites, and a Ca2+/Mg2+ competition experiment suggested that Mg2+ competes with Ca2+ for the same sites. For both CaM.Ca2(2)+ and TR1C, moderately large Ka (2000 and 3500 M-1, respectively) and moderate off-rates (koff = 2300 and 3000 s-1, respectively, at 25 degrees C) were observed. For both CaM.La2(3)+ and TR2C, binding of Mg2+ was weaker by a factor of ca. 10 (Ka = 300 and 200 M-1, respectively) while the off-rates were also moderate (koff = 3500 and 2200 s-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenothiazine-binding and attachment sites of CAPP1-calmodulin

In the presence of Ca2+ norchlorpromazine isothiocyanate forms a monocovalent complex with calmodulin: CAPP1-calmodulin (Newton et al, 1983). Trypsin digestion of [3H]CAPP1-calmodulin yields as the major radioactive peptide N epsilon-CAPP-Lys-Met-Lys, corresponding to residues 75-77 of calmodulin. Stoichiometric amounts of all other expected tryptic peptides are also found, indicating that norchlorpromazine isothiocyanate selectively acylates Lys 75. A second molecule of CAPP-NCS can react, albeit slowly, with calmodulin to form CAPP2-calmodulin. Fragments 38-74 and 127-148 are completely missing from the trypsin digests of CAPP2-calmodulin without deliberate exposure to UV irradiation. Possibly the lengthy preparation of CAPP2-calmodulin favors photolysis, caused by room lights, of the putative CAPP-binding domains located in these two peptides. Lys 148, the sole lysyl residue in fragment 127-148, is a probable site of attachment of the second molecule of CAPP. UV irradiation of CAPP1-calmodulin, followed by digestion with trypsin, results in the selective loss of 50% each of peptides containing residues 38-74 and 127-148, suggesting that these peptides contain the hydrophobic amino acids that form the phenothiazine-binding sites. The loss of peptides encompassing residues 38-74 and 127-148, located in the amino and carboxyl halves of calmodulin, respectively, suggests that the hydrophobic rings of CAPP can bind at either one of the two phenothiazine sites. Computer modeling of CAPP1-calmodulin with the X-ray coordinates of calmodulin (Babu et al., 1986) indicates that CAPP attached to Lys 75 cannot interact with the carboxyl-terminal phenothiazine-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of tryptic calmodulin fragments on guanylate cyclase activity from Paramecium tetraurelia

Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.     

Interactions of troponin I and its inhibitory fragment (residues 104-115) with troponin C and calmodulin

Fluorescent probes have been used to study the interaction of troponin I and its inhibitory peptide TnIp with troponin C, calmodulin, and the proteolytic fragments of calmodulin. The probes used included the noncovalently bound ligand TNS and the covalently attached labels dansyl and AEDANS. The fluorescence intensity of TNS bound to troponin C, calmodulin, or the calmodulin fragments was greatly enhanced by the presence of TnIp. This effect was used to estimate the corresponding binding constants. It was found that TnIp is bound by the C-terminal half-molecule of calmodulin, TR2C, with an affinity comparable to that of intact calmodulin or troponin C, while the binding affinity of the N-terminal half-molecule, TR1C, was an order of magnitude less, suggesting that the TnIp-containing portion of troponin I combines with the C-terminal half of calmodulin or troponin C. The fluorescence properties of an AEDANS group linked to Cys-98 of troponin C were modified by interaction with troponin I or TnIp. The fluorescence properties of the same group linked to Cys-27 of wheat germ calmodulin were affected by TnI, but not TnIp. TnI had a small effect upon the fluorescence of a dansyl group linked to Met-25 of troponin C. TnIp also inhibited the tryptic hydrolysis of the midpoint of the central connecting strand of calmodulin and troponin C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of a trifluoperazine binding site on troponin C

Trifluoperazine (TFP) was shown to interact with the cyanogen bromide fragment 9 (CB9) (residues 84-135) of rabbit skeletal troponin C and with a synthetic peptide representing the N-terminal region of CB9. The phenothiazine did not affect the calcium binding property of CB9 as observed by proton magnetic resonance and circular dichroism spectroscopies. The calculated calcium binding constants for CB9 in the presence and absence of trifluoperazine were identical (KCa2+ = 1.3 X 10(5) M-1). Localization of the trifluoperazine binding site was achieved by analyzing the 1H NMR spectrum of CB9 and of a synthetic fragment corresponding to residues 90-104 of CB9. Drug-induced shifting and broadening of the ring protons of phenylalanine residues and the methyl resonances of alanine, leucine, and isoleucine residues suggest that the segment 95-102 is in close proximity to the phenothiazine aromatic region. The neighboring negative side chains in the peptide sequence also suggest that the single positive charge present on the piperazine nitrogens of trifluoperazine may interact with them and sterically block a region of interaction of calmodulin (CaM) and troponin C (TnC) with modulated proteins such as phosphodiesterase. Primary sequence analysis of CaM and troponin C reveals that a homologous hydrophobic region to site 3 is also found in the N-terminal region of site 1 of both calcium binding proteins. Binding of TFP to CB9 occurs both in the presence and absence of calcium since the hydrophobic region in these small fragments is completely accessible to TFP whether calcium is present or not. The dissociation constant of the drug to apoCB9 (8 microM) was obtained by ellipticity measurements at 222 nm and was comparable to the 5 microM value obtained by Levin and Weiss [Levin, R. M., & Weiss, B. (1978) Biochim. Biophys. Acta 540, 197-204] for calcium-saturated rabbit skeletal troponin C.     

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.     

Modulation of the stability of rabbit skeletal muscle myosin light chain kinase through the calmodulin-binding domain

The binding of Ca2+(4).calmodulin (CaM) to rabbit skeletal muscle myosin light chain kinase (MLCK) is required for expression of the enzyme's activity. While both MLCK and CaM were stable at 30 degrees C, their complex was not. The binding of CaM to MLCK resulted in a time- and temperature-dependent inactivation that reflected an intrinsic instability of the complex. Separation of the components of the inactive complex yielded functional CaM, but catalytically inert MLCK, indicating that the site of the inactivating event was confined to MLCK. The behavior of proteolytic fragments further localized this event to the C-terminal 60% of the 603-residue protein. Changes in the tryptophan fluorescence and proteolytic susceptibility of MLCK-CaM indicated that a conformational change accompanied, and thus may have caused, inactivation. Substrates protected against inactivation, as did millimolar concentrations of Mg2+, Mn2+, and Ca2+. These metals appeared to bind to a site on MLCK distinct from that which recognized Mg2+.ATP. A proteolytic fragment of MLCK lacking the ability to bind CaM, C beta 35 (residues 255-584; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), was unstable at 30 degrees C, whereas a similar fragment which does bind CaM, T beta 40 (residues 236-595; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. (1985) J. Biol. Chem. 260, 11275-11285), was unstable only when CaM was bound.     

The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study.

Near-u.v. and far-u.v. c.d. spectra of bovine testis calmodulin and its tryptic fragments (TR1C, N-terminal half, residues 1-77, and TR2C, C-terminal half, residues 78-148) were recorded in metal-ion-free buffer and in the presence of saturating concentrations of Ca2+ or Cd2+ under a range of different solvent conditions. The results show the following: if there is any interaction between the N-terminal and C-terminal halves of calmodulin, it has not apparent effect on the secondary or tertiary structure of either half; the conformational changes induced by Ca2+ or Cd2+ are substantially greater in TR2C than they are in TR1C; the presence of Ca2+ or Cd2+ confers considerable stability with respect to urea-induced denaturation, both for the whole molecule and for either of the tryptic fragments; a thermally induced transition occurs in whole calmodulin at temperatures substantially below the temperature of major thermal unfolding, both in the presence and in the absence of added metal ion; the effects of Cd2+ are identical with those of Ca2+ under all conditions studied.     

Solution structures of the N-terminal domain of yeast calmodulin: Ca2+-dependent conformational change and its functional implication

We have determined solution structures of the N-terminal half domain (N-domain) of yeast calmodulin (YCM0-N, residues 1-77) in the apo and Ca(2+)-saturated forms by NMR spectroscopy. The Ca(2+)-binding sites of YCM0-N consist of a pair of helix-loop-helix motifs (EF-hands), in which the loops are linked by a short beta-sheet. The binding of two Ca(2+) causes large rearrangement of the four alpha-helices and exposes the hydrophobic surface as observed for vertebrate calmodulin (CaM). Within the observed overall conformational similarity in the peptide backbone, several significant conformational differences were observed between the two proteins, which originated from the 38% disagreement in amino acid sequences. The beta-sheet in apo YCM0-N is strongly twisted compared with that in the N-domain of CaM, while it turns to the normal more stable conformation on Ca(2+) binding. YCM0-N shows higher cooperativity in Ca(2+) binding than the N-domain of CaM, and the observed conformational change of the beta-sheet is a possible cause of the highly cooperative Ca(2+) binding. The hydrophobic surface on Ca(2+)-saturated YCM0-N appears less flexible due to the replacements of Met51, Met71, and Val55 in the hydrophobic surface of CaM with Leu51, Leu71, and Ile55, which is thought to be one of reasons for the poor activation of target enzymes by yeast CaM.     

Allosteric interactions among drug binding sites on calmodulin

Felodipine is a fluorescent dihydropyridine Ca²⁺-antagonist. It binds to calmodulin in a Ca²⁺-dependent manner, and undergoes a fluorescence increase which allows us to monitor its interaction with calmodulin. Hydrophobic ligands including the calmodulin antagonist, R24571 and Ca²⁺ antagonists, prenylamine and diltiazem, bind to calmodulin and potentiate felodipine binding by as much as 20 fold. These studies suggest that allosteric interactions occur among different drug binding sites on calmodulin. Our results are discussed in terms of the mechanism of action of calmodulin.     

Functional dynamics of the hydrophobic cleft in the N-domain of calmodulin

Molecular dynamics studies of the N-domain (amino acids 1-77; CaM(1-77)) of Ca2+-loaded calmodulin (CaM) show that a solvent exposed hydrophobic cleft in the crystal structure of CaM exhibits transitions from an exposed (open) to a buried (closed) state over a time scale of nanoseconds. As a consequence of burying the hydrophobic cleft, the R(g) of the protein is reduced by 1.5 A. Based on this prediction, x-ray scattering experiments were conducted on this domain over a range of concentrations. Models built from the scattering data show that the R(g) and general shape is consistent with the simulation studies of CaM(1-77). Based on these observations we postulate a model in which the conformation of CaM fluctuates between two different states that expose and bury this hydrophobic cleft. In aqueous solution the closed state dominates the population, while in the presence of peptides, the open state dominates. This inherent flexibility of CaM may be the key to its versatility in recognizing structurally distinct peptide sequences. This model conflicts with the currently accepted hypothesis based on observations in the crystal structure, where upon Ca2+ binding the hydrophobic cleft is exposed to solvent. We postulate that crystal packing forces stabilize the protein conformation toward the open configuration.