Selective Fe2+-catalyzed oxidative cleavage of gastric H+,K+-ATPase: implications for the energy transduction mechanism of P-type cation pumps

In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.     

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.     

Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca(2+)-ATPase.

The mechanism of inhibition of the sarcoplamc reticulum (SR) Ca(2+)-ATPase by the fluoroaluminate complexes was investigated. First, AlF4- was shown to bind to the Ca(2+)-free conformation of the enzyme by a slow quasi-irreversible process. The rate constants of the reaction are k+ = 16 x 10(3) M-1 s-1 and k- < 1.5 10(-3) s-1. We directly measured a stoichiometry of about 4.8 nmol of AlF4- bound/mg of protein. Mg2+ was a necessary cofactor for the reaction with a dissociation constant of 3 mM. It was demonstrated (Dupont, Y., and Pougeois, R. (1983) FEBS Lett. 156, 93-98) that phosphorylation by P(i) induced a dehydration of the catalytic site. The same process has been shown here to occur upon AlF4- binding either by the use of Me2SO or by demonstration of an increase of bound 2',3'-O-(2,4,6-trinitrocyclohexadienyldene)adenosine triphosphate fluorescence. Phosphorylation by P(i) is inhibited by the binding of AlF4-. Second, a fluoroaluminate complex, presumably AlF4-, was also shown to bind to the Ca(2+)-bound conformation of the Ca(2+)-ATPase in the presence of ADP and stabilize a E1.Ca2.ADP.AlFx complex. The dissociation constant of the nucleotidic site for ADP was shifted to the micromolar range. The Ca2+ ions bound on the external high affinity sites became occluded upon binding of (ADP + AlFx). We propose that AlF4- mimics P(i) binding to the Ca(2+)-free conformation of the ATPase and stabilizes an intermediate similar to the acyl-phosphate derivative; it also acts as an analogue of the gamma-phosphate of ATP and stabilizes an E1.[Ca2].ADP.AlF4 complex where the Ca2+ ions are occluded.     

Evidence for a global inhibitor-induced conformation change on the Ca(2+)-ATPase of sarcoplasmic reticulum from paired inhibitor studies

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of hydrophobic, hydroxy-containing molecules. A kinetic method has been used to study competition between binding of pairs of inhibitors to the ATPase. The presence of 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) decreases the affinity of the ATPase for 2,5-dipropyl-1,4-dihydroxybenzene (PHQ), suggesting that PHQ and BHQ bind to the same site on the ATPase. In contrast, the presence of BHQ increases the affinity of the ATPase for curcumin and vice versa. This suggests that BHQ and curcumin bind to separate sites on the ATPase and that binding of the first inhibitor to the ATPase results in a change to a conformation with higher affinity for the second inhibitor. This is consistent with previous experiments with BHQ and thapsigargin suggesting a conformation change on inhibitor binding, E2 + I <--> 2; E2I <--> 2; E2(A)I, with E2(A)I having a higher affinity for the second inhibitor than E2. The affinity for BHQ is also increased by binding of diethylstilbesterol, ellagic acid, or nonylphenol, and the affinity for curcumin is also increased by ellagic acid. These results showing that binding of a variety of inhibitors of very different structures all result in a general increase in inhibitor affinity point to a global conformational change on the Ca(2+)-ATPase caused by inhibitor binding, as well as any local, inhibitor-specific changes in conformation.     

Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.     

Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.     

Overlapping effects of S3 stalk segment mutations on the affinity of Ca2+-ATPase (SERCA) for thapsigargin and cyclopiazonic acid

Chimeric exchanges and mutations were produced in the Ca(2+)-ATPase (SERCA) to match (in the majority of cases) corresponding sequences of the Na(+),K(+)-ATPase. The effects of these mutations on the concentration dependence of the specific Ca(2+)-ATPase inhibition by thapsigargin (TG) and cyclopiazonic acid (CPA) were then determined. Extensive chimeric mutations on the large cytosolic loop, on the S4 stalk segment, and on the M3 transmembrane segments produced little or no modification of the Ca(2+)-ATPase sensitivity to either inhibitor. On the other hand, the presence of a six amino acid Na(+), K(+)-ATPase sequence within the S3 stalk segment of the Ca(2+)-ATPase raised 60-fold the apparent K(i) for TG and 250-fold the apparent K(i) for CPA. More limited mutations within the same S3 segment, however, affected differently the concentration dependence of the Ca(2+)-ATPase inhibition by TG or CPA. Specifically, single mutation of Phe256 to Val increased 20-fold the apparent K(i) for TG, while having very little effect on the apparent K(i) for CPA. These findings indicate significant overlap of the TG and CPA binding domains within the S3 stalk segment of the Ca(2+)-ATPase, where the contribution of each protein residue is dependent on the structures of the two inhibitors. Saturating concentrations of either or both TG and CPA produce an identical reduction of the affinity of the ATPase for ATP, suggesting that only one inhibitor can bind at any time due to significant overlap of their binding domains. It is suggested that perturbations produced by binding of either inhibitor within the stalk segment interfere with the long-range functional linkage between ATP utilization in the ATPase cytosolic region and Ca(2+) binding in the membrane-bound region.     

Inhibition of the intracellular Ca2+ transporter SERCA (Sarco-Endoplasmic Reticulum Ca2+-ATPase) by the natural polyphenol epigallocatechin-3-gallate

The use of a microsomal preparation from skeletal muscle revealed that both Ca(2+) transport and Ca(2+)-dependent ATP hydrolysis linked to Sarco-Endoplasmic Reticulum Ca(2+)-ATPase are inhibited by epigallocatechin-3-gallate (EGCG). A half-maximal effect was achieved at approx. 12?μM. The presence of the galloyl group was essential for the inhibitory effect of the catechin. The relative inhibition of the Ca(2+)-ATPase activity decreased when the Ca(2+) concentration was raised but not when the ATP concentration was elevated. Data on the catalytic cycle indicated inhibition of maximal Ca(2+) binding and a decrease in Ca(2+) binding affinity when measured in the absence of ATP. Moreover, the addition of ATP to samples in the presence of EGCG and Ca(2+) led to an early increase in phosphoenzyme followed by a time-dependent decay that was faster when the drug concentration was raised. However, phosphorylation following the addition of ATP plus Ca(2+) led to a slow rate of phosphoenzyme accumulation that was also dependent on EGCG concentration. The results are consistent with retention of the transporter conformation in the Ca(2+)-free state, thus impeding Ca(2+) binding and therefore the subsequent steps when ATP is added to trigger the Ca(2+) transport process. Furthermore, phosphorylation by inorganic phosphate in the absence of Ca(2+) was partially inhibited by EGCG, suggesting alteration of the native Ca(2+)-free conformation at the catalytic site.     

Deduced amino acid sequence and E1-E2 equilibrium of the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Comparison with the Ca(2+)-ATPase of rabbit fast twitch muscle.

The cDNA encoding a Ca(2+)-transport ATPase of frog (Rana esculenta) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca(2+)-ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca(2+)-ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca(2+)-ATPase were compared with those of the rabbit fast twitch muscle Ca(2+)-ATPase by functional expression of the cDNAs in COS-1 cells. The amphibian Ca(2+)-ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated in the frog Ca(2+)-ATPase than in the mammalian enzyme.     

Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase. Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed by transient kinetic measurements. In the Ca(2+)-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation "overshoot" was observed upon reaction of the Ca(2+)-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca(2+) binding transition (E2 --> E1 --> Ca(2)E1) as well as Ca(2+) dissociation from Ca(2+) site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca(2+) interaction at Ca(2+) site II. Because mutation of Asp(813) inhibited the Ca(2+) binding transition, but not Ca(2+) dissociation, and increased the apparent affinity for vanadate, the effect on the Ca(2+) binding transition seems in this case to be exerted by slowing the E2 --> E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca(2+) concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca(2+). Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P --> E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.     

Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism

We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).     

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.     

Ca2+ release to lumen from ADP-sensitive phosphoenzyme E1PCa2 without bound K+ of sarcoplasmic reticulum Ca2+-ATPase

During Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca(2+) release into the lumen. Here, we find that in the absence of K(+), Ca(2+) release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca(2+) release pathway is open to some extent in the K(+)-free E1PCa(2) structure. The Ca(2+) affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca(2+) binding sites are not disrupted. Thus, bound K(+) stabilizes the E1PCa(2) structure with occluded Ca(2+), keeping the Ca(2+) pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144-29155) that the K(+) bound in E2P reduces the Ca(2+) affinity essential for achieving the high physiological Ca(2+) gradient and to fully open the lumenal Ca(2+) gate for rapid Ca(2+) release (E2PCa(2) → E2P + 2Ca(2+)). These findings show that bound K(+) is critical for stabilizing both E1PCa(2) and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca(2+) handling.     

Side-chain protonation and mobility in the sarcoplasmic reticulum Ca2+-ATPase: implications for proton countertransport and Ca2+ release

Protonation of acidic residues in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca(2+)-bound state Ca(2)E1, in the Ca(2+)-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF(4)(2-) E2(TG+MgF(4)(2-)). Around physiological pH, all acidic Ca(2+) ligands (Glu(309), Glu(771), Asp(800), and Glu(908)) were unprotonated in Ca(2)E1; in E2(TG) and E2(TG+MgF(4)(2-)) Glu(771), Asp(800), and Glu(908) were protonated. Glu(771) and Glu(908) had calculated pK(a) values larger than 14 in E2(TG) and E2(TG+MgF(4)(2-)), whereas Asp(800) titrated with calculated pK(a) values near 7.5. Glu(309) had very different pK(a) values in the Ca(2+)-free states: 8.4 in E2(TG+MgF(4)(2-)) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu(309) can switch between a high and a low pK(a) mode, depending on the local backbone conformation. Protonated Glu(309) occupied predominantly two main, very differently orientated side-chain conformations in E2(TG+MgF(4)(2-)): one oriented inward toward the other Ca(2+) ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu(309) adopted completely the outwardly orientated side-chain conformation. The contact of Glu(309) with the cytoplasm in E2(TG+MgF(4)(2-)) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca(2+)-binding site I and the cytoplasm. Glu(771), Asp(800), and Glu(908) are proposed to take part in proton countertransport.     

Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 μM for the E2P ground state analog, 1 μM for the E2P transition state and product state analogs, and 11 μM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Energetics of the induced structural change in a Ca2+ regulatory protein: Ca2+ and troponin I peptide binding to the E41A mutant of the N-domain of skeletal troponin C

Structural studies have shown that the regulatory domains of skeletal and cardiac troponin C (sNTnC and cNTnC) undergo different conformational changes upon Ca(2+) binding; sNTnC "opens" with a large exposure of the hydrophobic surface, while cNTnC retains a "closed" conformation similar to that in the apo state. This is mainly due to the fact that there is a defunct Ca(2+)-binding site I in cNTnC. Despite the striking difference, the two proteins bind their respective troponin I (TnI) regions (sTnI(115-131) and cTnI(147-163), respectively) in a similar open fashion. Thus, there must exist a delicate energetic balance between Ca(2+) and TnI binding and the accompanying conformational changes in TnC for each system. To understand the coupling between Ca(2+) and TnI binding and the concomitant structural changes, we have previously engineered an E41A mutant of sNTnC and demonstrated that this mutation drastically reduced the Ca(2+)-binding affinity of site I in sNTnC, and as a result, E41A-sNTnC remains closed in the Ca(2+)-bound state. In the present work, we investigated the interaction of E41A-sNTnC with the sTnI(115-131) peptide and found that the peptide binds to the Ca(2+)-saturated E41A-sNTnC with a 1:1 stoichiometry and a dissociation constant of 300 +/- 100 microM. The peptide-induced chemical shift changes resemble those of Ca(2+) binding to sNTnC, suggesting that sTnI(115-131) induces the "opening" of E41A-sNTnC. In addition, the binding of sTnI(115-131) appears to be accompanied by a conformational change in site I of E41A-sNTnC so that the damaged regulatory site can bind Ca(2+) more tightly. Without Ca(2+), sTnI(115-131) only interacts with E41A-sNTnC nonspecifically. When Ca(2+) is titrated into E41A-sNTnC in the presence of sTnI(115-131), the Ca(2+)-binding affinity of site I was enhanced by approximately 5-fold as compared to when sTnI(115-131) was not present. These observations suggest that the binding of Ca(2+) and TnI is intimately coupled to each other. Together with our previous studies on Ca(2+) and TnI peptide binding to sNTnC and cNTnC, these results allow us to dissect the mechanism and energetics of coupling of ligand binding and structural opening intricately involved in the regulation of skeletal and cardiac muscle contraction.     

Effects of an ATP site affinity analog on some conformational and enzymatic properties of the canine kidney (Na+ + K+)-ATPase

We have shown previously that the canine kidney Na+,K+ pump [Na+ + K+)-ATPase) reacts with the ATP affinity analog p-fluorosulfonylbenzoyladenosine (FSBA). At 20 degrees C, we find the time-course of this reaction to be that predicted for a first-order reaction accompanied by competing solvolysis of the reagent. The FSBA-inactivated (Na+ + K+)-ATPase retains the ability to move between the E1 and E2 conformations that predominate in Na+ and K+ medium, respectively. Therefore, FSBA reaction with the enzyme does not interfere significantly with either its alkali metal cation binding or its conformational freedom. The ability of ATP to influence the enzyme's conformation by binding to the high-affinity nucleotide site is decreased, however, in proportion to the degree of inhibition of enzyme activity by FSBA. In addition, the ability of the enzyme to shift from the E1 to the E2 conformation through the (ATP + Na+)-dependent phosphorylation cycle is inhibited by FSBA treatment, as shown by the decreased ability of these substrates to stimulate the K+-dependent p-nitrophenylphosphatase activity. Both of these effects are consistent with specific reaction of FSBA with the ATP binding site of the enzyme. An additional effect of FSBA treatment is that it causes loss of p-nitrophenylphosphatase activity, but to a lesser extent than (Na+ + K+)-ATPase or Na+-ATPase activity. Binding of p-nitrophenylphosphate to the enzyme is apparently unaffected by FSBA treatment, since the Km for p-nitrophenylphosphate is not changed.     

The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements

The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.     

Heparin inhibits the reconstituted plasma membrane Ca2+-ATPase from porcine brain synaptosome

Heparin has been shown to be involved in the regulation of cellular Ca(2+) by binding to many proteins with high affinity. Here we examined the effects of heparin on the plasma membrane Ca(2+)-ATPase from porcine brain synaptosome. Our results showed that heparin dramatically inhibited the ATP hydrolysis and Ca(2+) uptake in the presence and absence of calmodulin. Together with controlled proteolysis by trypsin, we concluded that the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase was less important for the heparin inhibition. Excess phosphatidylserine was able to eliminate the heparin inhibition. We observed that Ca(2+) affinity kept no obvious changes, but the ATP affinity of plasma membrane Ca(2+)-ATPase was apparently decreased in the presence of heparin. Our results indicated that heparin had little effects on ATP or Ca(2+) binding sites of the enzyme.