Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p.

The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase.

Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

Antenna-based optical imaging of single Ca2+ transmembrane proteins in liquids.

Understanding the diversity of biological processes requires methods that can address single proteins in their natural environment and provide insights into structural and functional properties, as well as the local distribution of each individual protein. We use an optical antenna in the form of a single gold nanoparticle to localize incident laser radiation to 50 nm, significantly smaller than the diffraction limit of light. Our approach enables us to optically resolve individual plasma-membrane-bound Ca2+ pumps (PMCA4) immersed in aqueous environments and to determine the distribution of interprotein distances. We are able to correlate the protein maps with local topology. Improved antenna geometries will make it possible to resolve, identify, and probe single membrane proteins in live cells with true protein resolution of 5-10 nm.

Phosphatase actions at the site of appositional mineralization in bisphosphonate-affected bones of the rat.

Tissue-nonspecific alkaline phosphatase (TNSALP) and Ca-ATPase are known to play roles in bone mineralization, but how these enzymes contribute to appositional mineralization has been illusive. Here we examined the active sites of these enzymes in appositional mineralization using the bones of young rats being administered with 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 5 days. The doses of HEBP totally abolished mineralization of newly formed bone matrix except in matrix vesicles (MVs), and hence allowed precise localization of MVs and phosphatase reactions within non-mineralized extracellular matrix. Intense TNSALP and ATPase reactions were confirmed along the limited portions of osteoblast membranes where intimate cell-cell contacts were maintained. Diffuse reactions of these enzymes were throughout the osteoid implicating efflux of TNSALP and ATPase molecules into extracellular matrix from the osteoblast membranes. Phosphatase reactions associated with MVs varied both in intensity and location among the individual vesicles; newly formed MVs were almost free of reactions but appeared to gain those activities later in the osteoid. These data suggest that TNSALP and ATPase are released from the osteoblast membrane and later integrated into MVs within the osteoid. The osteoblasts may thus regulate appositional mineralization of bone from a distance at least in part by providing phosphatases via MVs.

Increased calcium deposits and decreased Ca2+-ATPase in right ventricular myocardium of ascitic broiler chickens.

Right ventricular hypertrophy and failure is an important step in the development of ascites syndrome (AS) in broiler chickens. Cytoplasmic calcium concentration is a major regulator of cardiac contractile function and various physiological processes in cardiac muscle cells. The purpose of this study was to measure the right ventricular pressure and investigate the precise ultrastructural location of Ca(2+) and Ca(2+)-ATPase in the right ventricular myocardium of chickens with AS induced by low ambient temperature. The results showed that the right ventricular diastolic pressure of ascitic broilers was significantly higher than that of control broilers (P < 0.01), and the maximum change ratio of right intraventricular pressure (RV +/- dp/dt(max)) of ascitic broilers was significantly lower than that of the controls (P < 0.01). Extensively increased calcium deposits were observed in the right ventricular myocardium of ascitic broilers, whereas in the age-matched control broilers, calcium deposits were much less. The Ca(2+)-ATPase reactive products were obviously found on the sarcoplasmic reticulum and mitochondrial membrane of the control right ventricular myocardium, but rarely observed in the ascitic broilers. The data suggest that in ascitic broilers there is the right ventricular diastolic dysfunction, in which the overload of intracellular calcium and the decreased Ca(2+)-ATPase activity might be the important factors.

Interactions between Ca2+-ATPase and the pentameric form of phospholamban in two-dimensional co-crystals.

Phospholamban (PLB) physically interacts with Ca(2+)-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca(2+)-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca(2+)-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22(1)2(1) symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca(2+)-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca(2+)-ATPase.

Structural role of countertransport revealed in Ca(2+) pump crystal structure in the absence of Ca(2+).

Ca(2+)-ATPase of sarcoplasmic reticulum is an ATP-powered Ca(2+) pump but also a H(+) pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-A-resolution crystal structure of the Ca(2+)-ATPase in the absence of Ca(2+) stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca(2+) binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca(2+)-release. They suggest that H(+) countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca(2+)-binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.

Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies.

Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca(2+)-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. PLB interacts with and regulates the cardiac Ca(2+)-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca(2+)-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca(2+)-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies.

Normal mode-based fitting of atomic structure into electron density maps: application to sarcoplasmic reticulum Ca-ATPase.

A method for the flexible docking of high-resolution atomic structures into lower resolution densities derived from electron microscopy is presented. The atomic structure is deformed by an iterative process using combinations of normal modes to obtain the best fit of the electron microscopical density. The quality of the computed structures has been evaluated by several techniques borrowed from crystallography. Two atomic structures of the SERCA1 Ca-ATPase corresponding to different conformations were used as a starting point to fit the electron density corresponding to a different conformation. The fitted models have been compared to published models obtained by rigid domain docking, and their relation to the known crystallographic structures are explored by normal mode analysis. We find that only a few number of modes contribute significantly to the transition. The associated motions involve almost exclusively rotation and translation of the cytoplasmic domains as well as displacement of cytoplasmic loops. We suggest that the movements of the cytoplasmic domains are driven by the conformational change that occurs between nonphosphorylated and phosphorylated intermediate, the latter being mimicked by the presence of vanadate at the phosphorylation site in the electron microscopy structure.

Structure and function of the calcium pump.

Active transport of cations is achieved by a large family of ATP-dependent ion pumps, known as P-type ATPases. Various members of this family have been targets of structural and functional investigations for over four decades. Recently, atomic structures have been determined for Ca2+-ATPase by X-ray crystallography, which not only reveal the architecture of these molecules but also offer the opportunity to understand the structural mechanisms by which the energy of ATP is coupled to calcium transport across the membrane. This energy coupling is accomplished by large-scale conformational changes. The transmembrane domain undergoes plastic deformations under the influence of calcium binding at the transport site. Cytoplasmic domains undergo dramatic rigid-body movements that deliver substrates to the catalytic site and that establish new domain interfaces. By comparing various structures and correlating functional data, we can now begin to associate the chemical changes constituting the reaction cycle with structural changes in these domains.

Endothelin-1 suppresses plasma membrane Ca++-ATPase, concomitant with contraction of hepatic sinusoidal endothelial fenestrae.

Intracytoplasmic free calcium ions (Ca++) are maintained at a very low concentration in mammalian tissue by extruding Ca++ from the cytoplasm against a steep extracellular Ca++ concentration gradient, mainly through the activity of plasma membrane Ca++ pump-ATPase. The present study aimed to elucidate how endothelin-1 (ET-1) affects the morphology of sinusoidal endothelial fenestrae and ultrastructural distribution of plasma membrane ATPases and intracytoplasmic free Ca++ in isolated rat hepatic sinusoidal endothelial cells. Sinusoidal endothelial fenestrae were observed by scanning electron microscope. Ando's electron cytochemical method was used for ultrastructural localization of Ca++-Mg++-ATPase activity, electron immunogold postembedding method for Ca++ pump-ATPase immunoactivity, and antimonate method for intracytoplasmic free Ca++. Addition of ET-1 to sinusoidal endothelial cells significantly decreased Ca++-Mg++-ATPase activity and Ca++ pump-ATPase expression and increased intracytoplasmic free Ca++ concentration, concomitant with a decrease in diameter of sinusoidal endothelial fenestrae. Co-treatment with Bosentan abolished the actions of ET-1. These results suggest that ET-1 suppresses Ca++-Mg++-ATPase activity and Ca++ pump-ATPase expression on the plasma membrane of sinusoidal endothelial fenestrae, thereby attenuating the extrusion of intracytoplasmic free Ca++ into the extracellular space, leading to an increased concentration of intracytoplasmic free calcium ions and contraction of sinusoidal endothelial fenestrae.

A calcium pump made visible.

The first high-resolution structure of a P-type ATPase, that of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, was published in 2000. This structure has provided many clues to how the Ca(2+)-ATPase might work, but no complete answers. The Ca(2+)-ATPase structure reveals no clear pathway from the cytoplasmic side of the membrane to the pair of high-affinity binding sites for Ca(2+) located in the transmembrane region of the ATPase and no clear pathway from these sites to the lumenal side of the membrane. The ATPase is therefore very unlike an ion channel in its construction. It is unclear from the crystal structure of the Ca(2+)-ATPase exactly how the protein sits within the lipid bilayer that surrounds it in the membrane. The Ca(2+)-ATPase is implicated in thermogenesis in some types of muscle; this could involve processes of slippage and leak modulated by interaction between the Ca(2+)-ATPase and sarcolipin.

Joint participation of mitochondria and sarcoplasmic reticulum in the formation of tubular aggregates in gastrocnemius muscle of CK-/- mice.

Tubular aggregates are specific subcellular structures that appear in skeletal muscle fibres under different pathological conditions. The origin of the tubular aggregates is generally ascribed to proliferating membranes of sarcoplasmic reticulum. There are, however, histochemical indications for the presence of mitochondrial enzymes in tubular aggregates suggesting contribution of mitochondria to the genesis of tubular aggregates. In this study we used an immunocytochemical detection technique to assess participation of mitochondria and of sarcoplasmic reticulum in derivation of tubular aggregates. The fast skeletal muscle fibres (m. gastrocnemius) of mice bearing the double invalidation for both the mitochondrial and the cytosolic isoforms of creatine kinase (CK), an enzyme involved in energetics of muscle cells, were employed as a model muscle with tubular aggregates (Steeghs et al., Cell 89, 93-103, 1997). Immunogold labelling of the bc1 complex, a specific integral protein of the inner mitochondrial membrane, provided strong signals in both the mitochondria and tubular aggregates but not in other ultrastructural components of muscle fibres. A similar strong immunogold signal was obtained when labelling for SERCA1, a specific enzyme of the sarcoplasmic reticulum membrane, in regions of typical occurrence of the sarcoplasmic reticulum and in tubular aggregates. In double labelling experiments, we found simultaneous labelling of tubular aggregates with both the bc1 and SERCA1 antibodies. It is concluded, that in CK-/- mouse both the inner mitochondrial membrane and the membrane of the sarcoplasmic reticulum participate in the formation of tubular aggregates.

A structural model for the catalytic cycle of Ca(2+)-ATPase.

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.

Locating phospholamban in co-crystals with Ca(2+)-ATPase by cryoelectron microscopy.

Phospholamban (PLB) is responsible for regulating Ca(2+) transport by Ca(2+)-ATPase across the sarcoplasmic reticulum of cardiac and smooth muscle. This regulation is coupled to beta-adrenergic stimulation, and dysfunction has been associated with end-stage heart failure. PLB appears to directly bind to Ca(2+)-ATPase, thus slowing certain steps in the Ca(2+) transport cycle. We have determined 3D structures from co-crystals of PLB with Ca(2+)-ATPase by cryoelectron microscopy of tubular co-crystals at 8--10 A resolution. Specifically, we have used wild-type PLB, a monomeric PLB mutant (L37A), and a pentameric PLB mutant (N27A) for co-reconstitution and have compared resulting structures with three control structures of Ca(2+)-ATPase alone. The overall molecular shape of Ca(2+)-ATPase was indistinguishable in the various reconstructions, indicating that PLB did not have any global effects on Ca(2+)-ATPase conformation. Difference maps reveal densities which we attributed to the cytoplasmic domain of PLB, though no difference densities were seen for PLB's transmembrane helix. Based on these difference maps, we propose that a single PLB molecule interacts with two Ca(2+)-ATPase molecules. Our model suggests that PLB may resist the large domain movements associated with the catalytic cycle, thus inhibiting turnover.

Phosphorylated Ca2+-ATPase stable enough for structural studies.

The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, J.-J. & McIntosh, D. B. (2000) J. Biol. Chem. 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved.

Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy.

Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.

Mitochondria-rich cells in gills and skin of an African lungfish, Protopterus annectens.

We used scanning electron microscopy, the vital dye DASPEI and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills and skin (opercular, dorsal and ventral) of the lungfish Protopterus annectens in its free-swimming conditions and at the beginning of aestivation. In the free-swimming period, the gills were short and thick and the pavement cells were extremely large (30-40 microns). The mitochondria-rich cells, which were distributed in the secondary and primary epithelium, occurred as two morphologically different types, i.e. elongated and oval, similar to the alpha and beta chloride cells of fresh water teleosts. In the skin, only one type of mitochondria-rich cells was found, resembling the alpha chloride cells. All the mitochondria-rich cells distributed in the gills and skin were labelled with anti Ca(2+)-ATPase serum indicating the possible uptake of Ca2+ at freshwater chloride cell level. At the start of aestivation, the skin and gills were covered by a thick layer of mucus and the epithelium of the gills was reduced. The mitochondria-rich cells were almost completely covered by the pavement cells.

Structure determination of tubular crystals of membrane proteins. II. Averaging of tubular crystals of different helical classes.

A set of programs has been developed for averaging the data from tubular crystals belonging to different helical classes. This was done either by (i) cutting out molecules constituting a unit cell from density maps, and aligning and averaging them in real space; (ii) transforming the densities in a unit cell to layer-line data according to a (possibly artificial) helical symmetry, aligning and averaging them in reciprocal space. These methods were applied to tubular crystals of Ca2+-ATPase. Either method worked well and substantially improved the data quality. Transforming the reconstructed images to the layer-line data has many advantages and is essential for fully exploiting the power of averaging.