Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs
Open Access
- 1 September 2010
- journal article
- research article
- Published by Elsevier BV in Journal of Biological Chemistry
- Vol. 285 (37), 28883-28892
- https://doi.org/10.1074/jbc.m110.136242
Abstract
Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca2+-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 Ca2+ 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.Keywords
This publication has 51 references indexed in Scilit:
- STIM and Orai: Dynamic Intermembrane Coupling to Control Cellular Calcium SignalsPublished by Elsevier BV ,2009
- Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPasePublished by Elsevier BV ,2009
- MolProbity: all-atom contacts and structure validation for proteins and nucleic acidsNucleic Acids Research, 2007
- Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitorsProceedings of the National Academy of Sciences of the United States of America, 2007
- Coot: model-building tools for molecular graphicsActa Crystallographica Section D-Biological Crystallography, 2004
- BAX and BAK Regulation of Endoplasmic Reticulum Ca 2+ : A Control Point for ApoptosisScience, 2003
- The Importance of the Hydroxyl Moieties for Inhibition of the Ca2+-ATPase by Trilobolide and 2,5-DI(TERT-Butyl)-1,4-BenzohydroquinoneBiochemical and Biophysical Research Communications, 1994
- The inhibitors thapsigargin and 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone favour the E2 form of the Ca2+, Mg2+‐ATPaseFEBS Letters, 1992
- Competition between cholesterol and phosphatidylcholine for the hydrophobic surface of sarcoplasmic reticulum calcium(2+) ATPaseBiochemistry, 1984
- Perturbation of the Structure and Function of a Membranous Ca2+ ‐ATPase by Non‐solubilizing Concentrations of a Non‐ionic DetergentEuropean Journal of Biochemistry, 1983