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.

FIGURE 1.

Structural formulas of thapsigargin and thapsigargin analogs used in this study.

FIGURE 2.

Structure of the Ca²⁺-ATPase thapsigargin-binding site. A, surface representation of the Tg Ca²⁺-ATPase-binding site of Tg (shown as green and red balls) with hydrophilic residues in blue, hydrophobic in orange, tryptophans highlighted in red. B and C, surface representation of Tg-bound E2-ATPase complex (PDB 1XP5) and with Tg docked into the uncomplexed structure (PDB 3B9R), respectively, with Tg shown as sticks. D, superimposition of structures of Ca²⁺-ATPase in the absence (E2-AlF₄⁻-AMPPCP, PDB code 3B9R) and presence of bound Tg (E2(Tg)- AlF₄⁻, PDB code 1XP5) with main chains shown as tubes in blue and yellow, respectively, and Tg as green sticks with carbon atoms colored green and oxygen atoms colored red. These two structures are representative of all known structures with and without Tg (supplemental Fig. S3). F, alignment of E2-AMPPCP-dOTg with E2-AMPPCP-Tg (PDB 2C88), flanked by structural formulas of Tg (E) and dOTg (G).

FIGURE 3.

Binding of thapsigargin by Ca²⁺-ATPase, phosphatidylcholine, and C₁₂E₈. To membranous Ca²⁺-ATPase, suspended at a protein concentration of 3 mg/ml in 100 mm KCl, 10 mm Tes/Tris (pH 7.5), and 1 mm Mg²⁺, was added 15 μm Tg together with 1 mm EGTA (A) or 0.5 mm Ca²⁺ (B). One-ml sample was applied to a 1 × 25-cm Sephadex G-50 coarse column pre-equilibrated and eluted at 1 ml/min with the same buffer as used for suspension of the ATPase. Both in the presence of EGTA and Ca²⁺, the protein (shown as blue lines) eluted at the void volume after 11–14 min without detectable loss of Tg (shown as red lines) from the membranes. C, there was also complete chromatographic retention of Tg after addition of 15 μm radiolabeled thapsigargin to unilamellar 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes (1.5 mg/ml), prepared by reconstitution from cholate and suspended in the same buffer as in B. D, 3 mg of Ca²⁺-ATPase was solubilized by 30 mg of C₁₂E₈ in 20 mm Tes/Tris (pH 7.0), 100 mm KCl, 1 mm Mg²⁺, 1 mm EGTA, and 20% (v/v) glycerol, and insoluble residues were removed by high speed centrifugation. To the supernatant was added 25 μm labeled Tg, and 0.4 ml was injected into a 0.75 × 30 cm TOSO Haas 3000SWxl HPLC column, equilibrated, and eluted at 0.5 ml/min with 1 mg of C₁₂E₈/ml in the same buffer as used for solubilization. D, peak 1 represents monomeric Ca²⁺-ATPase, and peak 2 represents the mixed micelles of C₁₂E₈ and sarcoplasmic reticulum lipid. Notice that Tg, which was added in about twice the stoichiometric concentration of active Ca²⁺-ATPase, is distributed about equally between the protein and mixed phospholipid/detergent peak.

FIGURE 4.

Structural alignments of the binding of long-chain thapsigargin analogs with that of thapsigargin. Alignments of E2-AMPPCP-Tg (2C8K) with E2-DTB (A) and E2-Boc-ϕTg (B). The Tg analogs are shown in wheat and Tg as green sticks. In addition, the figures show difference Fourier electron density maps of F_{obs(E2-analog} − F_{obs(E2-AMPPCP-Tg)}, at ±3σ in green and red mesh around the bound inhibitors and residues Leu-253 and Phe-256 in the E2-Tg-analog complexes. C, side view; D, top view of Boc-ϕTg binding to the Ca²⁺-ATPase. Boc-ϕTg is shown in blue sticks and SERCA as a wheat schematic with stick representations of some of the amino acid side chain residues surrounding the Boc-ϕTg ligand and the lipid (PTY) identified in a cavity between M2 and M4 (25). E, E2-Tg-BHQ (2AGV) taken from Obara et al. (33). Tg and BHQ is shown in yellow and Boc-ϕTg in light blue. F, E2-ADP-CPA (3FPS) with CPA shown in purple and Boc-ϕTg in light blue. Notice how the Boc-group of the E2-Boc-ϕTg linker is located in the same binding groove as the inhibitors BHQ (E) and CPA (F). The positioning of the phosphatidylethanolamine lipid (PTY) within the E2-Boc-ϕTg complex is indicated with a red broken line between the transmembrane helices M2 and M4. A Mg²⁺ ion that coordinates CPA (26) is shown as a green sphere.

FIGURE 5.

Effects of thapsigargin and thapsigargin analogs on the intrinsic fluorescence and Ca²⁺ binding of Ca²⁺-ATPase. The experiments were conducted with Ca²⁺-ATPase membranes, suspended at a protein concentration of 0.1 mg/ml in media containing 50 μm Ca²⁺, 5 mm Mg²⁺, 100 mm KCl, and 100 mm Mops/Tris (pH 7.2). Intrinsic fluorescence was recorded with a Shimadzu spectrofluorometer at 23 °C with excitation at 290 nm and emission recorded at 340 nm (A and B). A, two additions were made, the first one being 1 mm EGTA to convert the Ca²⁺-ATPase from the Ca²⁺-bound E1 form to E2, followed later by addition of 12 μm inhibitor. B, inhibitor (Tg, dOTg, DTB, or Boc-12ADT) was added, as indicated, at a concentration of 12 μm in the presence of the Ca²⁺-containing medium. C shows parallel experiments where changes in Ca²⁺ binding were measured by Millipore filtration with the aid of radiolabeled ⁴⁵Ca²⁺. D, comprehensive kinetic scheme is shown, with stepwise dissociation of Ca²⁺, used to evaluate the data as described in the text and to analyze the conversion of Ca₂E1 to E₂T with bound inhibitor (T) in terms of two apparent rate constants (k′ and k″) assembled in Table 2.