E2 Conformation Research Articles

Related flavonoids cause cooperative inhibition of the sarcoplasmic reticulum Ca2+ ATPase by multimode mechanisms

Flavonoids are group of plant-derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio-accumulate within humans and are considered to have beneficial health effects, including cancer chemoprotection. One mechanism proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca²⁺ ATPase. An investigation was undertaken with respect to the mechanism of inhibition for three flavonoids: quercetin, galangin and 3,6 dihydroxyflavone (3,6-DHF). Each inhibited the Ca²⁺ ATPase with K(i) values of 8.7, 10.3 and 5.4 μM, respectively, showing cooperative inhibition with n ~ 2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E₁ conformation and reduced [³²P]-ATP binding. However, both galangin and 3,6-DHF increased the affinity of Ca²⁺ for the ATPase by decreasing the Ca²⁺-dissociation rate constant, whereas quercetin had little effect. Ca²⁺-induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6-DHF and galangin (but not with quercetin), indicating that Ca²⁺-associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP-dependent phosphorylation and dephosphorylation, whereas 3,6-DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites: one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesize that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids.

Neutral Phospholipids Stimulate Na,K-ATPase Activity: A SPECIFIC LIPID-PROTEIN INTERACTION

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8-10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8-10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.

Phosphorylated Phospholamban Stabilizes a Distinct Compact Conformation of Sarco(endo)Plasmic Reticulum Ca-ATPase

Sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is inhibited by the 52 amino acid protein phospholamban (PLB). Inhibition of SERCA is relieved when PLB is phosphorylated by protein kinase A (PKA). To better understand the mechanism of this regulatory mechanism on SERCA structure, we designed a “2-color” SERCA labeled with donor and acceptor fluorescent proteins in the A- and N-domain. Intramolecular fluorescence resonance energy transfer (FRET) between these two domains was quantified as an index of SERCA conformation. Fluorescence lifetime distribution analysis (FLDA) showed that SERCA sampled three discrete conformations in the absence of PLB or in the presence of non-phosphorylatable PLB (S16A). Co-transfection of SERCA with phosphomimetic PLB (S16E) revealed an additional state characterized by a highly compact conformation. This unique state was only observed under conditions of high intracellular calcium. The data support the hypothesis that PLB remains bound to SERCA after phosphorylation by PKA, inducing the pump to assume a very compact E1 conformation.

Ion pumps as biological targets for decavanadate

The putative applications of poly-, oligo- and mono-oxometalates in biochemistry, biology, pharmacology and medicine are rapidly attracting interest. In particular, these compounds may act as potent ion pump inhibitors and have the potential to play a role in the treatment of e.g. ulcers, cancer and ischemic heart disease. However, the mechanism of action is not completely understood in most cases, and even remains largely unknown in other cases. In the present review we discuss the most recent insights into the interaction between mono- and polyoxometalate ions with ion pumps, with particular focus on the interaction of decavanadate with Ca(2+)-ATPase. We also compare the proposed mode of action with those of established ion pump inhibitors which are currently in therapeutic use. Of the 18 classes of compounds which are known to act as ion pump inhibitors, the complete mechanism of inhibition is only known for a handful. It has, however, been established that most ion pump inhibitors bind mainly to the E2 ion pump conformation within the membrane domain from the extracellular side and block the cation release. Polyoxometalates such as decavanadate, in contrast, interact with Ca(2+)-ATPase near the nucleotide binding site domain or at a pocket involving several cytoplasmic domains, and therefore need to cross through the membrane bilayer. In contrast to monomeric vanadate, which only binds to the E2 conformation, decavanadate binds to all protein conformations, i.e. E1, E1P, E2 and E2P. Moreover, the specific interaction of decavanadate with sarcoplasmic reticulum Ca(2+)-ATPase has been shown to be non-competitive with respect to ATP and induces protein cysteine oxidation with concomitant vanadium reduction which might explain the high inhibitory capacity of V10 (IC50 = 15 μM) which is quite similar to the majority of the established therapeutic drugs.

Structural Insights into the Conformation and Oligomerization of E2∼Ubiquitin Conjugates

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.

Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

Glutathionylation of cysteine 46 of the β1 subunit of the Na(+)-K(+) pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K(+)·P(i) configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na(+)-K(+) pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na(+)-K(+)-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na(+)-K(+)-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na(3) conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO(-)). Inhibition of Na(+)-K(+)-ATPase activity after exposure to ONOO(-) was greater when the enzyme had been in the E1Na(3) than the E2 conformation. We exposed myocytes to different extracellular K(+) concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K(+) concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO(-)-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na(+)-K(+) pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na(+)-K(+) pump.

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10nM Ang II for 5min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30mM NaCl and 3mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150mM NaCl and 3mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.

Crystal structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle

The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9Å resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.

Phospholamban does not Interact with E2 Intermediates of the Cardiac Calcium Pump Stabilized by Metal Fluorides

The phospholamban (PLB) monomer binds in the groove between M2, M4, and M9 of the cardiac calcium pump (SERCA2a) and inhibits enzyme activity. We previously demonstrated that cross-linking of PLB with engineered Cys-residues to this region is prevented by micromolar Ca, but is stimulated by the nucleotides ATP or ADP. We proposed that PLB inhibits the catalytic activity of the enzyme by stabilizing a unique, nucleotide bound, Ca-free intermediate of SERCA2a (E2•ATP). Stabilization of E2•ATP prevents Ca binding at the high affinity Ca binding sites and formation of E1, thus blocking the kinetic cycle. To further test our model, we have now checked if PLB interacts with other E2 conformations, notably, SERCA2a with the bound metal fluorides (MgF42-, AlF4-, or BeF3-), which mimic the E2•Pi, E2P, and E2-P states, respectively. Interactions between PLB and SERCA2a were examined by cross-linking of N30C-PLB to K328 of WT-SERCA2a at the cytoplasmic extension of M4 and of V49C-PLB to V89C-SERCA2a at the C-terminal end of M2. Insect cell microsomes co-expressing N30C-PLB/SERCA2a or V49C-PLB/V89C-SERCA2a were first incubated with the metal fluorides in Ca-free buffer, and then subjected to chemical cross-linking. All these metal fluorides prevented cross-linking of PLB to SERCA2a at both the cytoplasmic and transmembrane regions, suggesting that PLB does not interact with E2•Pi, E2P, and E2-P formed in the absence of nucleotide. Remarkably, however, the cross-linking of PLB to SERCA2a was completely restored upon addition of 3mM ATP, allowing formation of E2•ATP. These results strongly support our model in which E2•ATP is the single conformation binding PLB, and further suggest that the binding interaction between E2•ATP and PLB is very strong, sufficient to prevent formation of E2•Pi, E2P, or E2-P, even when stabilized by metal fluorides.

Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate

Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca 2 + -ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca 2+-ATPase, with the following IC 50 values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb 10), is the strongest P-type enzyme inhibitor, after decavanadate (V 10). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca 2 + -ATPase stoichiometry. Furthermore, V 10 binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H 2VO 4 2 − ; V 1) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate–Ca 2 + -ATPase, with a dissociation constant, k d of 1 μM − 1 . The interaction of decavanadate V 10O 28 6 − (V 10) with Ca 2 + -ATPase is prevented by the isostructural and isoelectronic decaniobate Nb 10O 28 6 − (Nb 10), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V 10 binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca 2 + -ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.

Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E2P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump

Nigella sativa seed oil was found to contain a modulator of Na,K-ATPase. Separation analyses combined with 1H NMR and GCMS identified the inhibitory fraction as a mixture of oleic and linoleic acids. These two fatty acids are specifically concentrated in several medicinal plant oils, and have particularly been implicated in decreasing high blood pressure. The ouabain binding site on Na,K-ATPase has also been implicated in blood pressure regulation. Thus, we aimed to determine how these two molecules modify pig kidney Na,K-ATPase. Oleic and linoleic acids did not modify reactions involving the E1 (Na+) conformations of the Na,K-ATPase. In contrast, K+ dependent reactions were strongly modified after treatment. Oleic and linoleic acids were found to stabilize a pump conformation that binds ouabain with high affinity, i.e., an ion free E2P form. Time-resolved binding assays using anthroylouabain, a fluorescent ouabain analog, revealed that the increased ouabain affinity is unique to oleic and linoleic acids, as compared with γ-linolenic acid, which decreased pump-mediated ATP hydrolysis but did not equally increase ouabain interaction with the pump. Thus, the dynamic changes in plasma levels of oleic and linoleic acids are important in the modulation of the sensitivity of the sodium pump to cardiac glycosides. Given the possible involvement of the cardiac glycoside binding site on Na,K-ATPase in the regulation of hypertension, we suggest oleic acid to be a specific chaperon that modulates interaction of cardiac glycosides with the sodium pump.

Dynamic lipid–protein stoichiometry on E1 and E2 conformations of the Na +/K +-ATPase

Annular lipid–protein stoichiometry in native pig kidney Na +/K +-ATPase preparation was studied by [ 125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na +/K +-ATPase in the E 1 state is less exposed to the lipids than in E 2, i.e. the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid–protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E 1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E 2 conformation.

Highly Specific, Conformationally-Dependent Cross-Linking of Lys27 of PLB to SERCA2a in Cardiac SR Vesicles from Humans

Phospholamban (PLB) inhibits SERCA2a, the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SR), by decreasing the apparent Ca2+ affinity of the enzyme. The mechanism of Ca2+ pump inhibition by PLB has been studied by our group using chemical cross-linking agents, which enable PLB-binding interactions with SERCA2a to be measured simultaneously with enzyme inhibition. Previously, cross-linking of canine PLB to SERCA2a was only attainable with Cys-scanning point mutants of PLB. For example, N27C of canine PLB cross-links exclusively to Lys328 of canine SERCA2a with heterobifunctional thiol-to-amine cross-linking agents after co-expression of the two proteins in Sf21 insect cells. Here, we show with SR vesicles prepared from human hearts, that PLB and SERCA2a are cross-linkable using DSG (disuccinimidyl glutarate), a 7.7 Å long, homobifunctional, amine-specific cross-linking agent. Cross-linking of human PLB to SERCA2a takes advantage of the unique Lys residue at position 27 of human PLB, making it susceptible to amine-specific cross-linkers without the need for mutagenesis. This was confirmed by testing SR vesicles prepared from both human and canine ventricles; DSG cross-linked human, but not canine, PLB to SERCA2a. Cross-linking of human PLB to SERCA2a was completely inhibited by either Ca2+ (Ki = 0.50 µM), or the Ca2+ pump inhibitor thapsigargin, but substantially augmented by ATP. This is the first demonstration that PLB binds exclusively to the E2 conformation of SERCA2a in SR vesicles, preferentially the state with bound nucleotide (E2·ATP), and not the state stabilized by thapsigargin (protonated E2). Importantly, similar results were obtained with SR vesicles prepared from both normal and failed human hearts, indicating that PLB-binding interactions with SERCA2a are unchanged in failing myocardium. Studies are in progress to demonstrate with human SR vesicles that Lys27 of PLB cross-links exclusively to Lys328 of SERCA2a.

Structure–function relationships in membrane segment 6 of the yeast plasma membrane Pma1 H+-ATPase

The crystal structures of the Ca2+- and H+-ATPases shed light into the membrane embedded domains involved in binding and ion translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure–function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20-fold increase in the IC50 for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the Km for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E2 conformation of the ATPase towards the E1 conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A) were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra et al., Biochim. Biophys. Acta 1768: 2383–2392, 2007) led to a dramatic increase in the apparent stoichiometry of H+ transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.

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

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.

Point mutations in Pma1 H+-ATPase of Saccharomyces cerevisiae: Influence on its expression and activity

Yeast Pma1 H+-ATPase is a key enzyme of cell metabolism generating electrochemical proton gradient across the plasma membrane, thus playing an important role in the maintenance of ion homeostasis in the cell. Using site-directed mutagenesis, we have previously replaced all 21 amino acid residues in the transmembrane segment M8 with Ala (Guerra et al. (2007) Biochim. Biophys. Acta, 1768, 2383-2392). In this work, we present new data on the role of these amino acid residues in the structure-function relationship in the enzyme and cell tolerance to heat shock. Mutations Q798A and I799A are lethal for cells regardless of expression of the enzyme in secretory vesicles or plasma membrane. The F796A mutation causes enzyme and cell sensitivity to heat shock when expressed in secretory vesicles. The I794A mutation increases temperature sensitivity of cells when the enzyme is expressed either in secretory vesicles or, to a lesser extent, in plasma membrane. The E803A mutation has no significant influence on the ATPase and cell sensitivity to heat shock; however, it causes a shift in the equilibrium between E1 and E2 conformations of the enzyme towards E1.

Interaction of sanguinarine and its dihydroderivative with the Na +/K +-ATPase. Complex view on the old problem

The effects of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on Na +/K +-ATPase were investigated using fluorescence spectroscopy. The results showed that the enzyme in E1 conformation can bind both charged and neutral (pseudobase) forms of SG with a K D = 7.2 ± 2.0 μM or 11.7 ± 0.9 μM, while the enzyme in E2 conformation binds only the charged form of SG with a K D = 4.7 ± 1.1 μM. Fluorescence quenching experiments suggest that the binding site in E1 conformation is located on the surface of the enzyme for both forms but the binding site in E2 conformation is protected from the solvent. We found no evidence for interaction of Na +/K +-ATPase and DHSG. This implies that any in vivo effect of SG attributable to inhibition of Na +/K +-ATPase can be considered only prior to SG → DHSG transformation in the gastro-intestinal tract and/or blood. Hence, Na +/K +-ATPase inhibition will be effective in SG topical application but its duration will be very limited in SG oral or parenteral administration.

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

Correlation of the virulence of CSFV with evolutionary patterns of E2 glycoprotein

Infection with classical swine fever virus (CSFV) is costly to the livestock industry. Several genomic sequences including velogenic strains and low virulent strains have been identified. However, the reasons for the virulence of the virus have remained unclear. Based on selective pattern and pressure strength, we classified all genes of CSFV into three classes. Among these genes, the E2 gene was under the strongest positive selection. Based on the analysis of 85 representative E2 gene sequences, the location and intensity of positive selection in CSFV isolates from group one and group two were identified. These results suggest that these two groups employ evolutionary difference. Moreover, the mutations, potentially driven by positive selection, can be correlated with the virulence of CSFV by altering the conformation and function of E2 and/or changing its glycosylation pattern. Based on these results, a model for the evolution of virulence of CSFV is proposed. The results provide a link between epidemiology and the gene function of CSFV, and may shed light on the molecular mechanism underlying the variation of CSFV virulence.

The Na/K-ATPase/Src Interaction and the E1/E2 Transition

In our previous studies, we have shown that Na+/K+-ATPase interacts directly with Src to form a signaling receptor complex. This complex is involved in control of basal Src activity and ouabain-induced signal transduction. The aim of this work is to demonstrate that Na+/K+-ATPase also regulates Src activity through its conformation-dependent domain movements during pumping cycles. It is known that the Na+/K+-ATPase transits from E1 to E2 conformation during an ion pumping cycle. Based on the known crystal structures of Na/K-ATPase, this conformational transition results in a 110 degree turn of the A domain of Na/K-ATPase where Src SH2 domain binds.