Interference with phosphoenzyme isomerization and inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene

ATP hydrolysis and Ca(2+) transport by the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) are inhibited by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br(2)-TITU) in the micromolar range (Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566). In a study of the mechanism of inhibition, we found that Br(2)-TITU allows the enzyme to bind Ca(2+) and undergo phosphorylation by ATP. The level of ADP-sensitive phosphoenzyme (i.e. E1P-2Ca(2+)) observed in the transient state following addition of ATP is much higher in the presence than in the absence of the inhibitor. Br(2)-TITU does not interfere with enzyme phosphorylation by P(i) in the reverse direction of the cycle (i.e. E2P) and produces only a slight inhibition of its hydrolytic cleavage. The inhibitory effect of Br(2)-TITU on steady state ATPase velocity is attributed to interference with the E1P-2Ca(2+) to E2P-2Ca(2+) transition. In fact, experiments on conformation-dependent protection from proteolytic digestion suggest that, in the presence of Br(2)-TITU, the loops connecting the "A" domain to the ATPase transmembrane region undergo greater fluctuation than expected in the E2 and E2P states. Optimal stability of the gathered headpiece domains is thereby prevented. These effects are opposite to those of thapsigargin, in which the mechanism of inhibition is related to stabilization of a highly compact ATPase conformation and interference with Ca(2+) binding and phosphoenzyme formation. Our experiments with Br(2)-TITU provide the first demonstration of a kinetic limit posed by an inhibitor on the E1P-2Ca(2+) to E2P-2Ca(2+) transition in the wild-type enzyme.     

Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca(2+) ATPase) was adsorbed on a solid supported membrane and then activated with Ca(2+) and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca(2+) jumps in the absence of ATP and following ATP jumps in the presence of Ca(2+). The related charge movements are attributed to Ca(2+) reaching its binding sites in the ground state of the enzyme (E(1)) and to its vectorial release from the enzyme phosphorylated by ATP (E(2)P). The Ca(2+) concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H(+) in exchange for Ca(2+) at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca(2+) and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca(2+)/H(+) exchange is lower in the E(1) than in the E(2)P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca(2+) affinity produced by ATP utilization and results in the countertransport of two Ca(2+) and two H(+) within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca(2+) binding domain, are involved in the observed Ca(2+)/H(+) exchange.     

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.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

Functional and structural roles of critical amino acids within the"N", "P", and "A" domains of the Ca2+ ATPase (SERCA) headpiece

Twenty five amino acids within the "N", "P", and "A" domains of the Ca(2+) ATPase (SERCA1) headpiece were subjected to site directed mutagenesis, taking advantage of a high yield expression system. Functional and conformational effects of mutations were interpreted systematically in the light of the high resolution WT structure, defining direct involvement in catalysis as well as in stabilization of various positions acquired by each domain upon substrate binding and utilization. Amino acids involved in binding of ATP (such as Phe487 and Arg560 in the N domain) or phosphate (such as Asp351, Thr625, Lys684, and Thr353 in the P domain) were characterized with respect to their binding mechanism. Further identified were "positional" roles of several amino acids that stabilize neighboring residues for optimal binding of substrate or Mg(2+), or interface between headpiece domains as they change their relative positions in the course of the catalytic cycle. These include cross-linking of the "N" and "P" domains (e.g., Arg560/Asp627 salt bridge to stabilize domain approximation by ATP binding), and stabilization of the "A", "N", and activated "P" domains in arrangements differing from the ground E2 state and driven by catalytic events. This stabilization is produced through hydrogen bonds at domain interfaces, which vary depending on the intermediate state (e.g., Glu486/T171 in E1P and E2P, as opposed to Glu486/H190 in E2). We demonstrate that specific arrangements of the headpiece domains shown in crystal structures are, in fact, required to trigger displacement of transmembrane segments during the enzyme cycle in solution, allowing long range linkage of catalytic and Ca(2+) binding functions.     

Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase

Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P(i), without a significant effect on Ca(2+) binding.     

Regulation of intracellular ATP concentration under conditions of reduced ATP consumption in pancreatic islets.

ATP is the most important factor in glucose-induced insulin secretion in pancreatic beta-cells, but examination of intracellular differences in ATP concentration is difficult because ATP production and consumption occur simultaneously. In the present study, we measured the ATP concentration under the condition of a reduced ATP requirement by omitting extracellular Ca(2+) and inhibiting Na-K ATPase. The ATP concentration in islets incubated with 16.7 mM glucose in the absence of Ca(2+) for 30 min was increased by about 1. 9-fold more than in the presence of Ca(2+). The increment was extracellular Ca(2+)-dependent, and was completely abolished by the metabolic inhibitors dinitrophenol and iodoacetic acid. The Ca channel blockers including nitrendipine and Ni(2+) did not affect the ATP concentration in islets incubated with 16.7 mM glucose in the presence of Ca(2+). However, when thapsigargin and suramin, inhibitors of Ca-ATPase at the endoplasmic reticulum, were added to Ca channel blockers in the presence of ambient Ca(2+), the intraislet ATP content was increased, similarly to that under Ca-free conditions. But thapsigargin did not further augment the ATP concentration in the islet with 16.7 mM glucose in the absence of Ca(2+). On the other hand, the suppression of Na-K ATPase by ouabain rather reduced the ATP concentration augmented by omission of extracellular Ca(2+). In addition, vanadate, a blocker of Ca-ATPase at the plasma membrane, failed to increase the ATP concentration in the islets. These data suggest that the increment of ATP concentration in the absence of Ca(2+) is attributable to the reduced ATP requirement due to stopping of the Ca-ATPase activity at the endoplasmic reticulum, and that the intracellular ATP concentration is differently regulated by Na-K ATPase at plasma membrane and by Ca-ATPase at endoplasmic reticulum.     

Substrate-induced conformational fit and headpiece closure in the Ca2+ATPase (SERCA)

Protection of the Ca2+ATPase (SERCA) from proteinase K digestion has been observed following the addition of Ca2+, Mg2+, and nucleotide and interpreted as a substrate-dependent conformational change (1). The protected digestion site is located on the loop connecting the A domain and the M3 transmembrane helix. We studied by mutational analysis the protective effect of AMP-PCP, an ATP analog that is not utilized for enzyme phosphorylation. We found that the nucleotide protective effect is interfered with by single mutations of Arg-560 and Glu-439 in the N domain and Lys-352, Lys-684, Thr-353, Asp-703, and Asp-707 in the P domain. This is consistent with a transition from the open to the compact configuration of the ATPase headpiece and approximation of the N and P domains by interactions with the nucleotide adenosine and phosphate moieties, respectively. The A domain-M3 loop is consequently involved. Protection by nucleotide substrate increased following the mutations of Asp-351 (the residue undergoing phosphorylation by ATP) and neighboring Asn-706 to Ala, underlying the importance of side chain specificity in positioning the nucleotide terminal phosphate and limiting the stability of the substrate-enzyme complex. Protection is not observed when AMP-PCP is added in the absence of Ca2+ or following mutations (E771Q or N796A) that interfere with Ca2+ binding. Therefore, nucleotide binds to the Ca2+-activated enzyme in the open headpiece conformation and the consequent approximation of the N and P domains occurs while the transmembrane domain is still in the Ca2+-bound conformation. Mg2+ is not required for the protective effect of nucleotide, even though it is specifically required for the subsequent catalytic reactions.     

Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.     

Deletions of any single residues in Glu40-Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate

Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.     

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.     

CRACM1 multimers form the ion-selective pore of the CRAC channel

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.     

The Ca2+-ATPase (SERCA1) is inhibited by 4-aminoquinoline derivatives through interference with catalytic activation by Ca2+, whereas the ATPase E2 state remains functional

Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to μm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the μm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.3 and 8.0 μm as demonstrated by measurements of steady state ATPase activity as well as single cycle charge transfer. Characterization of sequential reactions comprising the ATPase catalytic and transport cycle then demonstrated that NF1058, and similarly CLT, interferes with the mechanism of Ca(2+) binding and Ca(2+)-dependent enzyme activation (E(2) to E(1)·Ca(2) transition) required for formation of phosphorylated intermediate by ATP utilization. On the other hand, Ca(2+) independent phosphoenzyme formation by utilization of P(i) (i.e. reverse of the hydrolytic reaction in the absence of Ca(2+)) was not inhibited by NF1058 or CLT. Comparative experiments showed that the high affinity inhibitor thapsigargin interferes not only with Ca(2+) binding and phosphoenzyme formation with ATP but also with phosphoenzyme formation by utilization of P(i) even though this reaction does not require Ca(2+). It is concluded that NF1058 and CLT inhibit SERCA by stabilization of an E(2) state that, as opposed to that obtained with thapsigargin, retains the functional ability to form E(2)-P by reacting with P(i).     

Val200 residue in Lys189-Lys205 outermost loop on the A domain of sarcoplasmic reticulum Ca2+-ATPase is critical for rapid processing of phosphoenzyme intermediate after loss of ADP sensitivity

Possible roles of the Lys(189)-Lys(205) outermost loop on the A domain of sarcoplasmic reticulum Ca(2+)-ATPase were explored by mutagenesis. Both nonconservative and conservative substitutions of Val(200) caused very strong inhibition of Ca(2+)-ATPase activity, whereas substitutions of other residues on this loop reduced activity only moderately. All of the Val(200) mutants formed phosphoenzyme intermediate (EP) from ATP. Isomerization from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was not inhibited in the mutants, and a substantially larger amount of E2P actually accumulated in the mutants than in wild-type sarcoplasmic reticulum Ca(2+)-ATPase at steady state. In contrast, decay of EP formed from ATP in the presence of Ca(2+) was strongly inhibited in the mutants. Hydrolysis of E2P formed from P(i) in the absence of Ca(2+) was also strongly inhibited but was faster than the decay of EP formed from ATP, indicating that the main kinetic limitation of the decay comes after loss of ADP sensitivity but before E2P hydrolysis. On the basis of the well accepted mechanism of the Ca(2+)-ATPase, the limitation is likely associated with the Ca(2+)-releasing step from E2P.Ca(2). On the other hand, the rate of activation of dephosphorylated enzyme on high affinity Ca(2+) binding was not altered by the substitutions. In light of the crystal structures, the present results strongly suggest that Val(200) confers appropriate interactions of the Lys(189)-Lys(205) loop with the P domain in the Ca(2+)-released form of E2P. Results further suggest that these interactions, however, do not contribute much to domain organization in the dephosphorylated enzyme and thus would be mostly lost on E2P hydrolysis.     

The ATP-Mg2+ binding site and cytoplasmic domain interactions of Na+,K+-ATPase investigated with Fe2+-catalyzed oxidative cleavage and molecular modeling

This work utilizes Fe(2+)-catalyzed cleavages and molecular modeling to obtain insight into conformations of cytoplasmic domains and ATP-Mg(2+) binding sites of Na(+),K(+)-ATPase. In E(1) conformations the ATP-Fe(2+) complex mediates specific cleavages at 712VNDS (P domain) and near 440VAGDA (N domain). In E(2)(K), ATP-Fe(2+) mediates cleavages near 212TGES (A domain), near 440VAGDA, and between residues 460-490 (N domain). Cleavages at high ATP-Fe(2+) concentrations do not support suggestions for two ATP sites. A new reagent, fluorescein-DTPA, has been synthesized. The fluorescein-DTPA-Fe(2+) complex mediates cleavages similar to those mediated by ATP-Fe(2+). The data suggest the existence of N to P domain interactions in E(1)Na, with bound ATP-Fe(2+) or fluorescein-DPTA-Fe(2+), A-N, and A-P interactions in E(2)(K), and provide testable constraints for model building. Molecular models based on the Ca(2+)-ATPase structure are consistent with the predictions. Specifically, high-affinity ATP-Mg(2+) binding in E(1) is explained with the N domain tilted ca. 80 degrees toward the P domain, by comparison with well-separated N and P domains in the Ca-ATPase crystal structure. With ATP-Mg(2+) docked, bound Mg(2+) is close to both D710 (in 710DGVNDS) and D443 (in 440VAGDASE). D710 is known to be crucial for Mg(2+) binding. The cleavage and modeling data imply that D443 could also be a candidate for Mg(2+) binding. Comparison of E(1).ATP,Mg(2+) and E(2) models suggests an explanation of the high or low ATP affinities, respectively. We propose a scheme of ATP-Mg(2+) and Mg(2+) binding and N, P, and A domain interactions in the different conformations of the catalytic cycle.     

Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis

Partial proteolysis by exogenous proteases in the presence and absence of Ca(2+) was used to map the protease-resistant domains in m-calpain, and to obtain evidence for the conformational changes induced in this thiol protease by Ca(2+). The complication of autoproteolysis was avoided by using the inactive Cys105Ser calpain mutant. Both trypsin and chymotrypsin produced similar cleavage patterns from the large subunit (domains I-IV), while the small subunit (domain VI) was largely unaffected. N-Terminal sequencing of the major products showed that hydrolysis occurred in the N-terminal anchor peptide, which binds domain I to domain VI, at a site close to the C terminus of domain II, and at several sites within domain III. Of particular importance to the overall Ca(2+)-induced conformational changes was the increase in mobility and accessibility of domain III. The same sites were cleaved in the presence and absence of Ca(2+), but with one exception digestion was much more rapid in the presence of Ca(2+). The exception was a site close to residue 255 located within the active site cleft. This site was accessible to cleavage in the absence of Ca(2+), when the active site is not assembled, but was protected in the presence of Ca(2+). This result supports the hypothesis that Ca(2+) induces movement of domains I and II closer together to form the functional active site of calpain.     

Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.     

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.