Binding of trifluoperazine and fluorene-containing compounds to calmodulin and adducts (original) (raw)
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Trifluoperazine binding to calmodulin: A shift reagent 43Ca NMR study
Biochemical and Biophysical Research Communications, 1984
43Ca NMR experiments of Ca2+ binding to calmodulin (CaM) were performed in the presence and absence of the calmodulin antagonist trifluoperazine (TFP). By making use of the shift reagent Dy(PPP)z-(a I:2 complex of DyC13 and Na5P3010) we have succeeded in separating the "3Ca resonances of protein-bound Ca2+ and free Ca2+ in the otherwise unresolved spectra. This experimental strate$y has allowed us to demonstrate unequivocally that the affinity of CaM for Ca + is markedly increased in the presence of TFP. Thus Ca'+ is not liberated from the protein upon addition of TFP as had been suggested based on earlier 43Ca NMR experiments
Journal of Biological Chemistry
The Ca2+-dependent association of &endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing Ca2+ by ethylene glycol bis(8aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the t-amino groups of the seven lysyl residues on calmodulin by ['Hlacetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in Ca2+-saturated calmodulin. In the presence of Ca2+ and under conditions where &endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptide and protein, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in Ca2+-med1ated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the Ca2+-saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein (
Biochemistry, 1998
The modulatory action of Ca 2+ -calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 Å resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3′′-( -chloroethyl)-2′′,4′′-dioxo-3,5′′-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955-962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions with two molecules of either identical or different ligands.
Binding of Spin-Labeled Phenothiazines to Calmodulin
Biological chemistry Hoppe-Seyler
The binding to purified calmodulin of five spin-labelled derivatives of chlorophenothiazine was investigated by e.s.r. spectrometry and by the antagonizing potency on the calmodulin-dependent activation of myosin light chain kinase. The results of a comparative study and the influence of pH and ionic strength on the binding support the occurrence of an electrostatic binding involving the terminal amino group of the sidechain of the chlorophenothiazine. These results are discussed in relation to the specificity of the interaction that holds the antipsychotic drug-calmodulin complex together.
Biochemical Journal, 1992
An indirect enzyme-linked immunosorbent assay has been used to study the interactions between calmodulin and two calmodulin antagonists, trifluoperazine and a neuropeptide isolated from the hypothalamus. The binding of a monospecific anti-calmodulin antibody, raised in rabbit against dinitrophenylated calmodulin, to calmodulin was tested at various concentrations of these drugs under equilibrium conditions. Trifluoperazine at low concentrations stimulated, but at relatively high concentrations inhibited, immunocomplex formation. The neuropeptide displaced the antibody from calmodulin at nanomolar concentrations. Enzyme-linked immunosorbent assays were also carried out with the large tryptic fragments of calmodulin. The results suggest that (i) the C-terminal fragment binds the antibody with an affinity which is comparable with that of intact calmodulin; (ii) the neuropeptide can form complexes with both N-and C-terminal fragments, but with two orders of magnitude less activity in case of the C-terminal fragment; and (iii) trifluoperazine does not stimulate antibody binding to the C-terminal fragment. Therefore the tertiary structure of calmodulin must be intact to ensure long-distance interactions between the binding sites of trifluoperazine, the neuropeptide and the antibody. These interactions may produce distinct conformers of calmodulin which may exhibit altered potency, not only for antibody binding but also for stimulation/inhibition of target enzymes.
Binding of a spin-labelled chlorpromazine analogue to calmodulin
The Biochemical journal, 1984
The binding of a spin-labelled derivative of chlorpromazine to calmodulin was investigated by e.s.r. spectrometry. The completion of the spectroscopic changes requires the presence of 4 Ca2+ ions per calmodulin molecule. The influences of various physicochemical factors (pH, ionic strength) are discussed in relation to the nature (hydrophobic and polar) of the interactions that hold the drug-calmodulin complex together.
Journal of Protein Chemistry, 1987
These adducts are suitable for detailed characterization in an effort to provide information on calmoduhn structurefunction relationshlps. The adducts were incapable of or exhibited low potency (e.g., 0.1% that of calmoduhn) m, stimulating the activtty of an activatable bovine brain cyclic nucleotide phosphodtesterase (3 ; EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotatzonal correlation times of approximately 3-6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (-5-7 nsec). Thus, the nitroxtde reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestmgly, removal of the fluorenylmethyloxycarbonyl portton from the 1.1 adduct to give a "deprotected" 1 : I adduct resulted in apparent greater mobdity of the probe, since the rotational correlation coefficient was found to be --1 nsec. Circular dichrow spectra were obtained over the wavelength interval 200-250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmoduhn, exhtblted a Ca 2+-mediated increase in heliczty, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca 2+ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetzc resonance spectra were determined in the aromatic region (-6-8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide wtth ascorbate. The nitroxlde had httle effect on the chemical shifts of the two tyrosines and the single htstidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound 1I, o~-N-(9'fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct 1The Reproductive Plenum Pubhshmg Corporation
Localization of felodipine (dihydropyridine) binding site on calmodulin
Biochemistry, 1986
The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of C a M antagonist drugs through allosteric mechanisms [Mills, J. S., & Johnson, J. D. (1985) Biochemistry 24,48971. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to C A M and several of its proteolytic fragments. While TNS interacts with sites on both 'This work was supported by grants to
Interaction between calmodulin and five different spin-labelled chlorophenothiazines
The Biochemical journal, 1986
The binding to purified calmodulin of five spin-labelled derivatives of chlorophenothiazine was investigated by e.s.r. spectrometry and by the antagonizing potency on the calmodulin-dependent activation of myosin light chain kinase. The results of a comparative study and the influence of pH and ionic strength on the binding support the occurrence of an electrostatic binding involving the terminal amino group of the side-chain of the chlorophenothiazine. These results are discussed in relation to the specificity of the interaction that holds the antipsychotic drug-calmodulin complex together.