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1.
Prog Chem Org Nat Prod ; 115: 59-114, 2021.
Article in English | MEDLINE | ID: mdl-33797641

ABSTRACT

Thapsigargin, the first representative of the hexaoxygenated guaianolides, was isolated 40 years ago in order to understand the skin-irritant principles of the resin of the umbelliferous plant Thapsia garganica. The pronounced cytotoxicity of thapsigargin is caused by highly selective inhibition of the intracellular sarco-endoplasmic Ca2+-ATPase (SERCA) situated on the membrane of the endo- or sarcoplasmic reticulum. Thapsigargin is selective to the SERCA pump and to a minor extent the secretory pathway Ca2+/Mn2+ ATPase (SPCA) pump. Thapsigargin has become a tool for investigation of the importance of SERCA in intracellular calcium homeostasis. In addition, complex formation of thapsigargin with SERCA has enabled crystallization and structure determination of calcium-free states by X-ray crystallography. These results led to descriptions of the mechanism of action and kinetic properties of SERCA and other ATPases. Inhibition of SERCA depletes Ca2+ from the sarco- and endoplasmic reticulum provoking the unfolded protein response, and thereby has enabled new studies on the mechanism of cell death. Development of protocols for selective transformation of thapsigargin disclosed the chemistry and facilitated total synthesis of the molecule. Conversion of trilobolide into thapsigargin offered an economically feasible sustainable source of thapsigargin, which enables a future drug production. Principles for prodrug development were used by conjugating a payload derived from thapsigargin with a hydrophilic peptide selectively cleaved by proteases in the tumor. Mipsagargin was developed in order to obtain a drug for treatment of cancer diseases characterized by the presence of prostate specific membrane antigen (PSMA) in the neovascular tissue of the tumors. Even though mipsagargin showed interesting clinical effects the results did not encourage funding and consequently the attempt to register the drug has been abandoned. In spite of this disappointing fact, the research performed to develop the drug has resulted in important scientific discoveries concerning the chemistry, biosynthesis and biochemistry of sesquiterpene lactones, the mechanism of action of ATPases including SERCA, mechanisms for cell death caused by the unfolded protein response, and the use of prodrugs for cancer-targeting cytotoxins. The presence of toxins in only some species belonging to Thapsia also led to a major revision of the taxonomy of the genus.


Subject(s)
Biological Products , Cell Death , Drug Development , Male , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Thapsigargin/pharmacology
2.
IUCrJ ; 7(Pt 6): 1092-1101, 2020 Nov 01.
Article in English | MEDLINE | ID: mdl-33209320

ABSTRACT

The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein-vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein-vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.

3.
Cell Commun Signal ; 18(1): 12, 2020 Jan 27.
Article in English | MEDLINE | ID: mdl-31987044

ABSTRACT

BACKGROUND: Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive. METHODS: To systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling. RESULTS: In both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg. CONCLUSIONS: Together, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress. Video Abstract.


Subject(s)
Endoplasmic Reticulum Stress , Endoplasmic Reticulum/metabolism , Microtubule-Associated Proteins/metabolism , Thapsigargin/metabolism , Unfolded Protein Response , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolism , Autophagy , Caspase 8/genetics , Caspase 8/metabolism , Cell Line, Tumor , Endoplasmic Reticulum/genetics , Humans , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Microtubule-Associated Proteins/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , X-Box Binding Protein 1/genetics , X-Box Binding Protein 1/metabolism
4.
Acta Crystallogr D Struct Biol ; 74(Pt 12): 1208-1218, 2018 Dec 01.
Article in English | MEDLINE | ID: mdl-30605135

ABSTRACT

Neutron macromolecular crystallography (NMX) has the potential to provide the experimental input to address unresolved aspects of transport mechanisms and protonation in membrane proteins. However, despite this clear scientific motivation, the practical challenges of obtaining crystals that are large enough to make NMX feasible have so far been prohibitive. Here, the potential impact on feasibility of a more powerful neutron source is reviewed and a strategy for obtaining larger crystals is formulated, exemplified by the calcium-transporting ATPase SERCA1. The challenges encountered at the various steps in the process from crystal nucleation and growth to crystal mounting are explored, and it is demonstrated that NMX-compatible membrane-protein crystals can indeed be obtained.


Subject(s)
Crystallization/methods , Neutron Diffraction/methods , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Animals , Crystallography, X-Ray/methods , Models, Molecular , Protein Conformation , Rabbits
5.
J Biol Chem ; 292(17): 6938-6951, 2017 04 28.
Article in English | MEDLINE | ID: mdl-28264934

ABSTRACT

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.


Subject(s)
Calcium-Transporting ATPases/antagonists & inhibitors , Calcium-Transporting ATPases/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Thapsigargin/chemistry , Animals , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Calcium/chemistry , Cholesterol/chemistry , Drug Design , Female , Humans , Hydroquinones/chemistry , Indoles/chemistry , Linoleic Acids/chemistry , Liposomes/chemistry , Male , Mutagenesis, Site-Directed , Oleic Acids/chemistry , Phosphatidic Acids/chemistry , Prostatic Neoplasms/drug therapy , Protein Binding , Protein Conformation , Rabbits , Recombinant Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Sphingomyelins/chemistry , Structure-Activity Relationship
6.
PLoS One ; 11(9): e0163260, 2016.
Article in English | MEDLINE | ID: mdl-27644036

ABSTRACT

P-type ATPases catalyze the active transport of cations and phospholipids across biological membranes. Members of this large family are involved in a range of fundamental cellular processes. To date, a substantial number of P-type ATPase inhibitors have been characterized, some of which are used as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material.


Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Curcumin/analogs & derivatives , Enzyme Inhibitors/pharmacology , Saccharomyces cerevisiae/enzymology , Spinacia oleracea/enzymology , Curcumin/pharmacology , Diarylheptanoids
7.
Structure ; 24(4): 617-623, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-27050689

ABSTRACT

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.


Subject(s)
Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Vanadates/pharmacology , Animals , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Phosphorylation , Protein Conformation , Rabbits
10.
Mol Membr Biol ; 32(3): 75-87, 2015.
Article in English | MEDLINE | ID: mdl-26260074

ABSTRACT

Like other integral membrane proteins, the activity of the Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA) is regulated by the membrane environment. Cholesterol is present in the endoplasmic reticulum membrane at low levels, and it has the potential to affect SERCA activity both through direct, specific interaction with the protein or through indirect interaction through changes of the overall membrane properties. There are experimental data arguing for both modes of action for a cholesterol-mediated regulation of SERCA. In the current study, coarse-grained molecular dynamics simulations are used to address how a mixed lipid-cholesterol membrane interacts with SERCA. Candidates for direct regulatory sites with specific cholesterol binding modes are extracted from the simulations. The binding pocket for thapsigargin, a nanomolar inhibitor of SERCA, has been suggested as a cholesterol binding site. However, the thapsigargin binding pocket displayed very little cholesterol occupation in the simulations. Neither did atomistic simulations of cholesterol in the thapsigargin binding pocket support any specific interaction. The current study points to a non-specific effect of cholesterol on SERCA activity, and offers an alternative interpretation of the experimental results used to argue for a specific effect.


Subject(s)
Cholesterol/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Animals , Humans , Protein Binding , Thapsigargin/metabolism
11.
Steroids ; 97: 2-7, 2015 May.
Article in English | MEDLINE | ID: mdl-25065587

ABSTRACT

The skin irritating principle from Thapsia garganica was isolated, named thapsigargin and the structure elucidated. By inhibiting the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) thapsigargin provokes apoptosis in almost all cells. By conjugating thapsigargin to peptides, which are only substrates for either prostate specific antigen (PSA) or prostate specific membrane antigen (PSMA) prodrugs were created, which selectively affect prostate cancer cells or neovascular tissue in tumors. One of the prodrug is currently tested in clinical phase II. The prodrug under clinical trial has been named mipsagargin.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apiaceae/chemistry , Enzyme Inhibitors/pharmacology , Prodrugs/pharmacology , Prostatic Neoplasms/drug therapy , Soft Tissue Neoplasms/drug therapy , Thapsigargin/pharmacology , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Cell Proliferation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Humans , Male , Mice , Molecular Structure , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Prodrugs/chemistry , Prodrugs/isolation & purification , Prostatic Neoplasms/pathology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Soft Tissue Neoplasms/pathology , Thapsigargin/chemistry , Thapsigargin/isolation & purification
12.
J Biol Chem ; 289(49): 33850-61, 2014 Dec 05.
Article in English | MEDLINE | ID: mdl-25301946

ABSTRACT

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.


Subject(s)
Cysteine/chemistry , Muscle Proteins/chemistry , Muscle, Skeletal/metabolism , Oleic Acid/chemistry , Palmitic Acid/chemistry , Phenylalanine/chemistry , Protein Processing, Post-Translational , Proteolipids/chemistry , Amino Acid Sequence , Animals , Biological Evolution , Crystallography, X-Ray , Cysteine/metabolism , Gene Expression , Hydroxylamine/chemistry , Kinetics , Lipoylation , Models, Molecular , Molecular Sequence Data , Muscle Proteins/classification , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/chemistry , Oleic Acid/metabolism , Palmitic Acid/metabolism , Phenylalanine/metabolism , Phylogeny , Proteolipids/classification , Proteolipids/genetics , Proteolipids/metabolism , Rabbits , Sarcoplasmic Reticulum , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sequence Alignment , Species Specificity , Swine , Thermodynamics
13.
EMBO J ; 32(24): 3231-43, 2013 Dec 11.
Article in English | MEDLINE | ID: mdl-24270570

ABSTRACT

The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) couples ATP hydrolysis to transport of Ca(2+). This directed energy transfer requires cross-talk between the two Ca(2+) sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu(309) contributes to Ca(2+) coordination at site II, and a consensus has been that E309Q only binds Ca(2+) at site I. The crystal structure of E309Q in the presence of Ca(2+) and an ATP analogue, however, reveals two occupied Ca(2+) sites of a non-catalytic Ca2E1 state. Ca(2+) is bound with micromolar affinity by both Ca(2+) sites in E309Q, but without cooperativity. The Ca(2+)-bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an 'up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca(2+) site II.


Subject(s)
Calcium/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Adenosine Triphosphate/metabolism , Catalysis , Crystallography, X-Ray , Models, Molecular , Phosphorylation , Protein Conformation , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
14.
J Biol Chem ; 288(15): 10759-65, 2013 Apr 12.
Article in English | MEDLINE | ID: mdl-23400778

ABSTRACT

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.


Subject(s)
Calcium/metabolism , Protons , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Binding Sites , Calcium/chemistry , Cytosol/chemistry , Cytosol/metabolism , Humans , Ion Transport/physiology , Molecular Dynamics Simulation , Rabbits , Sarcoplasmic Reticulum/chemistry , Sarcoplasmic Reticulum/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics
15.
Article in English | MEDLINE | ID: mdl-21636921

ABSTRACT

Ca(2+)-ATPases are ATP-driven membrane pumps that are responsible for the transport of Ca(2+) ions across the membrane. The Listeria monocytogenes Ca(2+)-ATPase LMCA1 has been crystallized in the Ca(2+)-free state stabilized by AlF(4)(-), representing an occluded E2-P(i)-like state. The crystals belonged to space group P2(1)2(1)2 and a complete data set extending to 4.3 Šresolution was collected. A molecular-replacement solution was obtained, revealing type I packing of the molecules in the crystal. Unbiased electron-density features were observed for AlF(4)(-) and for shifts of the helices, which were indicative of a reliable structure determination.


Subject(s)
Calcium-Transporting ATPases/chemistry , Listeria monocytogenes/enzymology , Calcium-Transporting ATPases/isolation & purification , Crystallization , Crystallography, X-Ray , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Tertiary
16.
Nat Commun ; 2: 304, 2011.
Article in English | MEDLINE | ID: mdl-21556058

ABSTRACT

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.


Subject(s)
Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Models, Molecular , Molecular Conformation , Molecular Dynamics Simulation , Phosphorylation , Protein Structure, Secondary , Protein Structure, Tertiary
17.
J Biol Chem ; 286(2): 1609-17, 2011 Jan 14.
Article in English | MEDLINE | ID: mdl-21047776

ABSTRACT

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) ∼80 µm) and a high pH optimum (pH ∼9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.


Subject(s)
Calcium/metabolism , Listeria monocytogenes/enzymology , Listeria monocytogenes/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Adenosine Triphosphate/metabolism , Alkalies/metabolism , Amino Acid Sequence , Animals , Binding Sites/physiology , Biological Transport, Active/physiology , Electrochemical Techniques , Hydrogen-Ion Concentration , Molecular Sequence Data , Muscle, Skeletal/enzymology , Protein Structure, Tertiary , Rabbits , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
18.
Methods Mol Biol ; 654: 119-40, 2010.
Article in English | MEDLINE | ID: mdl-20665264

ABSTRACT

Improvements in the handling of membrane proteins for crystallization, combined with better synchrotron sources for X-ray diffraction analysis, are leading to clarification of the structural details of an ever increasing number of membrane transporters and receptors. Here we describe how this development has resulted in the elucidation at atomic resolution of a large number of structures of the sarcoplasmic Ca(2+)-ATPase (SERCA1a) present in skeletal muscle. The structures corresponding to the various intermediary states have been obtained after stabilization with structural analogues of ATP and of metal fluorides as mimicks of inorganic phosphate. From these results it is possible, in accordance with previous biochemical and molecular biology data, to give a detailed structural description of both ATP hydrolysis and Ca(2+) transport through the membrane, to serve as the starting point for a fuller understanding of the pump mechanism and, in future studies, on the regulatory role of this ubiquitous intracellular Ca(2+)-ATPase in cellular Ca(2+) metabolism in normal and pathological conditions.


Subject(s)
Calcium-Transporting ATPases/chemistry , Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/metabolism , X-Ray Diffraction/methods , Biological Transport , Calcium/metabolism , Fluorides/metabolism , Models, Biological , Protein Structure, Secondary
19.
Bioorg Med Chem ; 18(15): 5634-46, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20615710

ABSTRACT

Removal of each of the acyl groups of thapsigargin at O-3, O-8 and O-10 significant reduces the affinity of the inhibitors to the SERCA1a pump. Replacement of the acyl groups at O-3 and O-10 with flexible residues could be performed with only a minor decrease of the affinity, whereas introduction of voluminous stiff residues caused dramatic reduction of the affinity. The results can be rationalized on the basis of the interactions of thapsigargin with the SERCA1a pump as revealed from 3D X-ray structural models of thapsigargin bound to the SERCA1a. In conclusion the results confirm and elaborate the previously suggested pharmocophore model of thapsigargin.


Subject(s)
Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Thapsigargin/chemistry , Animals , Binding Sites , Computer Simulation , Rabbits , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Thapsigargin/pharmacology
20.
J Biol Chem ; 284(20): 13513-13518, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19289472

ABSTRACT

We have determined the structure of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in an E2.P(i)-like form stabilized as a complex with MgF(4)(2-), an ATP analog, adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5A 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 Ca(2+)-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 Phe(93) 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.


Subject(s)
Cations, Divalent/chemistry , Indoles/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Animals , Binding Sites/physiology , Calcium/chemistry , Calcium/metabolism , Cations, Divalent/metabolism , Crystallography, X-Ray , Fluorides/chemistry , Fluorides/metabolism , Magnesium Compounds/chemistry , Magnesium Compounds/metabolism , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiology , Rabbits , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
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