Effects of trifluoperazine on calcium binding by calmodulin. Heat capacity and entropy changes

The possible structural changes of the calmodulin-trifluoperazine (TFP) complex caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of calmodulin with Ca2+ in the presence of 8-fold molar excess TFP have been made both in the absence and presence of Mg2+, at pH 7.0, and at 5, 15, and 25 degrees C. At high concentrations of TFP calmodulin forms a complex with TFP even in the absence of Ca2+. The reaction of the calmodulin-TFP complex with Ca2+ is exothermic, both in the presence and absence of Mg2+. In the presence of Mg2+ the reaction is driven almost entirely by a favorable enthalpy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of this complex on Ca2+ binding have been estimated by the empirical method of Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). In the presence of Mg2+, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner by Ca2+ binding to each of the binding sites. In contrast, when Mg2+ is absent, the hydrophobic entropy gradually increases on Ca2+ binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the overall structure is loosened in the presence of Mg2+, but tightened in the absence of Mg2+.     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interaction of trifluoperazine with Tetrahymena calmodulin. A 19F NMR study

We have used 19F NMR to study interactions of trifluoperazine (TFP), a potent calmodulin (CaM) antagonist, with Tetrahymena calmodulin (Tet. CaM). Changes in chemical shift and bandwidth of TFP caused by adding Tet. CaM in the presence of excess Ca2+ were much smaller than those by adding porcine CaM. The spectral features of the TFP-Tet. CaM solution in the presence of excess Ca2+ were quite similar to those of the TFP-porcine CaM solution in the absence of Ca2+. The exchange rate of TFP from Tet. CaM was estimated to be nearly 20 s-1. The TFP-Tet. CaM solution in the absence of Ca2+ showed a pronounced pH dependence of the 19F NMR chemical shift, whereas the solution in the presence of excess Ca2+ showed a smaller pH dependence. Thus, it was suggested that TFP is located near a hydrophilic region of the Tet. CaM molecule in the absence of Ca2+, while TFP is located near a hydrophobic region of the Tet. CaM in the presence of excess Ca2+.     

The nature of the trifluoperazine binding sites on calmodulin and troponin-C

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.     

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.     

Quantitative aspects of the development of a hydrophobic binding site on calmodulin by calcium binding

The interactive binding by calmodulin of Ca²⁺ and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been examined. In the presence of saturating levels of Ca²⁺, calmodulin develops one moderately strong binding site for 1,8-ANS, plus one or more weaker sites. The binding of 1,8-ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca²⁺ ions. Little further change occurs on binding additional Ca²⁺ ions.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Multiple calcium binding sites make calmodulin multifunctional

Protein-protein or protein-ion interactions with multisite proteins are essential to the regulation of intracellular and extracellular events. There is, however, limited understanding of how ligand-multisite protein interactions selectively regulate the activities of multiple protein targets. In this paper, we focus on the important calcium (Ca(2+)) binding protein calmodulin (CaM), which has four Ca(2+) ion binding sites and regulates the activity of over 30 other proteins. Recent progress in structural studies has led to significant improvements in the understanding of Ca(2+)-CaM-dependent regulation mechanisms. However, no quantitative model is currently available that can fully explain how the structural diversity of protein interaction surfaces leads to selective activation of protein targets. In this paper, we analyze the multisite protein-ligand binding mechanism using mathematical modelling and experimental data for Ca(2+)-CaM-dependent protein targets. Our study suggests a potential mechanism for selective and differential activation of Ca(2+)-CaM targets by the same CaM molecules, which are involved in a variety of intracellular functions. The close agreement between model predictions and experimental dose-response curves for CaM targets available in the literature suggests that such activation is due to the selective activity of CaM conformations in complexes with variable numbers of Ca(2+) ions. Although the paper focuses on the Ca(2+)-CaM pair as a particularly data rich example, the proposed model predictions are quite general and can easily be extended to other multisite proteins. The results of the study may therefore be proposed as a general explanation for multifunctional target regulation by multisite proteins.     

Effects of calcium binding on the internal dynamic properties of bovine brain calmodulin, studied by NMR and optical spectroscopy.

The dynamic properties of bovine brain calmodulin have been studied as a function of binding calcium ions, using a number of complementary spectroscopic methods. Rotational correlation times for proton-proton vectors within tyrosine and phenylalanine residues of calmodulin have been determined from time-dependent NOE measurements. In the presence of Ca2+, a range of rotational correlation times is observed. The longest value is consistent with Ca4-calmodulin having a markedly nonspherical shape in solution. In the absence of Ca2+, the rotational correlation times of all vectors are significantly shorter, indicating that several phenylalanine side chains in apocalmodulin have increased internal dynamics. Time-resolved tyrosine fluorescence anisotropy shows global correlation times broadly in agreement with the NMR results, but with an additional faster correlation time [approximately 600 ps]. Tyrosine residues in apocalmodulin have substantial segmental motion, which becomes significantly reduced, but not eliminated, when Ca2+ is bound. The correlation time for global rotation of Ca4-calmodulin increases from pH 7 to 4.5, indicating increased overall molecular asymmetry. This occurs without a significant change in total alpha-helix content as measured by circular dichroism. These results are consistent with the central region of Ca4-calmodulin being relatively flexible in solution at pH 7, but with the molecule adopting a more extended shape under more acidic conditions. The Ca(2+)-induced change in alpha-helix content can be mimicked by protonation. The alpha-helix content of Ca4-calmodulin in solution appears less than in the crystal structure; additional alpha-helix is induced in partially nonaqueous solutions, particularly at acidic pH, as used in crystallization conditions.     

Estimation of pH-dependence of calcium binding with calmodulin

A methodical approach to estimating calmodulin Ca(2+)-binding properties based on its interaction with highly porous watman and consequent 45Ca2+ binding was proposed. At changing pH from 6.5 until 7.5 the affinity of Ca2+ to calmodulin increases in 4.3-fold. The article displays a model of mechanism for Ca(2+)-binding with calmodulin where the dissociation of H+ from Ca(2+)-binding sites is a limited stage of the process.     

Cyclic nucleotide phosphodiesterase of rat pancreatic islets. Effects of Ca2+, calmodulin and trifluoperazine.

Cyclic nucleotide phosphodiesterase activity towards cyclic AMP and cyclic GMP was studied in extracts of rat islets of Langerhans. Biphasic Eadie plots [Eadie (1942) J. Biol. Chem. 146, 85-93] were obtained with either substrate suggesting the presence of both 'high'- and 'low'-Km components. The apparent Km values were 6.2 +/- 0.5 (n = 8) microM and 103.4 +/- 13.5 (6) microM for cyclic AMP and 3.6 +/- 0.3 (12) microM and 61.4 +/- 7.5 (13) microM for cyclic GMP. With cyclic AMP as substrate, phosphodeisterase activity was increased by calmodulin and Ca2+ and decreased by trifluoperazine, a specific inhibitor of calmodulin. With cyclic GMP as substrate, phosphodiesterase activity was decreased by omission of Ca2+ or addition of trifluoperazine. Addition of exogenous calmodulin had no effect on activity. The data suggest that Ca2+ may influence the islet content of cyclic AMP and cyclic GMP via effects on calmodulin-dependent cyclic nucleotide phosphodiesterase(s).     

Effect of phosphorylation of calmodulin on calcium binding affinity as estimated by terbium fluorescence

The effect of phosphorylation of calmodulin by casein kinase 2 on the calcium binding of the former was studied by measurement of terbium fluorescence. The binding of Tb3+ to calmodulin was followed by an increase in Tb3+ fluorescence at 545 nm. The terbium fluorescence of phosphorylated calmodulin increased at a lower concentration of Tb3+ than that of non-phosphorylated calmodulin, indicating that Tb3+ binding affinity of calmodulin was increased by phosphorylation. Our results suggest that the interaction between calcium and binding domain becomes stronger by phosphorylation.     

Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody.

The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4.     

Effect of trifluoperazine and calmodulin on catecholamine secretion by saponin-skinned cultured chromaffin cells

We have examined the effect of trifluoperazine on catecholamine secretion by chemically skinned, cultured adrenal chromaffin cells. These cells require only ATP and calcium for secretion. Catecholamine secretion was unaffected by the drug in the presence or absence of calcium and ATP over the range 0.1 to 10 microM. At 100 microM trifluoperazine, catecholamine release was calcium and ATP independent and represented 70-80% of the total cellular content. High concentrations of exogenous calmodulin had no effect on secretion in the presence or absence of calcium. We conclude that low concentrations of the drug have no effect on secretion, while high concentrations cause non-physiological catecholamine release.     

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation.

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Antitubercular activity of trifluoperazine, a calmodulin antagonist

Trifluoperazine, a calmodulin antagonist, completely inhibited the growth of mycobacteria. The minimum inhibitory concentrations in shake cultures in a synthetic medium containing 0.2% Tween 80 were 5 and 8 micrograms/ml, respectively, for the human pathogenic strain Mycobacterium tuberculosis H37Rv and M. tuberculosis resistant to isoniazid. When added to a growing culture of M. tuberculosis H37Rv on the 10th day (mid exponential phase), trifluoperazine 50 micrograms/ml further arrested growth of this organism. It is suggested that trifluoperazine or similar calmodulin antagonists might be useful as antitubercular drugs.     

Effects of opiates on synaptosomal calmodulin and calcium uptake

Acute opiate administration in vivo increases the level of cytoplasmic calmodulin in isolated rat brain synaptosomes. These synaptosomes do not, however, display decreased K⁺-stimulated⁴⁵Ca uptake in vitro. Opiates affect neither cytoplasmic calmodulin nor Ca uptake after incubation of synaptosomes with the drugs in vitro. In contrast to the interpretation of electrophysiological data, these results suggest that the observed inhibition by opiates of the release of several transmitters may not be mediated by presynaptic opiate receptors that inhibit Ca uptake.     

Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.