Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Xu, Cheng; Ma, Hailun; Inesi, Giuseppe; Al‐Shawi, Marwan K.; Toyoshima, Chikashi

doi:10.1074/jbc.m313263200

Cited by 60 publications

(50 citation statements)

References 44 publications

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“…This finding is in line with the results of limited proteolysis, which demonstrate that the A-domain-M3 and the A-domain-M2 loops are flexible and susceptible to proteinase K attack in E2. If the M3 helix is stabilized with TG, the A-domain-M3 loop is largely protected from proteinase K attack, making the A-domain-M2 link the predominant target (27). Our present results show that the A-domain-M1Ј link is also flexible as well as the M1-M1Ј link (Figs.…”

Section: Discussionsupporting

confidence: 60%

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Takahashi

Kondou²,

Toyoshima³

2007

Proc. Natl. Acad. Sci. U.S.A.

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111

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Ca 2؉ -ATPase of skeletal muscle sarcoplasmic reticulum is an ATPdriven Ca 2؉ pump consisting of three cytoplasmic domains and 10 transmembrane helices. In the absence of Ca 2؉ , the three cytoplasmic domains gather to form a compact headpiece, but the ATPase is unstable without an inhibitor. Here we describe the crystal structures of Ca 2؉ -ATPase in the absence of Ca 2؉ stabilized with cyclopiazonic acid alone and in combination with other inhibitors. Cyclopiazonic acid is located in the transmembrane region of the protein near the cytoplasmic surface. The binding site partially overlaps with that of 2,5-di-tert-butyl-1,4-dihydroxybenzene but is separate from that of thapsigargin. The overall structure is significantly different from that stabilized with thapsigargin: The cytoplasmic headpiece is more upright, and the transmembrane helices M1-M4 are rearranged. Cyclopiazonic acid primarily alters the position of the M1 helix and thereby M2 and M4 and then M5. Because M5 is integrated into the phosphorylation domain, the whole cytoplasmic headpiece moves. These structural changes show how an event in the transmembrane domain can be transmitted to the cytoplasmic domain despite flexible links between them. They also reveal that Ca 2؉ -ATPase has considerable plasticity even when fixed by a transmembrane inhibitor, presumably to accommodate thermal fluctuations.Ca 2ϩ -ATPase ͉ ion pump ͉ membrane protein C a 2ϩ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) is structurally and functionally the best studied member of the P-type ion-translocating ATPases (1, 2). Located in the sarcoplasmic reticulum membrane, SERCA1 pumps Ca 2ϩ released into muscle cells during contraction and thereby causes relaxation. The reaction cycle consists of at least eight intermediates, and crystal structures have been determined for six of them (3-9). These crystal structures show that SERCA1 consists of three cytoplasmic domains (A, actuator; N, nucleotide binding; and P, phosphorylation) and 10 (M1-M10) transmembrane helices (3). They also demonstrate that ATPase undergoes large and global conformational changes during the reaction cycle. In conventional E1/E2 theory (10-12), active transport of Ca 2ϩ is achieved by alternating the affinity and accessibility of the transmembrane Ca 2ϩ -binding sites. That is, in E1, the binding sites have high affinity and face the cytoplasm; in E2, the binding sites have low affinity to Ca 2ϩ and face the lumen of the sarcoplasmic reticulum. Although H ϩ and water, instead of Ca 2ϩ , stabilize the binding sites in E2 (13), this form of the ATPase is unstable, particularly when solubilized (14). Therefore, so far all successful crystallization experiments in the absence of Ca 2ϩ used inhibitors that stabilize the transmembrane domain.Several inhibitors specific to SERCA1 are available (15-17). By far the best known and most potent inhibitor is thapsigargin (TG), a plant sesquiterpene lactone (18), with the dissociation constant in the subnanomolar range (15). Another commonly used inhibito...

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Section: Discussionsupporting

confidence: 60%

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Takahashi

Kondou²,

Toyoshima³

2007

Proc. Natl. Acad. Sci. U.S.A.

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111

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“…In this regard, it is similar to thapsigargin, a very high affinity [K d of pM range (26)] transmembrane inhibitor of Ca 2ϩ -ATPase. It forms only 1 hydrogen bond with Ca 2ϩ -ATPase (27), and the substitution of Phe (Phe-256) that forms a part of the complementary surface largely reduces its affinity (28). Similarity with the binding pockets of steroid receptors (29) is also evident.…”

Section: Resultsmentioning

confidence: 74%

Crystal structure of the sodium-potassium pump (Na ⁺ ,K ⁺ -ATPase) with bound potassium and ouabain

Ogawa

Shinoda

Cornelius

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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315

274

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The sodium-potassium pump (Na ؉ ,K ؉ -ATPase) is responsible for establishing Na ؉ and K ؉ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na ؉ ,K ؉ -ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2⅐2K ؉ ⅐Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K ؉ , in marked contrast to previous models. Due to antagonism between ouabain and K ؉ , the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.cardiac glycosides ͉ crystallography

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“…In the presence of Tg, the binding cavity is slightly larger because of movements of some of the amino acid side chains to accommodate the bound inhibitor. Thus, the side chain of Phe-256, which from mutational evidence has been identified as a key residue in the binding of Tg (26,27), is rotated backward into the transmembrane region, to establish the correct position for stacking of the phenyl group against the central cycloheptane and ␥-lactone ring of the Tg tricyclic guaianolide nucleus. Furthermore, Gln-259 is rotated 180°outward to allow for accommodation of Tg inside the cavity, although Phe-834 undergoes a slight rotation in the opposite direction, combined with a small cytosolic shift, probably to realize a favorable interaction between the phenyl ring and the carbonyl group of the acetoxy group of Tg at O-10.…”

Section: Methodsmentioning

confidence: 99%

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

Winther

Liu

Sonntag

et al. 2010

Journal of Biological Chemistry

100

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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...

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Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Cited by 60 publications

References 44 publications

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Crystal structure of the sodium-potassium pump (Na ⁺ ,K ⁺ -ATPase) with bound potassium and ouabain

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

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Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Cited by 60 publications

References 44 publications

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Crystal structure of the sodium-potassium pump (Na + ,K + -ATPase) with bound potassium and ouabain

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

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Crystal structure of the sodium-potassium pump (Na ⁺ ,K ⁺ -ATPase) with bound potassium and ouabain