Simultaneous Binding of Drugs with Different Chemical Structures to Ca²⁺-Calmodulin:  Crystallographic and Spectroscopic Studies^,

Abstract

The modulatory action of Ca2+-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 A 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"-(beta-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.

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

“…For example, troponin C binds two TFP molecules (PDB 1WRK) and calmodulin binds TFP with a range of stoichiometries; 1∶1 (PDB 1CTR), 1∶2 (PDB 1A29), and 1∶4 (PDB 1LIN) (28)(29)(30). Even though S100A4, troponin C, and calmodulin are built upon the same basic four-helical structural module, the architectures of the TFP binding pockets, and the positions and orientations of the bound TFP molecules are quite different amongst the three proteins (Fig.…”

The Secretory Response of the Rat Colon to the Flavonol Quercetin is Dependent on Ca2+‐Calmodulin

“…The observation that two different Ca¥-calmodulin antagonists, like trifluoperazine and ophiobolin A, inhibited the quercetin-induced increase in Isc strongly supports the hypothesis that the flavonol elicits its secretory activity in rat colon via a Ca¥-and calmodulin-dependent mechanism (Ilund ain & Naftalin, 1979;Leung et al 1984). Both antagonists bind to calmodulin in a different and very specific manner (Au & Leung, 1998;Vertessy et al 1998). Therefore, a non-specific effect of both trifluoperazine and ophiobolin A on the flavonol-induced secretion seems unlikely, but the possibility cannot be excluded.…”

Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine

“…Although anti-CaM drugs can interact with CaM in a versatile manner (9,10), the binding to apoCaM has only been demonstrated in case of KAR-2. Natural products with anti-CaM properties also exist, but can mainly be found in certain plant species and animal venoms (11).…”