Two trifluoperazine-binding sites on calmodulin predicted from comparative molecular modeling with troponin-C

Among the known regulatory proteins that are conformationally sensitive to the binding of calcium ions, calmodulin and troponin-C have the greatest primary sequence homology. This observation has led to the conclusion that the most accurate predicted molecular model of calmodulin would be based on the X-ray crystallographic coordinates of the highly refined structure of turkey skeletal troponin-C. This paper describes the structure of calmodulin built from such a premise. The resulting molecular model was subjected to conjugate gradient energy minimization to remove unacceptable intramolecular non-bonded contacts. In the analysis of the resulting structure, many features of calmodulin, including the detailed conformation of the Ca2+-binding loops, the amino- and carboxy-terminal hydrophobic patches of the Ca2+-bound form, and the several clusters of acidic residues can be reconciled with much of the previously published solution data. Calmodulin is missing the N-terminal helix characteristic of troponin-C. The deletion of three residues from the central helical linker (denoted D/E in troponin-C) shortens the molecule and changes the orientation of the two domains of calmodulin by 60 degrees relative to those in troponin-C. The molecular model has been used to derive two possible binding sites for the antipsychotic drug trifluoperazine, a potent competitive inhibitor of calmodulin activity.     

Effects of trifluoperazine on calcium binding by calmodulin. A microcalorimetric study

Microcalorimetric titrations of calmodulin with Ca2+ and trifluoperazine (TFP) at various molar ratios have been carried out at 25 degrees C and at pH 7.0. Ca2+ binding to calmodulin produces heat (-delta H) in the presence of TFP, while heat is absorbed in the absence of TFP. The total heat produced by Ca2+ binding to all four sites is increased at increasing TFP-to-calmodulin ratios, attaining a plateau at about 7. These results indicate that at the higher ratios, the enthalpy changes (delta H) associated with Ca2+ binding are affected by TFP molecules bound at both high- and low-affinity sites. In addition, the Ca2+ binding reaction of the calmodulin-TFP complex is driven solely by a favorable enthalpy change of -27 kJ/mol of site; the entropy change (delta S) is -35 J/mol/K. These thermodynamic changes are opposite to those for TFP-free calmodulin and distinctly different from other Ca2+ binding proteins such as skeletal and cardiac troponin C and parvalbumin, where the reaction is driven by favorable changes of entropy as well as enthalpy.     

Direct identification of the high and low affinity calcium binding sites of troponin-C

Covalent labelling of the calcium ligands of intact troponin-C (0.1 M KCl, pH 6.0) with [³H] -ethanolamine, at various ratios of calcium to troponin-C followed by analysis of the two separated cleavage products, shows that the first and second calcium binding sites of the sequence are the low affinity sites and that the third and fourth sites are the high affinity or structure defining sites of troponin-C.     

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.     

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+.     

The effect of calmodulin and troponin-C on activities of homogeneous liver phosphoprotein phosphatases

The effect of calmodulin was determined on activities of two homogeneous liver phosphoprotein phosphatases with phosphorylase a and phosphorylated histones as substrates. Calmodulin in the absence or presence of calcium had no effect on the dephosphorylation of phosphorylase a by either phosphatases. However, calmodulin inhibited the dephosphorylation of histones catalyzed by both phosphatases. No difference was found whether the reactions were carried out in the absence or presence of calcium. The effect of calmodulin on histone dephosphorylation was variable depending on (i) the absence or presence of KCl and Mg²⁺, and (ii) the concentration of histone in the reaction mixture. In the presence of KCl and Mg²⁺ at a histone concentration of 0.1 mg/ml, calmodulin inhibited the enzyme activity. At 1 mg/ml histone, lower concentrations of calmodulin activated whereas higher concentrations of calmodulin inhibited the enzyme activity. Similar, but relatively less, effect was observed with troponin-C. In the absence of KCl and Mg²⁺, calmodulin as well as troponin-C activated the enzyme activity. The optimal concentration of calmodulin (or troponin-C) was approximately 30–50% of histone concentration in the reaction mixture. Calcium alone or with calmodulin (or troponin-C) had no additional effect on enzyme activities. DNA and RNA, two negatively charged nucleic acids, also showed similar effects on histone dephosphorylation. Depending on whether KCl and Mg²⁺ were absent or present in the reaction mixtures, both nucleic acids either activated or inhibited the dephosphorylation of histones.     

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.     

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.     

The regulation of muscle phosphorylase kinase by calcium ions,calmodulin and troponin-C

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase $\text{in vivo}$ is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism for coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the δ - subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state.

Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45–49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.     

Calcium binding to calmodulin. Cooperativity of the calcium-binding sites

The effects of Mg2+ ion, pH, and KCl concentration on Ca2+ binding to calmodulin were studied by using a Ca2+ ion-sensitive electrode. The Ca2+ ion affinity of calmodulin increased with increasing pH or decreasing KCl concentration. Cooperativity between the Ca2+-binding sites was observed, and increased with decreasing pH or increasing KCl concentration. Free Ca2+ ion concentration was decreased by adding MgCl2 ion at low Mg2+ concentration and increased at higher concentrations in the presence of small amounts of Ca2+ ion. The decrease of free Ca2+ ion concentration by Mg2+ ion strongly suggests cooperativity between the Ca2+-binding sites, and it is difficult to explain the decrease in terms of the ordered binding models previously proposed. These results can be explained by a simple model which has four equivalent binding sites that bind Ca2+ and Mg2+ competitively, and showing cooperativity when either Ca2+ or Mg2+ is bound. Mg2+ ion binding to calmodulin was measured in the presence or absence of Ca2+ to confirm the validity of this model, and no Mg2+-specific site was observed.     

Calcium-dependent hydrophobic chromatography of calmodulin, S-100 protein and troponin-C

We have demonstrated calcium-dependent hydrophobic interactions among calmodulin, S-100 protein and troponin-C and a homologous series of omega-aminoalkyl-agaroses. The three Ca2+-binding proteins were retained on the column of agarose substituted with omega- aminooctyl or even longer with alkylamine, in the presence of Ca2+ and 0.15 M NaCl. As these proteins were not retained on the column with shorter alkylamine 'arms' (N = 2, 4), they are probably successively absorbed with a higher affinity to the hydrophobic agarose column. Calmodulin and S-100 protein were eluted from the aminoocytl -agarose column with 1 mM EGTA in the presence of 0.15 M NaCl and the elution of troponin-C was Ca2+-independently carried out with 0.3 M NaCl. On the other hand, S-100 and troponin-C were eluted Ca2+-dependently from aminodecyl -agarose in the presence of 1 M NaCl and half the amount of the calmodulin applied was eluted with 1 M NaCl. As there are obvious differences among the three Ca2+-binding proteins with regard to chromatographic behavior on omega-aminoalkyl-agarose columns, our results suggest that these three proteins expose different hydrophobic regions following Ca2+-induced conformational changes and, if so, such would explain the interaction with aminoalkyl-agaroses.     

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites

Due to Ca²⁺‐dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet‐lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet‐lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large‐margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM‐binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome‐wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif‐based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub‐sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels .     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

Sites of interaction of calmodulin with trifluoperazine and glucagon

The combination of glucagon with calmodulin alters the microenvironment of Tyr-99, but not Tyr-138, and is blocked by the binding of trifluoperazine by calmodulin. Trp-25 of glucagon is probably involved in the zone of interaction, which may also overlap one or more strong binding sites for trifluoperazine. From energy transfer measurements, one strong binding site for trifluoperazine probably involves the N-terminal region of binding domain III. Energy transfer and other evidence suggest that the zone of contact with glucagon involves the N-terminal region of binding domain III.     

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.     

On the nature of antibiotic binding sites in ribosomes

The ribosome is an enzyme and enzymes have active sites. Antibiotics that affect ribosomal function can be considered as enzyme inhibitors (or regulators) and it is therefore pertinent to identify their molecular targets as a means of studying the active sites of the particle. The methods available for doing this are considered and, in general terms, the data are evaluated. The conclusion is reached that there exists virtually no compelling evidence that antibiotics bind primarily to ribosomal proteins. Rather, studies of antibiotic resistance in various systems strongly suggest that ribosomal RNA is the primary target for a number of drugs. Moreover, in at least one case (relating to the antibiotic thiostrepton), such an effect can be demonstrated directly. These conclusions imply a fundamental role for RNA in ribosomal function.     

The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845-2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca(2+)-concentrations.     

Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

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.