Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs

Anne-Marie Lund Winther; Huizhen Liu; Yonathan Sonntag; Claus Olesen; Marc le Maire; Helmer Søhoel; C Erik Olsen; Søren Brøgger Christensen; Poul Nissen; Jesper Vuust Møller

doi:10.1074/jbc.M110.136242

Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca²⁺-ATPase with thapsigargin and thapsigargin analogs

Anne-Marie Lund Winther, Huizhen Liu, Yonathan Sonntag, Claus Olesen, Marc le Maire, Helmer Søhoel, C Erik Olsen, Søren Brøgger Christensen, Poul Nissen, Jesper Vuust Møller

70 Citations (Scopus)

Abstract

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

Original language	English
Journal	Journal of Biological Chemistry
Volume	285
Issue number	37
Pages (from-to)	28883-28892
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M110.136242
Publication status	Published - 10 Sept 2010

Keywords

Former Faculty of Life Sciences

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1074/jbc.M110.136242

Cite this

Winther, A.-M. L., Liu, H., Sonntag, Y., Olesen, C., le Maire, M., Søhoel, H., Olsen, C. E., Christensen, S. B., Nissen, P., & Møller, J. V. (2010). Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca²⁺-ATPase with thapsigargin and thapsigargin analogs. Journal of Biological Chemistry, 285(37), 28883-28892. https://doi.org/10.1074/jbc.M110.136242

Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca²⁺-ATPase with thapsigargin and thapsigargin analogs. / Winther, Anne-Marie Lund; Liu, Huizhen; Sonntag, Yonathan et al.
In: Journal of Biological Chemistry, Vol. 285, No. 37, 10.09.2010, p. 28883-28892.

Research output: Contribution to journal › Journal article › Research › peer-review

Winther, A-ML, Liu, H, Sonntag, Y, Olesen, C, le Maire, M, Søhoel, H, Olsen, CE, Christensen, SB, Nissen, P & Møller, JV 2010, 'Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca²⁺-ATPase with thapsigargin and thapsigargin analogs', Journal of Biological Chemistry, vol. 285, no. 37, pp. 28883-28892. https://doi.org/10.1074/jbc.M110.136242

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abstract = "Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.",

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T1 - Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs

AU - Winther, Anne-Marie Lund

AU - Liu, Huizhen

AU - Sonntag, Yonathan

AU - Olesen, Claus

AU - le Maire, Marc

AU - Søhoel, Helmer

AU - Olsen, C Erik

AU - Christensen, Søren Brøgger

AU - Nissen, Poul

AU - Møller, Jesper Vuust

PY - 2010/9/10

Y1 - 2010/9/10

N2 - Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

AB - Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

KW - Former Faculty of Life Sciences

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DO - 10.1074/jbc.M110.136242

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VL - 285

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JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

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