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J. Biol. Chem., Vol. 280, Issue 9, 8266-8274, March 4, 2005

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The Structure of the Complex of Calmodulin with KAR-2

A NOVEL MODE OF BINDING EXPLAINS THE UNIQUE PHARMACOLOGY OF THE DRUG^*

István Horváth, Veronika Harmat¶, András Perczel||, Vill Pálfi||, László Nyitray**, Attila Nagy**, Emma Hlavanda, Gábor Náray-Szabó, and Judit Ovádi

From the Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29 Budapest, H-1113 Hungary, ^¶Protein Modeling Group, Hungarian Academy of Sciences, Eötvös Loránd University, Pázmány sétány 1A, Budapest, H-1117 Hungary, ^||Department of Organic Chemistry, Eötvös Loránd University, Pázmány sétány 1A, Budapest, H-1117 Hungary, and ^**Departments of Biochemistry and Theoretical Chemistry, Eötvös Loránd University, Pázmány sétány 1A, Budapest, H-1117 Hungary

3'-(-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca²⁺-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation (¹H-¹⁵N heteronuclear single quantum coherence) spectra of ¹⁵N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-Å resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.

Received for publication, September 9, 2004 , and in revised form, December 2, 2004.

The atomic coordinates and structure factors (code 1XA5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Hungarian National Scientific Research Fund Grants OTKA T-046071 and TS-044730, OTKA T-43746 (to L. N.), and T047186 (to A. P.), Hungarian Ministry of Education Grants OMFB-00701/2003 and FP6–2003-LIFESCIHEALTH-I: BioSim, NKFP Medi Chem2 1/A/005/2004, and a Charles Simonyi fellowship (to J. O.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

To whom correspondence should be addressed: Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29, Budapest, H-1113 Hungary. Tel.: 36-1-279-3129; Fax: 36-1-466-5465; E-mail: judit{at}enzim.hu.

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