Crystals of sarcoplasmic reticulum Ca(2+)-ATPase

High-resolution structures of the Ca(2+)-ATPase have over the last 5 years added a structural dimension to our understanding of the function of this integral membrane protein. The Ca(2+)-ATPase is now by far the membrane protein where the most functionally different conformations have been described in precise structural detail. Here, we review our experience from solving Ca(2+)-ATPase structures: a purification scheme involving minimum handling of the protein to preserve natural and essential lipids, a rational approach to screening for crystals based on a limited number of polyethyleneglycols and many different salts, improving crystal quality using additives, collecting the data and finally solving the structures. We argue that certain of the lessons learned in the present study are very likely to be useful for crystallisation of eukaryotic membrane proteins in general.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Vectorially oriented monolayers of detergent-solubilized Ca(2+) -ATPase from sarcoplasmic reticulum.

A method for tethering proteins to solid surfaces has been utilized to form vectorially oriented monolayers of the detergent-solubilized integral membrane protein Ca(2+) -ATPase from the sarcoplasmic reticulum (SR). Bifunctional, organic self-assembled monolayers (SAMs) possessing "headgroup" binding specificity for the substrate and "endgroup" binding specificity for the enzyme were utilized to tether the enzyme to the substrate. Specifically, an amine-terminated 11-siloxyundecaneamine SAM was found to bind the Ca(2+)-ATPase primarily electrostatically. The Ca(2+)-ATPase was labeled with the fluorescent probe 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid before monolayer formation. Consequently, fluorescence measurements performed on amine-terminated SAM/enzyme monolayers formed on quartz substrates served to establish the nature of protein binding. Formation of the monolayers on inorganic multilayer substrates fabricated by molecular beam epitaxy made it possible to use x-ray interferometry to determine the profile structure for the system, which was proved correct by x-ray holography. The profile structures established the vectorial orientation of the Ca(2+)-ATPase within these monolayers, to a spatial resolution of approximately 12 A. Such vectorially oriented monolayers of detergent-solubilized Ca(2+)-ATPase from SR make possible a wide variety of correlative structure/function studies, which would serve to elucidate the mechanism of Ca(2+) transport by this enzyme.     

Proton paths in the sarcoplasmic reticulum Ca(2+) -ATPase

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.     

Characterisation of thapsigargin-releasable Ca(2+) from the Ca(2+)-ATPase of sarcoplasmic reticulum at limiting [Ca(2+)

The Ca(2+) binding sites of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using (45)Ca(2+), have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca(2+)-limited state, [Ca(2)](lim), where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48-60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca(2+)-ATPase, released a fraction of total Ca(2+) at [Ca(2+)](lim) that accumulated during active transport. Maximal Ca(2+) release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca(2+) resistant to TG. The amount of Ca(2+) released depended on the incubation time at [Ca(2+)](lim), being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0. 13 to 0.03 s(-1). The rapidly released early fraction declined with time and k=0.13 min(-1). Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca(2+) followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 microM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca(2+) release mechanism. It is concluded that the Ca(2+) released by TG is from the occluded Ca(2+) fraction. The Ca(2+) occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite 'in line' sequential binding mechanism for Ca(2+) transport.     

Inhibition by A23187 of conformational changes involved in the Ca(2+)-induced activation of sarcoplasmic reticulum Ca(2+)-ATPase.

We previously showed that A23187 in high ionophore/protein ratios almost completely inhibits the sarcoplasmic reticulum Ca(2+)-ATPase [Hara, H. & Kanazawa, T. (1986) J. Biol. Chem. 261, 16584-16590]. In an attempt to obtain information on the mechanism of this inhibition, the effects of A23187 on conformational changes involved in the Ca(2+)-induced activation of the enzyme were investigated. The purified enzyme from sarcoplasmic reticulum of rabbit skeletal muscle as well as the purified enzyme labeled with fluorescein 5-isothiocyanate (FITC) were preincubated with A23187 in the absence of Ca2+ at pH 7.0 and 0 degrees C for 45 min. The activation of the enzyme following addition of CaCl2 was assessed by determining the capacity for rapid formation of phosphoenzyme from ATP. This activation was strongly inhibited by the preincubation with A23187. This indicates that the previously observed inhibition of the Ca(2+)-ATPase is mostly due to hindrance of the Ca(2+)-induced activation of the enzyme. In the control, in which the FITC-labeled enzyme was preincubated without A23187, the fluorescence intensity of the bound FITC decreased in a biphasic manner upon addition of CaCl2. The first rapid phase of this fluorescence drop was unaffected by A23187, whereas its second slow phase was almost completely inhibited by this drug. These results show that the Ca(2+)-dependent conformational change is biphasic and that the second slow phase (but not the first rapid phase) of this conformational change is inhibited by A23187. This suggests that the observed inhibition of Ca2+ activation is attributed to hindrance of the second slow phase of the Ca(2+)-dependent conformational change.     

Interaction of an aromatic dibromoisothiouronium derivative with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum

Isothiouronium compounds [Hoving, S., Bar-Shimon, M., Tijmes, J. J., Goldshleger, R., Tal, D. M., and Karlish, S. J. (1995) J. Biol. Chem. 270, 29788-29793] act as high-affinity competitive antagonists for Na(+) and K(+) (Rb(+)) on the renal Na(+)/K(+)-ATPase where they favor the E1 conformation. We have now characterized the effects of 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene (Br(2)-TITU) on the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum. Br(2)-TITU inhibited the Ca(2+)-ATPase, both transport and catalytic activity, with a K(0.5) of 5-15 microM. Maximum inhibition was at 10 min with t(0.5) of 3-5 min. Br(2)-TITU, 100 microM, quenched Trp autofluorescence by 80%, but the residual signal still responded to Ca(2+) binding. Maximum quenching of fluorescence was at pH 9.0. Total E-P levels, during the steady state of turnover of the Ca(2+)-ATPase, were increased from 0.5 to 5.8 nmol x mg(-1) by Br(2)-TITU at pH 6.8. Trinitrophenyl-ATP (TNP-ATP) superfluorescence, which monitors hydrophobicity of the ATP site, was increased 3-4-fold, suggesting that Br(2)-TITU favors an "E2"-like state. Fluorescence was also increased 3-5-fold when E-P was induced with P(i) plus EGTA. Br(2)-TITU increased the rate constants of induction of superfluorescence with ATP plus Ca(2+) from 0.32 to 0.69 s(-1) and with P(i) plus EGTA from 0.84 to 7.45 s(-1). Br(2)-TITU also decreased rate constants for "off" reactions from 2.9 to 0.66 s(-1) and from 10.9 to 0.73 s(-1) for the ATP and P(i) reactions, respectively. Br(2)-TITU, which competitively inhibits the Na(+)/K(+)-ATPase, has a novel effect on the Ca(2+)-ATPase. It promotes accumulation of E2-P species due to increased rate of formation and decreased rate of hydrolysis and quenches tryptophan autofluorescence. Br(2)-TITU could be a useful inhibitor to probe intermediate reactions of the Ca(2+)-ATPase that link catalysis with Ca(2+) translocation.     

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum.

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.     

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Interactions of hexachlorocyclohexanes with the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Hexachlorocyclohexanes have been shown to inhibit the (Ca2+ + Mg2+)-ATPase of muscle sarcoplasmic reticulum reconstituted into bilayers of dioleoylphosphatidylcholine. However, for the ATPase reconstituted into bilayers of dimyristoleoylphosphatidylcholine, a pattern of activation at low concentration followed by inhibition at higher concentration is seen for hexachlorocyclohexanes and alkanes such as decane and hexadecane. The ATPase in sarcoplasmic reticulum vesicles is also inhibited by the hexachlorocyclohexanes. The effects of hexachlorocyclohexanes on activity are largely independent of concentrations of Ca2+ and ATP. Inhibition is more marked at lower temperatures. The hexachlorocyclohexanes quench the tryptophan fluorescence of the ATPase, and the quenching can be used to obtain partition coefficients into the membrane system. As for simple lipid bilayers, partition exhibits a negative temperature coefficient. Binding is related to effects on ATPase activity.     

Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum

Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.     

Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.     

Characterization of phenylmaleimide inhibition of the Ca(2+)-ATPase from skeletal-muscle sarcoplasmic reticulum

The Ca(2+)-ATPase from sarcoplasmic reticulum reacts with phenylmaleimide, producing the inhibition of the ATPase activity following a pseudo-first-order kinetic with a rate constant of 19 M(-1) s(-1). Calcium and ATP binding are not altered upon phenylmaleimide inhibition. However, the presence of millimolar calcium, and to a lesser extent magnesium, in the inhibition medium enhances the effect of phenylmaleimide, causing a higher degree of inhibition. Solubilization with C(12)E(8) does not affect the ATPase inhibition, excluding any kind of participation of the lipid bilayer. Phosphorylation with ATP in steady-state conditions as well as phosphorylation with inorganic phosphate in equilibrium conditions were strongly inhibited. Conversely, we have found that the occupancy of the phosphorylation site by ortovanadate fully protects against the inhibitory effect of phenylmaleimide, indicating a conformational transition associated with the phosphorylation reaction.     

A phosphorylated conformational state of the (Ca2+-Mg2+)-ATPase of fast skeletal muscle sarcoplasmic reticulum can mediate rapid Ca2+ release

A rapid Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles from fast skeletal muscle can be induced under conditions which permit the formation of a stable phosphorylated intermediate of the (Ca2+-Mg2+)-ATPase. Such a state can be achieved experimentally by phosphorylating the ATPase in the absence of Mg2+ ions, which otherwise would stimulate the dephosphorylation step(s). Also, quercetine stimulates the rapid release of Ca2+ if used in the concentration range which does not produce inhibition of phosphoenzyme formation, but which inhibits phosphoenzyme dephosphorylation. The rapid efflux of Ca2+ ions proceeds as long as the low affinity Ca2+-binding sites facing the lumen of the vesicles are saturated and as long as Ca2+ is removed from the catalytic sites facing the cytosol. A molecular mechanism of the phosphoenzyme-mediated Ca2+ release is proposed. This mechanism is based on a rapid shuttling of the ATPase molecules between an ADP-sensitive and an ADP-insensitive phosphorylated state.     

CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.     

Arsenate-induced fluorescence changes in the Ca(2+)-ATPase of sarcoplasmic reticulum membranes.

Arsenate, an analogue of inorganic phosphate, causes an increase in the intrinsic fluorescence of the Ca(2+)-ATPase of sarcoplasmic reticulum membranes. This increase in fluorescence is observed regardless of whether Ca(2+)-loaded or leaky vesicles are assayed. The maximal fluorescence change (2-3%) is observed at pH 6.0 in the presence of Mg2+ and is abolished by the addition of micromolar Ca2+ concentrations. Dimethyl sulfoxide (20% v/v) increases the enzyme's affinity for arsenate one order of magnitude. It is concluded that arsenate, after binding, promotes the same conformational change of the enzyme as that produced by Pi.     

Oligomerisation of sarcoplasmic reticulum Ca2+-ATPase monomers from skeletal muscle

The fast-twitch SERCA1 isoform of the sarcoplasmic reticulum Ca(2+)-ATPase was purified to homogeneity and conjugated to peroxidase. The SERCA1 probe showed high affinity binding to the immobilized monomeric enzyme, but not crosslinker-stabilized oligomers. This suggests a preferential complex formation via homo-dimerization, rather than interactions with established oligomeric structures.     

Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum

Favero, Terence G., David Colter, Paul F. Hooper, andJonathan J. Abramson. Hypochlorous acid inhibitsCa²⁺-ATPase from skeletal musclesarcoplasmic reticulum. J. Appl. Physiol. 84(2): 425-430, 1998.Hypochlorous acid(HOCl) is produced by polymorphonuclear leukocytes that migrate andadhere to endothelial cells as part of the inflammatory response totissue injury. HOCl is an extremely toxic oxidant that can react with avariety of cellular components, and concentrations reaching 200 µMhave been reported in some tissues. In this study, we show that HOClinteracts with the skeletal sarcoplasmic reticulumCa²⁺-adenosinetriphosphatase(ATPase), inhibiting transport function. HOCl inhibits sarcoplasmicreticulum Ca²⁺-ATPase activity ina concentration-dependent manner with a concentration required toinhibit ATPase activity by 50% of 170 µM and with completeinhibition of activity at 3 mM. A concomitant reduction infree sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluoresceinisothiocyanate to the ATPase protein, indicating some structural damagemay have occurred. These findings suggest that the reactive oxygenspecies HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygenspecies disrupt the normalCa²⁺-handling kinetics in musclecells.