Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

We have determined the structure of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in an E2·P_i-like form stabilized as a complex with , an ATP analog, adenosine 5′-(β,γ-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca²⁺-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na⁺,K⁺-ATPase reveals that the Phe⁹³ side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na⁺,K⁺-ATPase.The Ca²⁺-ATPase from sarco(endo)plasmic reticulum of rabbit skeletal muscle (SERCA,⁵ isoform 1a) is a thoroughly studied member of the P-type ATPase family (1). SERCA possesses 10 transmembrane helices (M1 through M10) with both the N terminus and the C terminus facing the cytoplasmic side and three cytoplasmic domains, inserted in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain) (2). The enzyme mediates the uptake of Ca²⁺ ions into the lumen of the sarcoplasmic reticulum (SR) after their release into the cytoplasm through calcium release channels during muscle contraction (3). SERCA, plasma membrane Ca²⁺-ATPase, and a third, Golgi-located secretory pathway Ca²⁺-ATPase are important factors in calcium and manganese homeostasis, transport, signaling, and regulation (4, 5).Crystal structures of all major states in the reaction cycle of SERCA have been determined. These include the Ca₂E₁·ATP state (6, 7) with high affinity Ca²⁺ binding sites accessible from the cytoplasmic side of the SR membrane, the calcium-occluded transition state (6), the open E2P state with luminal facing ion binding sites that have low affinity for Ca²⁺ and high affinity for protons (8) and the proton-occluded H_2–3E2ATP] state with a bound modulatory ATP (9). This considerable amount of structural information has turned the Ca²⁺-ATPase into a valuable model system for studies on structural rearrangements that take place during the catalytic cycle of P-type ATPases. SERCA is considered a promising drug target in medical research, with a particular focus on prostate cancer and infectious diseases. Several compounds have already been shown to bind and inhibit SERCA by stabilizing the enzyme in a particular conformational state. Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl) hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri (methylisothiouronium)benzene stabilizes an E1-P-like conformation (10–13). CPA is a toxic indole tetramic acid first isolated from Penicillium cyclopium (14) and later found to be produced by Aspergillus versicolor and Aspergillus flavus. Like TG, CPA specifically binds to and inhibits SERCA with nanomolar affinity (15). Indeed, CPA is widely used in biochemical and physiological studies on Ca²⁺ signaling and muscle function, where it causes Ca²⁺ store depletion due to specific inhibition of Ca²⁺ reuptake by SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA (16), but recent crystal structures have shown a distinct site of interaction (17, 18). Despite these structural insights, a previously demonstrated magnesium dependence of CPA binding (19) remained unexplained, and opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural derivatives have been isolated from bacterial and fungal species (reviewed in Ref. 20). Tetramic acids possessing a 3-acyl group have the ability to chelate divalent metal ions. For instance, tenuazonic acid from the fungus Phoma sorghina has been shown to form complexes with Ca²⁺ and Mg²⁺ (21), as well as heavier metals such as Cu(II), Ni(II), and Fe(III) (22).Previously published crystallographic structures of the SERCA·CPA complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed calcium access channel of SERCA. However, the structures did not reveal a role for magnesium, and the orientation of CPA within this binding site differed in the two studies (17, 18). To address these ambiguities, we have determined the crystal structure of SERCA in complex with , AMPPCP (an ATP analog), and Mn²⁺·CPA. The structure reveals novel insight into CPA binding, which we find to be mediated by a divalent cation, as demonstrated by means of the anomalous scattering properties of Mn²⁺. Further and improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in light of our new findings, also revealed a strong plausibility for a magnesium ion bound at this site. Furthermore, we find a new configuration of the bound AMPPCP nucleotide, addressing the modulatory role of ATP binding to the E2·P_i occluded conformation of SERCA.