SIMULTANEOUS BINDING OF DRUGS WITH DIFFERENT CHEMICAL STRUCTURES TO CA2-CALMODULIN - CRYSTALLOGRAPHIC AND SPECTROSCOPIC STUDIES()

The modulatory action of Ca2+-calmodulin on multiple targets is inhibi ted by trifluoperazine, which competes with target proteins for calmod ulin binding. The structure of calmodulin crystallized with two triflu operazine molecules is determined by X-ray crystallography at 2.74 Ang strom 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 o f two trifluoperazine molecules to calmodulin, Accordingly, the bindin g of trifluperazine to the C-terminal hydrophobic Docket is followed b y the interaction of the second drug molecule with an interdomain sire , Recently, we demonstrated that the two bisindole derivatives, vinbla stine and KAR-2 [3 ''-(beta-chloroethlyl)-2 '',4 ''-dioxo-3,5 ''-spiro oxazolidino-4-deacetoxyvinblastine], interact with calmodulin with com parable affinity; however, they display different functional effects [ Orosz et al, (1997) British J. Pharmacol, 121, 955-962]. The structura l basis responsible for these effects were investigated by circular di chroism 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 vinblastin e. The combination of the binding and structural data suggests that di stinct binding sites exist on calmodulin for vinblastine and KAR-2 whi ch correspond, at least partly, to that of trifluoperazine at the C-te rminal hydrophobic pocket and at an interdomain site, respectively. Th is structural arrangement can explain why these drugs display differen t anticalmodulin activities. Calmodulin complexed with melittin is als o able to bind two trifluoperazine molecules, the binding of which app ears to be cooperative. Results obtained with intact and proteolytical ly cleaved calmodulin reveal that the central linker region of the pro tein is indispensable for simultanous interactions with two molecules of either identical or different ligands.