The sarcoplasmic reticulum Ca2+-ATPase

Summary This review summarizes studies on the structural organization of Ca²⁺-ATPase in the sarcoplasmic reticulum membrane in relation to the function of the transport protein. Recent advances in this field have been made by a combination of protein-chemical, ultrastructural, and physicochemical techniques on membraneous and detergent solubilized ATPase. A particular feature of the ATPase (Part I) is the presence of a hydrophilic head, facing the cytoplasm, and a tail inserted in the membrane. In agreement with this view the protein is moderately hydrophobic, compared to many other integral membrane proteins, and the number of traverses of the 115 000 Dalton peptide chain through the lipid may be limited to 3–4.There is increasing evidence (Part II) that the ATPase is self-associated in the membrane in oligomeric form. This appears to be a common feature of many transport proteins. Each ATPase peptide seems to be able to perform the whole catalytic cycle of ATP hydrolysis and Ca²⁺ transport. Protein-protein interactions seem to have a modulatory effect on enzyme activity and to stabilize the enzyme against inactivation.Phospholipids (Part III) are not essential for the expression of enzyme activity which only requires the presence of flexible hydrocarbon chains that can be provided e.g. by polyoxyethylene glycol detergents. Perturbation of the lipid bilayer by the insertion of membrane protein leads to some immobilization of the lipid hydrocarbon chains, but not to the extent envisaged by the annulus hypothesis. Strong immobilization, whenever it occurs, may arise from steric hindrance due to protein-protein contacts. Recent studies suggest that breaks in Arrhenius plots of enzyme activity primarily reflect intrinsic properties of the protein rather than changes in the character of lipid motion as a function of temperature.     

Heterologous expression of sarcoplasmic reticulum Ca2+-ATPase

In recent years, expression of rabbit sarcoplasmic reticulum (SR) Ca²⁺-ATPase in heterologous systems has been a widely used strategy to study altered enzymes generated by site-directed mutagenesis. Various eukaryotic expression systems have been tested, all of them yielding comparable amounts of recombinant protein. However, the relatively low yield of recombinant protein obtained so far suggests that novel purification techniques will be required to allow further characterization of this enzyme based on direct ligand-binding measurements.     

Affinity labeling of the active site of the Ca2+-ATPase of sarcoplasmic reticulum

The inactivation of sarcoplasmic reticulum ATPase by fluorescein isothiocyanate (FITC) was shown to have a hyperbolic dependence on the concentration of FITC. The results were quantitatively accounted for by a model in which the reagent first binds reversibly (Kf = 70 microM) to the ATPase and then reacts irreversibly (kmax = 0.8 and 2 min-1 in the absence and presence of 1 mM Mg2+, respectively) to form inactive enzyme. Comparison with the rate constant for the reaction of the model compound alpha-acetyllysine with FITC showed that the FITC-reactive lysyl side-chain of the ATPase is not unusually reactive, indicating that the specificity of the reaction is due to affinity labeling behavior of the reagent. This was supported by protection experiments using ATP, ADP, AdoPP[NH]P, ITP, and TNP-ATP, all of which displayed protection constants similar to their known binding constants to the active site of the ATPase. Both inorganic phosphate and orthovanadate were effective in preventing inactivation by FITC, and calcium only partially reversed the effect of these anions, implying the existence of a ternary complex such as Ca2.E.Pi. Since all ligands (ATP, ADP and Pi) which bind or react at the catalytic site protect it, only the unliganded form appears to bind and react with FITC. Addition of calcium to the MgATP complex of the ATPase caused an increase in the FITC inactivation rate, implying that during turnover there is a larger fraction of unliganded enzyme present, i.e., substrate binding is weaker (Ks is larger). Protection was also observed with fluorescein and two related dyes, eosin and erythrosin. Like FITC, the isothiocyanates of these dyes were effective inactivators. In separate experiments, these two dyes were shown to promote photoinactivation of the ATPase. ATP exerted a protective effect with a concentration dependence consistent with high-affinity active-site binding.     

Occlusion of Ca2+ in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum Ca2+-ATPase solubilized in monomeric form by nonionic detergent was reacted with CrATP in the presence of 45Ca2+. A Ca2+-occluded complex formed, which was stable during high performance liquid chromatography in the presence of excess non-radioactive Ca2+. The elution position corresponded to monomeric Ca2+-ATPase. It is concluded that a single Ca2+-ATPase polypeptide chain provides the full structural basis for Ca2+ occlusion.     

Crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum

Microcrystalline arrays of Ca2+-transporting ATPase (EC 3.6.1.38) develop in detergent-solubilized sarcoplasmic reticulum upon exposure to 10-20 mM CaCl2 at pH 6.0 for several weeks at 2 degrees C, in a crystallization medium that preserves the ATPase activity for several months. Of 48 detergents tested, optimal crystallization was obtained with Brij 36T, Brij 56, and Brij 96 at a detergent:protein weight ratio of 4:1 and with octaethylene glycol dodecyl ether at a ratio of 2:1. Similar Ca2+-induced crystalline arrays were obtained with the purified or delipidated Ca2+-ATPase of sarcoplasmic reticulum but at lower detergent:protein ratios. The crystals are stabilized by fixation with glutaraldehyde and persist even after the removal of phospholipids by treatment with phospholipases A or C and by extraction with organic solvents. The crystals obtained so far can be used only for electron microscopy, but ongoing experiments suggest that under similar conditions large ordered arrays may develop that are suitable for x-ray diffraction analysis.     

The structure of the Ca2+-ATPase of sarcoplasmic reticulum

In this article the morphology of sarcoplasmic reticulum, classification of Ca(2+)-ATPase (SERCA) isoenzymes presented in this membrane system, as well as their topology will be reviewed. The focus is on the structure and interactions of Ca(2+)-ATPase determined by electron and X-ray crystallography, lamellar X-ray and neutron diffraction analysis of the profile structure of Ca(2+)-ATPase in sarcoplasmic reticulum multilayers. In addition, targeting of the Ca(2+)-ATPase to the sarcoplasmic reticulum is discussed.     

Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.     

Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by 2-mercaptoethanol

The Ca²⁺-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0°C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22°C was considerably lower than that at 0°C. Ca²⁺-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate.     

Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

Changes in the fluoresence ofN-acetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca²⁺-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca²⁺ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes andEP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitiveEP, and transition of ADP-sensitiveEP to ADP-insensitiveEP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca²⁺-binding sites in the transmembrane domain in these latter two steps.Abbreviations SR sarcoplasmic reticulum - EP phosphoenzyme - EDANS N-acetyl-N-(5-sulfo-1-naphthyl)ethylenediamine - AMP-PCP adenosine 5-(, -methylene)triphosphate - NEM N-ethylmaleimide     

Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains.     

Ca2+-ATPase from sarcoplasmic reticulum: acylphosphatase activity

Fractionation of sarcoplasmic reticulum vesicles from rabbit skeletal muscle was performed by solubilization of the vesicles in the presence of deoxycholate, followed by sucrose density gradient centrifugation and gel filtration chromatography. This procedure permitted the isolation of essentially pure Ca²⁺-ATPase; this enzyme showed ATPase as well as acylphosphatase activity, both activities being clearly enhanced by deoxycholate. The acylphosphatase activity of the purified Ca²⁺-ATPase was characterized with regard to some kinetic properties, such as pH, Mg²⁺, Ca²⁺, and deoxycholate dependence, and substrate affinity, determined in the presence of acetylphosphate, succinylphosphate, carbamylphosphate, and benzoylphosphate; in addition, the stability of both activities was checked in time-course experiments. The main similarities between the two activities, such as the Mg²⁺ requirement, the deoxycholate activation, and the pH dependence, together with the competitive inhibition of the benzoylphosphatase activity by ATP, the inhibition of both activities by tris(bathophenanthroline)-Fe²⁺, and the relief of this inhibitory effect by carbonylcyanide-4-trifluoromethoxyphenyl hydrazone support the hypothesis that acylphosphatase and ATPase activities of sarcoplasmic reticulum vesicles reside in the same active site of the enzyme. With regard to possible relationships between acylphosphatase activity of the purified Ca²⁺-ATPase and “soluble” acylphosphatase present in the 100,000g supernatant fraction, comparison of some kinetic and structural parameters indicate that these two activities are supported by quite different enzymes.     

The binding of ATP to the catalytic and the regulatory site of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum

Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca²⁺, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca²⁺. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP.     

Localization of a K+ -binding site involved in dephosphorylation of the sarcoplasmic reticulum Ca2+ -ATPase

K+ plays an important role for the function of the sarco(endo)plasmic reticulum Ca2+ -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 Glu732 define the K+/Rb+ site in the Ca2+ -ATPase conformation with bound Ca2+, ADP, and AlF4-. Functional analysis of Ca2+ -ATPase mutants with alterations to Glu732 shows that this site is indeed important for the stimulatory effect of K+ on the dephosphorylation rate. Comparison with the Ca2+ -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.     

Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase

A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibition has been found. Phospholamban decreases the number of activatable Ca2+ATPase without affecting substrate affinity or the ability of nucleotide to serve its dual modulatory roles, i.e., catalytic and regulatory. Low concentrations of certain nucleotide mimetics, quercetin, tannin, and ellagic acid, with structural similarity to adenine can unmask phospholamban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approximately 0.3 microM) and ellagic acid (EC50 approximately 3 microM) stimulated Ca2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was followed by pronounced inhibiton at only slightly higher tannin concentrations (IC50 approximately 3 microM), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 microM) than the EC50. A complex relationship between compound, SR protein, and MgATP concentration is a major determining factor in the observed effects. Stimulation was only observed under conditions of phospholamban regulation, while the inhibitory effects were observed in cardiac SR at micromolar Ca2+ and in skeletal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ATPase was identical to that observed with the anti-phospholamban monoclonal antibody 1D11. Both compounds appear to relieve the Ca2+ATPase from phospholamban inhibition, thereby increasing the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibition of nucleotide activation. The data suggest that nucleotide activation of Ca2+ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulation. Clearly, this portion of Ca2+ATPase needs further study to elucidate its role in phospholamban inhibition.     

Steroid-induced conformational changes of FITC-labelled sarcoplasmic reticulum Ca2+-ATPase

Interactions between transmembrane and cytoplasmic domains of Ca2+-ATPase from sarcoplasmic reticulum (SR) have been studied. To affect the hydrophobic transmembrane domain, we used four amphiphilic steroids - esters of a dibasic acid and 20-oxypregnene. All four steroids contained cholesterol-like nuclei and differed by the structure of side chains. Steroids with carboxyl groups in the side chains inhibited the rates of ATP hydrolysis and Ca2+ transport, whereas a steroid without the carboxyl group did not appreciably affect Ca2+-ATPase function. Fluorimetric titration of FITC-labelled Ca2+-ATPase in SR vesicles by Nd3+ showed that steroids increased the apparent dissociation constant for Nd3+ bound to the hydrolytic site, the potency order of the steroids being the same as for the sterol-induced inhibition of the hydrolytic activity of Ca2+-ATPase. These results suggest structural changes in the active site. Ca2+ transport was inhibited more efficiently by steroids than the hydrolytic activity of the enzyme. This could be partially due to the increase of the membrane passive permeability induced by steroids, which, in turn, reflected the efficiency of the interaction of the steroids with lipid bilayers. The effects of the steroids were largely dependent on their amphiphilicity (the availability of polar groups in regions A and D), the structure of the side chains, and, possibly, on the distance between the molecular polar groups. We suggest that the inhibition of hydrolytic and transport functions of Ca2+-ATPase in the SR membrane is due to the interaction of the steroids with the transmembrane alpha-helical segments.     

Properties of deoxycholate solubilized sarcoplasmic reticulum Ca2+-ATPase.

The Ca2+-dependent ATPase of sarcoplasmic reticulum after solubilization with deoxycholate and removal of lipid by gel chromatography exists as a mixture of monomer, dimer, and smaller amounts of higher molecular weight aggregates. The binding capcity of deoxycholate by monomeric and oligomeric forms of the ATPase is 0.3 g/g of protein at pH 8 and ionic strength 0.11. Examination in the analytical ultracentrifuge results in estimates of protein molecular weight of monomer of 115 000 +/- 7000 and of Stokes radius of 50-55 A. The results indicate an asymmetric shape of both delipidated monomer and dimer. Solubilization of ATPase vesicles by deoxycholate at high protein dilutions leads to almost instantaneous loss of ATPase activity. However, ATPase may be solubilized by deoxycholate in presence of phospholipid and sucrose in a temporarily active state. Inactivation appears to be accompanied by delipidation and conformational changes of the protein as evidenced by circular dichroism measurements. Sedimentation velocity examination of enzymatically active preparations of soluble ATPase in presence of phospholipid and sucrose strongly suggests that the major part of enzymatic activity is derived from a monomer with an asymmetric shape. The extent of formation of soluble oligomers by column chromatography was dependent on the exact conditions used for initial solubilization of ATPase. No evidence for differences among monomer and dimer fractions was obtained by isoelectric focusing and amino acid analysis. The results of these studies are compatible with electron-microscopic studies by other authors which suggest that the ATPase has an elongated shape with limited hydrophobic contact with the membrane lipid. A resemblance of delipidated oligomers with the form in which ATPase occurs in the membrane is conjectural at present.     

Structure of the vanadate-induced crystals of sarcoplasmic reticulum Ca2+-ATPase

The projected structure of the vanadate-induced crystalline aggregates of Ca2+-ATPase molecules in isolated sarcoplasmic reticulum membranes has been determined. The molecules form tubular crystals with an oblique surface lattice having cell dimensions a = 65.9 A, b = 114.4 A and gamma = 77.9 degrees. The space group is P2. The crystalline tubules are formed through lateral aggregation of chains made up of dimers of Ca2+-ATPase molecules.