Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: Crystallographic and spectroscopic studies

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Abstract

The modulatory action of Ca2+-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-(β-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 simultaneous interactions with two molecules of either identical or different ligands.

Original languageEnglish
Pages (from-to)15300-15310
Number of pages11
JournalBiochemistry
Volume37
Issue number44
DOIs
Publication statusPublished - Nov 3 1998

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Trifluoperazine
Calmodulin
Vinblastine
Pharmaceutical Preparations
Molecules
Circular Dichroism
Melitten
Circular dichroism spectroscopy
Calmodulin-Binding Proteins
X ray crystallography
X Ray Crystallography
Fluorescence Spectrometry
Fluorescence spectroscopy
Drug Interactions
Binding Sites
X-Rays
Ligands
Derivatives
X rays
deacetoxyvinzolidine

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: Crystallographic and spectroscopic studies",
abstract = "The modulatory action of Ca2+-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 {\AA} 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-(β-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 simultaneous interactions with two molecules of either identical or different ligands.",
author = "B. V{\'e}rtessy and V. Harmat and Zsolt B{\"o}cskei and G. N{\'a}ray-Szab{\'o} and F. Orosz and J. Ov{\'a}di",
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T1 - Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin

T2 - Crystallographic and spectroscopic studies

AU - Vértessy, B.

AU - Harmat, V.

AU - Böcskei, Zsolt

AU - Náray-Szabó, G.

AU - Orosz, F.

AU - Ovádi, J.

PY - 1998/11/3

Y1 - 1998/11/3

N2 - The modulatory action of Ca2+-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-(β-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 simultaneous interactions with two molecules of either identical or different ligands.

AB - The modulatory action of Ca2+-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-(β-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 simultaneous interactions with two molecules of either identical or different ligands.

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