Acta Crystallographica Section D Biological Crystallography, 2002
ABSTRACT The 11 kDa C-terminal fragment of the proteolyticly matured surface antigen, PfMSP1, fro... more ABSTRACT The 11 kDa C-terminal fragment of the proteolyticly matured surface antigen, PfMSP1, from Plasmodium falciparum is a promising malaria vaccine candidate. The soluble recombinant form of this naturally occurring fragment has been crystallized as a complex with the Fab of a specific murine monoclonal antibody. The crystals belong to the space group P2(1), with unit-cell parameters a = 51.8, b = 213.5,c = 60.0 A, beta =101.0 degrees, and with Z = 4. Diffraction data have been measured to 2.9 A resolution and a preliminary model of the complex has been determined by molecular replacement. The epitope recognised by G17.12 is located on the N-terminal EGF-like domain of the antigen.
Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca 2+ -ATPases (SERCA), binds with h... more 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 intramembraneous 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 guianolide 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 (PSA) to cleave peptide conjugated prodrugs targeting SERCA of cancer cells (Denmeade et al. 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 which is sensitive to e.g. cholesterol.
Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureu... more Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.
Membrane proteins are largely dependent for their function on the phospholipids present in their ... more Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly depende...
This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire ... more This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains ...
Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of it... more 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 gr...
The sarco(endo)plasmic reticulum Ca 2 þ-ATPase (SERCA) couples ATP hydrolysis to transport of Ca ... more The sarco(endo)plasmic reticulum Ca 2 þ-ATPase (SERCA) couples ATP hydrolysis to transport of Ca 2 þ. This directed energy transfer requires cross-talk between the two Ca 2 þ 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 þ coordination at site II, and a consensus has been that E309Q only binds Ca 2 þ at site I. The crystal structure of E309Q in the presence of Ca 2 þ and an ATP analogue, however, reveals two occupied Ca 2 þ sites of a non-catalytic Ca 2 E1 state. Ca 2 þ is bound with micromolar affinity by both Ca 2 þ sites in E309Q, but without cooperativity. The Ca 2 þ-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 þ site II.
The present study characterizes the effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca... more The present study characterizes the effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca2+-ATPase membranes, prepared from sarcoplasmic reticulum (SR). At low concentrations C12E8 is incorporated into the membrane (less than or equal to 0.2 g/g protein), without any solubilization or appreciable morphological changes of freeze-fracture replica. Binding studies of C12E8 to ATPase membranes and SR lipid liposomes suggest that the major part of the detergent interacts with lipid. Solubilization of ATPase membranes occurs at a free concentration of C12E8 close to the critical micellar concentration (c.m.c); at low temperatures (2 degrees C) phospholipid is extracted somewhat more easily than ATPase. Electron-spin resonance (ESR) spectra of appropriate spin labels, incorporated into ATPase membranes, show that C12E8 strongly increases the fluidity of the lipid phase and the rotational diffusion of ATPase in the membrane. The effect of C12E8 on the ESR spectra is indistinguishable from that produced by a rise in temperature. Incorporation of C12E8 alters the functional properties of Ca2+-ATPase in a characteristic way: V is decreased and the modulatory effect of high ATP concentrations is reduced, in contrast to what occurs by a rise of temperature. The intrinsic fluorescence of the protein is increased, especially in the absence of Ca2+, suggesting that C12E8 modified in particular the E* form (Ca2+-depleted conformation) of the enzyme. Furthermore, stopped-flow data indicate that C12E8 strongly activates the E* to E transition, which may account for the effect of the detergent on ATP modulation during steady-state ATP hydrolysis. It is concluded that C12E8 perturbs ATPase turnover by direct interaction with the enzyme, rather than by an indirect effect exerted via a change in the lipid phase or protein aggregation.
We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca 2 þ-A... more We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca 2 þ-ATPase, stabilized by the inhibitor thapsigargin and the ATP analog AMPPCP. The structures allow us to describe the ATP binding site in a modulatory mode uncoupled from the Asp351 phosphorylation site. The Glu439 side chain interacts with AMPPCP via an Mg 2 þ ion in accordance with previous Fe 2 þ-cleavage studies implicating this residue in the ATPase cycle and in magnesium binding. Functional data on Ca 2 þ mediated activation indicate that the crystallized state represents an initial stage of ATP modulated deprotonation of E2, preceding the binding of Ca 2 þ ions in the membrane from the cytoplasmic side. We propose a mechanism of Ca 2 þ activation of phosphorylation leading directly from the compact E2-ATP form to the Ca 2 E1-ATP state. In addition, a role of Glu439 in ATP modulation of other steps of the functional cycle is suggested.
Proceedings of the National Academy of Sciences, 1985
The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state ... more The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state of membrane-bound and soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum have been examined at pH 8.0, 2 degrees C. The transition from ADP-sensitive to ADP-insensitive phosphoenzyme occurs in the soluble monomer as well as in membranous Ca2+-ATPase and is accompanied by an increase in fluorescence from 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine diphosphate bound to the catalytic site and change in tryptic cleavage pattern. A decrease of Ca2+ affinity occurs simultaneously with the fluorescence rise, suggesting a single-step mechanism for energy transfer between the catalytic site and the Ca2+ transport sites. This is in accordance with the tryptic degradation pattern that suggests proximity between the phosphorylation site and Ca2+ transport sites on the peptide. The structural changes occurring in the soluble monomeric Ca2+-ATPase show that a single polypept...
The sarcoplasmic reticulum Ca 21-ATPase, a P-type ATPase, has a critical role in muscle function ... more The sarcoplasmic reticulum Ca 21-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca 21-ATPase, representing the phosphoenzyme intermediates associated with Ca 21 binding, Ca 21 translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca 21-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca 21 release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.
The recently determined crystal structure of the sarcoplasmic reticulum Ca 2+-ATPase (SERCA1a) wi... more The recently determined crystal structure of the sarcoplasmic reticulum Ca 2+-ATPase (SERCA1a) with a bound ATP analogue (AMPPCP) reveals a compact state, similar to that found in the presence of ADP and aluminium fluoride. However, although the two Ca 2+-binding sites in the membrane are known to be occluded in the latter state, in the AMPPCP-bound state the Ca 2+-binding sites are not occluded under conditions with physiological levels of Mg 2+ and Ca 2+. It has been shown that the high concentration (10 mM) of Ca 2+ used for crystallization (in the presence of Mg 2+) may be responsible for the discrepancy. To determine whether Ca 2+ competes with Mg 2+ and affects the nucleotide-binding site, we have subjected the AMPPCP and ADP:AlF 4 − bound forms to crystallographic analysis by anomalous difference Fourier maps, and we have compared AMPPCP-bound forms crystallized in the absence or in the presence of Mg 2+. We found that Ca 2+ rather than Mg 2+ binds together with AMPPCP at the phosphorylation site, whereas the ADP:AlF 4 − complex is associated with two magnesium ions. These results address the structure of the phosphorylation site before and during phosphoryl transfer. The bound CaAMPPCP nucleotide may correspond to the activated pre-complex, formed immediately before phosphorylation, whereas the Mg 2 ADP:AlF 4 − transition state complex reflects the preference for Mg 2+ in catalysis. In addition, we have identified a phosphatidylcholine lipid molecule bound at the cytosol-membrane interface.
K ؉ plays an important role for the function of the sarco(endo)plasmic reticulum Ca 2؉-ATPase (SE... more K ؉ plays an important role for the function of the sarco(endo)plasmic reticulum Ca 2؉-ATPase (SERCA), but its binding site within the molecule has remained unidentified. We have located the binding site for a K ؉ ion in the P-domain by means of x-ray crystallography using crystals prepared in the presence of the K ؉ congener Rb ؉. Backbone carbonyls from the loop containing residues 711-715 together with the side chain of Glu 732 define the K ؉ /Rb ؉ site in the Ca 2؉-ATPase conformation with bound Ca 2؉ , ADP, and AlF 4 ؊. Functional analysis of Ca 2؉-ATPase mutants with alterations to Glu 732 shows that this site is indeed important for the stimulatory effect of K ؉ on the dephosphorylation rate. Comparison with the Ca 2؉-ATPase in a dephosphorylated E2 conformation suggests that the K ؉ site is involved in the correct movement and positioning of the A-domain during translocation and dephosphorylation. The Ca 2ϩ-ATPase of sarco(endo)plasmic reticulum (SERCA) 1 is a P-type ATPase (1) that actively transports Ca 2ϩ
Limited proteolysis by proteinase K of rabbit SERCA1 Ca 2؉-ATPase generates a number of fragments... more Limited proteolysis by proteinase K of rabbit SERCA1 Ca 2؉-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca 2؉ , as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca 2؉ as well as from labeling with 45 Ca 2؉ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the Nterminal side, did not bind Ca 2؉ when submitted to the same experiments. Two cluster mutants of Ca 2؉-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca 2؉-ATPase activity. Region 808-818 is thus essential for both Ca 2؉ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H ؉ , K ؉-ATPase (Swarts, H. G. P.,
We have characterized a putative Ca 2؉-ATPase from the pathogenic bacterium Listeria monocytogene... more 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 ϳ80 M) and a high pH optimum (pH ϳ9). 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.
Acta Crystallographica Section D Biological Crystallography, 2002
ABSTRACT The 11 kDa C-terminal fragment of the proteolyticly matured surface antigen, PfMSP1, fro... more ABSTRACT The 11 kDa C-terminal fragment of the proteolyticly matured surface antigen, PfMSP1, from Plasmodium falciparum is a promising malaria vaccine candidate. The soluble recombinant form of this naturally occurring fragment has been crystallized as a complex with the Fab of a specific murine monoclonal antibody. The crystals belong to the space group P2(1), with unit-cell parameters a = 51.8, b = 213.5,c = 60.0 A, beta =101.0 degrees, and with Z = 4. Diffraction data have been measured to 2.9 A resolution and a preliminary model of the complex has been determined by molecular replacement. The epitope recognised by G17.12 is located on the N-terminal EGF-like domain of the antigen.
Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca 2+ -ATPases (SERCA), binds with h... more 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 intramembraneous 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 guianolide 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 (PSA) to cleave peptide conjugated prodrugs targeting SERCA of cancer cells (Denmeade et al. 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 which is sensitive to e.g. cholesterol.
Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureu... more Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.
Membrane proteins are largely dependent for their function on the phospholipids present in their ... more Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly depende...
This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire ... more This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains ...
Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of it... more 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 gr...
The sarco(endo)plasmic reticulum Ca 2 þ-ATPase (SERCA) couples ATP hydrolysis to transport of Ca ... more The sarco(endo)plasmic reticulum Ca 2 þ-ATPase (SERCA) couples ATP hydrolysis to transport of Ca 2 þ. This directed energy transfer requires cross-talk between the two Ca 2 þ 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 þ coordination at site II, and a consensus has been that E309Q only binds Ca 2 þ at site I. The crystal structure of E309Q in the presence of Ca 2 þ and an ATP analogue, however, reveals two occupied Ca 2 þ sites of a non-catalytic Ca 2 E1 state. Ca 2 þ is bound with micromolar affinity by both Ca 2 þ sites in E309Q, but without cooperativity. The Ca 2 þ-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 þ site II.
The present study characterizes the effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca... more The present study characterizes the effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca2+-ATPase membranes, prepared from sarcoplasmic reticulum (SR). At low concentrations C12E8 is incorporated into the membrane (less than or equal to 0.2 g/g protein), without any solubilization or appreciable morphological changes of freeze-fracture replica. Binding studies of C12E8 to ATPase membranes and SR lipid liposomes suggest that the major part of the detergent interacts with lipid. Solubilization of ATPase membranes occurs at a free concentration of C12E8 close to the critical micellar concentration (c.m.c); at low temperatures (2 degrees C) phospholipid is extracted somewhat more easily than ATPase. Electron-spin resonance (ESR) spectra of appropriate spin labels, incorporated into ATPase membranes, show that C12E8 strongly increases the fluidity of the lipid phase and the rotational diffusion of ATPase in the membrane. The effect of C12E8 on the ESR spectra is indistinguishable from that produced by a rise in temperature. Incorporation of C12E8 alters the functional properties of Ca2+-ATPase in a characteristic way: V is decreased and the modulatory effect of high ATP concentrations is reduced, in contrast to what occurs by a rise of temperature. The intrinsic fluorescence of the protein is increased, especially in the absence of Ca2+, suggesting that C12E8 modified in particular the E* form (Ca2+-depleted conformation) of the enzyme. Furthermore, stopped-flow data indicate that C12E8 strongly activates the E* to E transition, which may account for the effect of the detergent on ATP modulation during steady-state ATP hydrolysis. It is concluded that C12E8 perturbs ATPase turnover by direct interaction with the enzyme, rather than by an indirect effect exerted via a change in the lipid phase or protein aggregation.
We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca 2 þ-A... more We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca 2 þ-ATPase, stabilized by the inhibitor thapsigargin and the ATP analog AMPPCP. The structures allow us to describe the ATP binding site in a modulatory mode uncoupled from the Asp351 phosphorylation site. The Glu439 side chain interacts with AMPPCP via an Mg 2 þ ion in accordance with previous Fe 2 þ-cleavage studies implicating this residue in the ATPase cycle and in magnesium binding. Functional data on Ca 2 þ mediated activation indicate that the crystallized state represents an initial stage of ATP modulated deprotonation of E2, preceding the binding of Ca 2 þ ions in the membrane from the cytoplasmic side. We propose a mechanism of Ca 2 þ activation of phosphorylation leading directly from the compact E2-ATP form to the Ca 2 E1-ATP state. In addition, a role of Glu439 in ATP modulation of other steps of the functional cycle is suggested.
Proceedings of the National Academy of Sciences, 1985
The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state ... more The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state of membrane-bound and soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum have been examined at pH 8.0, 2 degrees C. The transition from ADP-sensitive to ADP-insensitive phosphoenzyme occurs in the soluble monomer as well as in membranous Ca2+-ATPase and is accompanied by an increase in fluorescence from 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine diphosphate bound to the catalytic site and change in tryptic cleavage pattern. A decrease of Ca2+ affinity occurs simultaneously with the fluorescence rise, suggesting a single-step mechanism for energy transfer between the catalytic site and the Ca2+ transport sites. This is in accordance with the tryptic degradation pattern that suggests proximity between the phosphorylation site and Ca2+ transport sites on the peptide. The structural changes occurring in the soluble monomeric Ca2+-ATPase show that a single polypept...
The sarcoplasmic reticulum Ca 21-ATPase, a P-type ATPase, has a critical role in muscle function ... more The sarcoplasmic reticulum Ca 21-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca 21-ATPase, representing the phosphoenzyme intermediates associated with Ca 21 binding, Ca 21 translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca 21-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca 21 release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.
The recently determined crystal structure of the sarcoplasmic reticulum Ca 2+-ATPase (SERCA1a) wi... more The recently determined crystal structure of the sarcoplasmic reticulum Ca 2+-ATPase (SERCA1a) with a bound ATP analogue (AMPPCP) reveals a compact state, similar to that found in the presence of ADP and aluminium fluoride. However, although the two Ca 2+-binding sites in the membrane are known to be occluded in the latter state, in the AMPPCP-bound state the Ca 2+-binding sites are not occluded under conditions with physiological levels of Mg 2+ and Ca 2+. It has been shown that the high concentration (10 mM) of Ca 2+ used for crystallization (in the presence of Mg 2+) may be responsible for the discrepancy. To determine whether Ca 2+ competes with Mg 2+ and affects the nucleotide-binding site, we have subjected the AMPPCP and ADP:AlF 4 − bound forms to crystallographic analysis by anomalous difference Fourier maps, and we have compared AMPPCP-bound forms crystallized in the absence or in the presence of Mg 2+. We found that Ca 2+ rather than Mg 2+ binds together with AMPPCP at the phosphorylation site, whereas the ADP:AlF 4 − complex is associated with two magnesium ions. These results address the structure of the phosphorylation site before and during phosphoryl transfer. The bound CaAMPPCP nucleotide may correspond to the activated pre-complex, formed immediately before phosphorylation, whereas the Mg 2 ADP:AlF 4 − transition state complex reflects the preference for Mg 2+ in catalysis. In addition, we have identified a phosphatidylcholine lipid molecule bound at the cytosol-membrane interface.
K ؉ plays an important role for the function of the sarco(endo)plasmic reticulum Ca 2؉-ATPase (SE... more K ؉ plays an important role for the function of the sarco(endo)plasmic reticulum Ca 2؉-ATPase (SERCA), but its binding site within the molecule has remained unidentified. We have located the binding site for a K ؉ ion in the P-domain by means of x-ray crystallography using crystals prepared in the presence of the K ؉ congener Rb ؉. Backbone carbonyls from the loop containing residues 711-715 together with the side chain of Glu 732 define the K ؉ /Rb ؉ site in the Ca 2؉-ATPase conformation with bound Ca 2؉ , ADP, and AlF 4 ؊. Functional analysis of Ca 2؉-ATPase mutants with alterations to Glu 732 shows that this site is indeed important for the stimulatory effect of K ؉ on the dephosphorylation rate. Comparison with the Ca 2؉-ATPase in a dephosphorylated E2 conformation suggests that the K ؉ site is involved in the correct movement and positioning of the A-domain during translocation and dephosphorylation. The Ca 2ϩ-ATPase of sarco(endo)plasmic reticulum (SERCA) 1 is a P-type ATPase (1) that actively transports Ca 2ϩ
Limited proteolysis by proteinase K of rabbit SERCA1 Ca 2؉-ATPase generates a number of fragments... more Limited proteolysis by proteinase K of rabbit SERCA1 Ca 2؉-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca 2؉ , as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca 2؉ as well as from labeling with 45 Ca 2؉ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the Nterminal side, did not bind Ca 2؉ when submitted to the same experiments. Two cluster mutants of Ca 2؉-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca 2؉-ATPase activity. Region 808-818 is thus essential for both Ca 2؉ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H ؉ , K ؉-ATPase (Swarts, H. G. P.,
We have characterized a putative Ca 2؉-ATPase from the pathogenic bacterium Listeria monocytogene... more 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 ϳ80 M) and a high pH optimum (pH ϳ9). 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.
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Papers by Jesper Møller