Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs

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.Understanding protein-membrane interaction and the activity of lipophilic drugs and specific lipids like sterols attracts increasing attention and represents a challenge to our current models of protein-solvent interactions and protein dynamics. Sarco/endoplasmic Ca 2ϩ -ATPase (SERCA) 6 offers an attractive model for the analysis of such interactions due to favorable biochemical properties with sensitive assays of function, a large resource of inhibitors, and a wealth of crystal structures related to the functional cycle. In conjunction with plasma membrane Ca 2ϩ -ATPases and Ca 2ϩ channel proteins, SERCA orchestrates spatiotemporal changes in the concentration of Ca 2ϩ inside the cell, providing the background essential for the function of Ca 2ϩ as a second messenger in the regulation of numerous cellular processes (1-3). At the same time, high cytosolic (Ͼ1 M) concentrations of Ca 2ϩ , induced by depletion of the endoplasmic Ca 2ϩ stores and uptake of extracellular Ca 2ϩ by the endoplasmic store-regulated mechanism (4), are involved as essential factors in the induction of apoptotic processes, leading to mitochondrial disruption with release of cytochrome c, activation of intracellular caspases, and cell death (5). As a consequence, interference of cellular function by inhibition of SERCA may lead to apoptotic cell death irrespective of the...

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes

“…X-ray structures of SERCA, corresponding to the E2 and the E2P forms, respectively, were used as starting structures for the protein. For the E2 conformation, a structure for a similar crystal form with a nortrilobolide inhibitor, refined at a higher resolution, was applied (PDB 3NAL 41 ). This structure has a Cα-RMSD of 0.5 Å to the E2 crystal structure reported here.…”

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

“…A similar mechanism has been derived from structural data of Ca-ATPase. In E 2 , the headgroup of a PE molecule was identified between TM2, -4, -6, and -9 (58,59). In E 1 , this space is occupied by TM2, and the phospholipid is bound more superficially between TM2 and -9 (60).…”

HBV suppresses thapsigargin-induced apoptosis via inhibiting CHOP expression in hepatocellular carcinoma cells

“…Thapsigargin (TG) is a natural product isolated from the seeds of Thapsia garganica L, which binds tightly to and inhibits the function of the transmembrane portion of the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase pump, inducing apoptosis (1,2). TG initiates endoplasmic reticulum (ER) stress via the unfolded protein response (UPR), which is initiated by 3 ER transmembrane proteins termed protein kinase-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor (ATF) 6 (3,4).…”