Simultaneous Binding of Drugs with Different Chemical Structures to Ca 2+ -Calmodulin: Crystallographic and Spectroscopic Studies ²,‡ Beata G. Vertessy, § Veronika Harmat, | Zsolt Bo ¨cskei, |,⊥ Ga ´bor Na ´ray-Szabo ´, | Ferenc Orosz, § and Judit Ova ´di* ,§ Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Department of Structural Chemistry, Eo ¨ tVo ¨ s Lora ´ nd UniVersity, and Chinoin Pharmaceuticals, Budapest, Hungary ReceiVed April 9, 1998; ReVised Manuscript ReceiVed July 13, 1998 ABSTRACT: 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. The small acidic protein calmodulin (CaM) 1 can be considered as a main intracellular “switch” in many cellular processes controlled by Ca 2+ -dependent signaling pathways. CaM controls the activity of several kinases and phos- phatases, enzymes of cyclic nucleotide metabolism, and the Ca 2+ -transport pump and interacts with the cytoskeleton (1, 2). Mostly, CaM induces an increase in the activity of its target enzymes; however, in the case of the glycolytic enzyme 1-phosphofructokinase, enzymatic activity is de- creased upon interaction with CaM (3, 4). The crystal structure of CaM shows an elongated dumbbell-shaped molecule with two structurally similar globular domains separated by the central helix region (5-7); however, in solution this region is flexible (8, 9). The protein can be selectively cleaved at residue Lys 77 of this segment by limited trypsinolysis (10). The resulting globular domains are homologous, and each contains a pair of the Ca 2+ -binding helix-loop-helix motifs, termed EF-hands. Ca 2+ -induced protein conformational changes lead to the exposure of hydrophobic surfaces on both domains, which are responsible for binding of various target peptides as determined by NMR spectroscopy in solution (11) or by X-ray crystallography (12, 13). Anti-CaM drugs of strikingly heterogeneous chemical structure prevent or modify the interactions of CaM with target enzymes. Presently, a detailed structural description of drug binding to CaM is available only in the case of trifluoperazine (TFP), a phenothiazine derivative. CaM has been crystallized with one and four TFP molecules, and the structures of CaM-drug complexes were determined at 2.45 or 2.0 Å resolution, respectively (14, 15). Both crystal structures suggest a unique site for TFP binding in the C-terminal globular domain: this is the only site occupied in the 1:1 CaM-TFP complex, while this is the best-defined site in the 1:4 CaM-TFP complex (14, 15). In the latter structure two TFP molecules are accommodated at the sequentially homologous hydrophobic pockets of the C- and ² This study was supported by grants from the Hungarian National Science Foundation and the Ministry for Education: OTKA F-017392 and F-020862 to B.G.V., T-17830 and T-025291 to J.O., and T-022191 to G.N.-S. ‡ X-ray structural data have been deposited in the Brookhaven Protein Data Bank under Accession Number 1a29. * Correspondence should be addressed to this author at the Hungarian Academy of Sciences, P.O. Box 7, H-1518, Budapest, Hungary. Phone: +361 1665 923. Fax: +361 1665 465. E-mail: ovadi@enzim.hu. § Hungarian Academy of Sciences. | Eo ¨tvo ¨s Lora ´nd University. ⊥ Chinoin Pharmaceuticals. 1 Abbreviations: CaM, calmodulin; TFP, trifluoperazine; KAR-2, 3′′-(-chloroethyl)-2′′,4′′-dioxo-3,5′′-spirooxazolidino-4-deacetoxy- vinblastin; CD, circular dichroism. 15300 Biochemistry 1998, 37, 15300-15310 10.1021/bi980795a CCC: $15.00 © 1998 American Chemical Society Published on Web 10/20/1998