The Structure of the Complex of Calmodulin with KAR-2

• Published in: The Journal of biological chemistry Vol. 280; no. 9; pp. 8266 - 8274
• Main Authors: István Horváth, Veronika Harmat, András Perczel, Villő Pálfi, László Nyitray, Attila Nagy, Emma Hlavanda, Gábor Náray-SzabÃ, Judit Ovádi
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 04-03-2005
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M410353200

3′-(β-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 Ca 2+ -calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation
( 1 H- 15 N heteronuclear single quantum coherence) spectra of 15 N-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.