Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca2+-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca2+ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu51, Glu55, or Glu58, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp59, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca2+ dissociation toward the cytoplasmic side. Mutation of Leu60 to alanine or proline and of Val62 to alanine also enhanced Ca2+ dissociation, whereas an 11-fold reduction of the rate of Ca2+ dissociation was observed upon alanine substitution of Leu65, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca2+ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp59, and alanine substitution of Leu65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca2+ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

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.     

Mutational analysis of the role of Glu309 in the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle.

Site-specific mutagenesis was used to analyse the role of the residue, Glu309, in the function of the Ca(2+)-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 microM Ca2+ was unable to prevent phosphorylation from Pi, consistent with previous observations on the Ca(2+)-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476-478]. At neutral pH, however, micromolar concentrations of Ca2+ were sufficient to inhibit phosphorylation of the Glu309----Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca2+ site was relatively intact in this mutant. The Glu309----Lys mutant was unable to form a phosphoenzyme from ATP at all Ca2+ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu309----Ala mutant occurred at 12.5 mM Ca2+, but not at Ca2+ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E2P intermediate in the Glu309----Lys mutant. A less pronounced stabilization of E2P was observed with the Glu309----Ala mutant, suggesting a possible role of the charge at the position of Glu309 in phosphoenzyme hydrolysis.     

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase

Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca(2+) coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca(2+) concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 --> Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca(2+) was also impaired, thereby explaining the stronger inhibition of Ca(2+) transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca(2+) following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.     

Critical interaction of actuator domain residues arginine 174, isoleucine 188, and lysine 205 with modulatory nucleotide in sarcoplasmic reticulum Ca2+-ATPase

ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.     

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.     

Critical role of Glu40-Ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme

The functional importance of the length of the A/M1 linker (Glu(40)-Ser(48)) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase was explored by its elongation with glycine insertion at Pro(42)/Ala(43) and Gly(46)/Lys(47). Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa(2) possessing two occluded Ca(2+) ions at the transport sites, and the Ca(2+) deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca(2+)-free form of E2P produced from P(i) without Ca(2+) was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa(2) accumulated is an intermediate state between E1PCa(2) and the Ca(2+)-released E2P state. Namely in E2PCa(2), the actuator domain is already largely rotated from its position in E1PCa(2) and associated with the phosphorylation domain as in the Ca(2+)-released E2P state; however, in E2PCa(2), the hydrophobic interactions among these domains and Leu(119)/Tyr(122) on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr(122) are critical for formation of the Ca(2+)-released E2P structure. Results showed that the EP isomerization/Ca(2+)-release process consists of the following two steps: E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+); and the intermediate state E2PCa(2) was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa(2), is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca(2+) deocclusion and release.     

Functional consequences of alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal part of transmembrane helix M3 of the Na+,K(+)-ATPase

Mutations Ile279 --> Ala, Ile283 --> Ala, Glu284 --> Ala, His285 --> Ala, His285 --> Lys, His285 --> Glu, Phe286 --> Ala, and His288 --> Ala in transmembrane helix M3 of the Na+,K(+)-ATPase were studied. Except for His285 --> Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K(+)-transport cycle. In Ile279 --> Ala and Ile283 --> Ala, the E1 form accumulated, whereas in His285 --> Lys and His285 --> Glu, E1P accumulated. Phe286 --> Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P --> E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1-E2 and E1P-E2P conformational changes. Because the E1-E2 and E1P-E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na(+)-activated ATPase activity in the absence of K+ ("Na(+)-ATPase activity"). Ile279 --> Ala, Ile283 --> Ala, and His288 --> Ala showed reduced Na+ affinity of the E1 form. The rate of Na(+)-activated phosphorylation from ATP was reduced in Ile279 --> Ala and Ile283 --> Ala, and these mutants showed evidence similar to Glu329 --> Gln of destabilization of the Na(+)-occluded state.     

Functional consequences of alterations to Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum with their corresponding amides. These residues are predicted to lie in the transmembrane domain and have been suggested as oxygen ligands for Ca2+ binding at high affinity sites (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). The Glu309----Gln and Asp800----Asn mutants were unable to form a phosphoenzyme from ATP at the Ca2+ concentrations examined (up to 12.5 mM), whereas the Glu771----Gln mutant phosphorylated from ATP at 2.5 mM Ca2+. In all three mutants, Ca2+ at concentrations well below 12.5 mM prevented or inhibited phosphorylation with Pi, suggesting that at least one calcium-binding site was functioning in each mutant. In the mutants Glu309----Gln and Glu771----Gln, the ADP-insensitive phosphoenzyme intermediate was unusually stable, as indicated by a very low rate of dephosphorylation observed in kinetic experiments and by an increased apparent affinity for Pi determined in equilibrium phosphorylation experiments. These data indicate a central role of Glu309 and Glu771 in the energy-transducing conformational changes and/or in the activation of phosphoenzyme hydrolysis.     

Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.     

Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum

Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+ transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+ transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+ transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+ transport activity to alterations of Asn111 and Asn114. The mutant Asn114 to alanine retained about 50% activity, whereas the change Asn111 to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+ uptake observed in the Asn111 and Asn114 mutants was not due to a defect in enzyme activation by Ca2+ or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+ following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+ and to channel it to domains that are involved in enzyme activation and cation translocation. Simultaneous mutation of 4 glutamate residues to alanine in the lumenal loop between transmembrane sequences M1 and M2 did not affect Ca2+ transport activity, indicating that acidic residues in this lumenal loop do not play an essential role in Ca2+ transport. Similarly, mutation of Glu192 and Asp196 in the beta-strand domain between stalk helices 2 and 3 did not affect Ca2+ transport activity, although mutation of Asp196 did diminish expression of the protein.     

Critical hydrophobic interactions between phosphorylation and actuator domains of Ca2+-ATPase for hydrolysis of phosphorylated intermediate

Functional roles of seven hydrophobic residues on the interface between the actuator (A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca2+-ATPase were explored by alanine and serine substitutions. The residues examined were Ile179/Leu180/Ile232 on the A domain, Val705/Val726 on the P domain, and Leu119/Tyr122 on the loop linking the A domain and M2 (the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr122 in the crystal structures of Ca2+-ATPase in Ca2+-unbound E2 (unphosphorylated) and E2P (phosphorylated) states but are far apart in those of Ca2+-bound E1 (unphosphorylated) and E1P (phosphorylated) states. The substitution-effects were also compared with those of Ile235 on the A domain/M3 linker and those of T181GE of the A domain, since they are in the immediate vicinity of the Tyr122-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca2+-activated E1P formation from ATP. Substitutions of Ile235 and T181GE blocked the E1P to E2P transition, whereas those in the Tyr122-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile235 and Glu183 also blocked EP hydrolysis. Results indicate that the Tyr122-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu183 properly for hydrolyzing the acylphosphate. Ile235 on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr122-cluster, Ile235, and T181GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca2+.     

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.     

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.     

Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites

The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca(2+)-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca(2+) dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca(2+) at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His resulted in a conspicuous increase in the rate of Ca(2+) dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca(2+) dissociation, whereas a conspicuous decrease both in the rate of Ca(2+) dissociation and in the rate of Ca(2+) binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH(2)-terminal half of M3 is involved in control of the gateway to the Ca(2+) sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.     

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.     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.