The structure of the complex of calmodulin with KAR-2: A novel mode of binding explains the unique pharmacology of the drug

István Horváth, V. Harmat, A. Perczel, Villo Pálfi, L. Nyitray, A. Nagy, Emma Hlavanda, G. Náray-Szabó, J. Ovádi

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Abstract

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 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.

Original languageEnglish
Pages (from-to)8266-8274
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number9
DOIs
Publication statusPublished - Mar 4 2005

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Calmodulin
Pharmacology
Pharmaceutical Preparations
Trifluoperazine
Diffraction
X-Rays
Amino Acids
X rays
deacetoxyvinzolidine
Poisons
Mitosis
Microtubules
Nuclear magnetic resonance spectroscopy
Conformations
Magnetic Resonance Spectroscopy
Crystal structure
Cells
Nuclear magnetic resonance
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "The structure of the complex of calmodulin with KAR-2: A novel mode of binding explains the unique pharmacology of the drug",
abstract = "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 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-{\AA} 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.",
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T1 - The structure of the complex of calmodulin with KAR-2

T2 - A novel mode of binding explains the unique pharmacology of the drug

AU - Horváth, István

AU - Harmat, V.

AU - Perczel, A.

AU - Pálfi, Villo

AU - Nyitray, L.

AU - Nagy, A.

AU - Hlavanda, Emma

AU - Náray-Szabó, G.

AU - Ovádi, J.

PY - 2005/3/4

Y1 - 2005/3/4

N2 - 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 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.

AB - 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 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.

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