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J. Biol. Chem., Vol. 282, Issue 47, 34429-34447, November 23, 2007

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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^*

From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan

Received for publication, September 12, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The functional importance of the length of the A/M1 linker (Glu⁴⁰-Ser⁴⁸) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca²⁺-ATPase was explored by its elongation with glycine insertion at Pro⁴²/Ala⁴³ and Gly⁴⁶/Lys⁴⁷. 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 E2PCa₂ possessing two occluded Ca²⁺ ions at the transport sites, and the Ca²⁺ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca²⁺-free form of E2P produced from P_i without Ca²⁺ was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa₂ accumulated is an intermediate state between E1PCa₂ and the Ca²⁺-released E2P state. Namely in E2PCa₂, the actuator domain is already largely rotated from its position in E1PCa₂ and associated with the phosphorylation domain as in the Ca²⁺-released E2P state; however, in E2PCa₂, the hydrophobic interactions among these domains and Leu¹¹⁹/Tyr¹²² on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr¹²² are critical for formation of the Ca²⁺-released E2P structure. Results showed that the EP isomerization/Ca²⁺-release process consists of the following two steps: E1PCa₂ E2PCa₂ E2P + 2Ca²⁺; and the intermediate state E2PCa₂ 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 E2PCa₂, is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca²⁺ deocclusion and release.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a)² is a representative member of P-type ion transporting ATPases and catalyzes Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (Refs. 1, 2 and for recent reviews see Refs. 3-7). In the catalytic cycle, the enzyme is activated by binding of two Ca²⁺ ions to the transport sites (E2to E1Ca₂, steps 1 and 2) and then autophosphorylated at Asp³⁵¹ with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which can react with ADP to regenerate ATP in the reverse reaction. Upon formation of this EP, the bound Ca²⁺ ions are occluded in the transport sites (E1PCa₂). The subsequent isomeric transition to the ADP-insensitive form (E2P), i.e. the loss of the ADP sensitivity at the catalytic site, results in rearrangements of the Ca²⁺-binding sites to deocclude Ca²⁺, reduce the affinity, open the luminal gate, and thus release Ca²⁺ into the lumen (steps 4 and 5). As an intermediate state in the EP isomerization/Ca²⁺-release process, E2PCa₂ has been postulated (e.g. see Ref. 8), although this state has never been identified. Finally, the E2P hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca²⁺-unbound form (E2, steps 6 and 7). The transport cycle is totally reversible, e.g. E2P can be formed from P_i in the presence of Mg²⁺ and the absence of Ca²⁺ by reversal of its hydrolysis, and the subsequent addition of high concentrations of Ca²⁺ to E2P reverse the Ca²⁺-releasing step and the E1P to E2P isomerization.

The enzyme has three cytoplasmic domains as follows: the nucleotide binding (N), phosphorylation (P), and actuator (A) domains, and 10 transmembrane helices M1-M10 (Fig. 2). During the EP isomerization/Ca²⁺-release E1PCa₂ E2P + 2Ca²⁺, the A domain largely rotates (by 110°) parallel to the membrane and associates with the P domain (see Refs. 9-17) (see E1·AlF_x·ADP (the E1PCa₂·ADP analog) (the E2·P_i analog) in Fig. 2). The interactions of the A domain with the P domain in the E2P state occur at three regions (Fig. 2, semitransparent purple, blue, and orange on ): i.e. at the T¹⁸¹GES loop with the residues of the P domain around Asp³⁵¹; at the Val²⁰⁰ loop (Asp¹⁹⁶-Asp²⁰³) with the polar residues of the P domain (Arg⁶⁷⁸/Glu⁶⁸⁰/Arg⁶⁵⁶/Asp⁶⁶⁰); and at the Tyr¹²²-hydrophobic cluster formed by seven hydrophobic residues gathered from the A domain (Ile¹⁷⁹/Leu¹⁸⁰/Ile²³²), the P domain (Val⁷⁰⁵/Val⁷²⁶), and the top part of M2 (the A/M2 linker region, Leu¹¹⁹/Tyr¹²²). The formation of the A-P domain interaction at the T¹⁸¹GES loop has been predicted to be critical for the loss of ADP sensitivity at the catalytic site, i.e. the E1P to E2P isomerization, by the structural and mutation studies (18-20). The mutations at the latter two interaction regions were shown not to inhibit the E1P to E2P isomerization but to markedly retard the subsequent EP decay (19, 21, 22). Its kinetics were consistent with the view that there is a Ca²⁺-releasing step from E2PCa₂(E2PCa₂ E2P + 2Ca²⁺) before the E2P hydrolysis and that this Ca²⁺-releasing step is blocked and became the kinetic limit for the EP decay by the disruption of the A-P domain interactions at each of the latter two regions (19, 21, 22). It is therefore very interesting to know how the motions and interactions of the A and P domains progress during the postulated successive steps E1PCa₂ E2PCa₂ E2P + 2Ca²⁺ as the key structural events in the energy coupling between the cytoplasmic and transmembrane domains. In this respect, it is also critical to clarify and distinguish the structural roles of the three linkers connecting the A domain with M1'/M1, M2, and M3 (A/M1, A/M2, and A/M3 linkers). Tyr¹²²/Leu¹¹⁹ involved in the aforementioned Tyr¹²²-hydrophobic cluster is at the A/M2 linker region. The A/M3 linker, because of its strain, has been predicted to be important for the large rotation of the A domain in the EP isomerization (12, 13).

View larger version (7K): [in this window] [in a new window]   FIGURE 1. Ca2+ transport cycle of SERCA.

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Structure of SERCA1a. a, structural model for the change E1PCa2 E2P + 2Ca2+ was depicted with E1·AlFx·ADP (E1PCa2·ADP analog) and (E2·Pi analog) (Protein Data Bank accession codes 1WPE and 1WPG, respectively; see Refs. 13 and 17). The cytoplasmic three domains N, P, and A are green, pink, and yellow. The transmembrane helices (M1-M10) are numbered, and some are colored. The two structures were manually fitted with M8 -M10, which virtually do not move in the two structures. In E1·AlFx·ADP, the two Ca2+ ions bound at their binding (transport) sites are depicted (red spheres); the sites consist of the residues on M4, M5, M6, and M8. The linkers connecting the A domain with M1 (A/M1 linker with red), with M2 (A/M2 linker with green), and with M3 (A/M3 linker with gray) are indicated. The arrows on E1·AlFx·ADP with yellow, pink, and blue show the movements of the A domain, the P domain, and M1', respectively, in the change E1·AlFx·ADP . The phosphorylation site Asp351, AlFx, and MgF2-4 are shown, but ADP was not depicted for simplicity. The specific cleavage sites for trypsin (T2, Arg198 on the Val200 loop (Asp196-Asp203); T1, Arg505) and proteinase K (PrtK, Leu119) are indicated on E1·AlFx·ADP. The semitransparent spheres on with purple, blue, and orange indicate the three contact regions between the A and P domains, i.e. at the T181GES loop (purple loop with TGES) with the residues around Asp351, at the Val200 loop, including Arg198 (dark blue loop) with the polar residues of the P domain (Arg678/Glu680/Arg656/Asp660), and at Tyr122/Leu119. Tyr122 and Leu119 on the top part of M2 (A/M2 linker region) form the interaction network "Tyr122-hydrophobic cluster" with Ile179/Leu180/Ile232 of the A domain and Val705/Val726 of the P domain (see Fig. 13). Details of the other two interaction regions are also not depicted for simplicity (see supplemental Figs. II and III in Ref. 19 for the details). b, in , the regions at the A/M1 linker (Glu40-Ser48, red), M1' (Leu49-Asp59, blue), M1 (blue), and the N-terminal neighbor of the A/M1 linker (yellow) are depicted. The sites for the insertion mutations made in this study are at Gly46/Lys47 (between Gly46 and Lys47) and Pro42/Ala43 on the A/M1 linker and at Thr22/Gly23 (as indicated with the -carbons in pink spheres), and at Val53/Ile54 on M1'.

Results demonstrated that the elongation of the linker markedly accelerates the E1PCa₂ to E2PCa₂ isomerization, strongly stabilizes E2PCa₂ that possesses two occluded Ca²⁺ ions at the transport sites, and blocks the Ca²⁺ deocclusion and release from E2PCa₂. Thus, for the first time, the intermediate state E2PCa₂ was identified and trapped in this study. We were then able to characterize the structure of this state. Results revealed that the correct length of the A/M1 linker is critical for structural events in each of successive steps in E1PCa₂ E2PCa₂ E2P + 2Ca²⁺ and E2P + H₂O E2 + P_i, and they further suggested how the motions and interactions of the properly positioned A and P domains progress with the critical contribution of the linker to accomplish the successive structural events in these steps. Our study also revealed the importance of M1' directly connected with the A/M1 linker likely for forming the base of this linker.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mutagenesis and Expression—The QuikChange^TM site-directed mutagenesis method (Stratagene, La Jolla, CA) was utilized for the insertions and substitutions of residues in the rabbit SERCA1a cDNA. The ApaI-KpnI restriction fragments with the desired mutation were excised from the plasmid and ligated back into the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector (24). The pMT2 DNA was transfected into COS-1 cells by the liposome-mediated transfection method. Microsomes were prepared from the cells as described previously (25). The "control microsomes" were prepared from COS-1 cells transfected with the pMT2 vector containing no SERCA1a cDNA. The amount of expressed SERCA1a was quantified by a sandwich enzyme-linked immunosorbent assay as described previously (26). Expression levels of the wild-type SERCA1a and mutants examined in this study were 2-3% of total microsomal proteins, except a mutant 4950525354S for M1' with serine substitutions of Leu⁴⁹/Trp⁵⁰/Leu⁵²/Val⁵³/Ile⁵⁴, which showed markedly reduced expression (only 15-20% of the wild type).

Ca²⁺-ATPase Activity—The rate of ATP hydrolysis was determined at 25 °C in a mixture containing 1 µg of microsomal protein, 0.1 mM [-³²P]ATP, 1 µM A23187 [GenBank] , 0.1 M KCl, 7 mM MgCl₂, 0.55 mM CaCl₂, 0.5 mM EGTA, and 50 mM MOPS/Tris (pH 7.0). The Ca²⁺-ATPase activity was obtained by subtracting the Ca²⁺-independent ATPase activity, which was determined in the presence of 5 mM EGTA without added CaCl₂, otherwise as above. The ATPase activity/mg of expressed SERCA1a protein was calculated from the amount of expressed SERCA1a and the Ca²⁺-ATPase activity of expressed SERCA1a, which was obtained by subtracting the Ca²⁺-ATPase activity of the control microsomes from that of the microsomes expressing SERCA1a. This background level with the control microsomes was as low as 3% of the activity of microsomes expressing the wild-type SECRA1a.

Formation and Hydrolysis of EP—Phosphorylation of SERCA1a in microsomes with [-³²P]ATP or ³²P_i and dephosphorylation of ³²P-labeled SERCA1a were performed under conditions described in the figure legends. The reactions were quenched with ice-cold trichloroacetic acid containing P_i. Rapid kinetics measurements of phosphorylation and dephosphorylation were performed with a handmade rapid mixing apparatus (27), otherwise as above. The precipitated proteins were separated by 5% SDS-PAGE at pH 6.0 according to Weber and Osborn (28). The radioactivity associated with the separated Ca²⁺-ATPase was quantitated by digital autoradiography as described (29). The amount of EP formed with the expressed SERCA1a was obtained by subtracting the background radioactivity with the control microsomes. This background was less than 5% of the radioactivity of EP formed with the expressed wild-type SERCA1a. The amount of EP/mg of SERCA1a protein was calculated from the amount of EP thus obtained and the amount of expressed SERCA1a.

Ca²⁺ Occlusion in EP—As described in Fig. 8 legend, the expressed mutant SERCA1a in microsomes was phosphorylated with ATP and ⁴⁵CaCl₂, and then the mixture was diluted by a "washing solution" containing excess EGTA and immediately filtered through a 0.45-µm nitrocellulose membrane filter (Millipore). The filter was washed four times with 2 ml of the washing solution, and ⁴⁵Ca²⁺ remaining on the filter was quantitated. The amount of Ca²⁺ specifically bound to the transport sites of EP in the expressed SERCA1a was obtained by subtracting the amount of nonspecific Ca²⁺ binding, which was determined by including 1 µM thapsigargin (TG) in the phosphorylation mixture, otherwise as above. This background subtraction is ensured by the fact that TG inhibits the Ca²⁺ binding at the transport sites and the EP formation (30). The background level thus determined was 60% of the total amount of ⁴⁵Ca²⁺ remaining on the filter when the maximum amount of EP was present (i.e. at the zero time of EP decay in Fig. 8). It should be noted that the specifically bound Ca²⁺ in EP thus determined represents the occluded one because it is not released even after the extensive washing by EGTA. The Ca²⁺ occluded/mg of expressed SERCA1a protein was calculated from the amount of expressed SERCA1a and the amount of occluded Ca²⁺. The Ca²⁺ occlusion resulted from Ca²⁺ binding to E2P in the reverse reaction of the Ca²⁺-release process was also determined. In this case, E2P was first formed from P_i in the absence of Ca²⁺, and ⁴⁵Ca²⁺ was then added to E2P otherwise as described in Fig. 10 legend, and the amount of occluded ⁴⁵Ca²⁺ was determined as above.

Limited Proteolysis of Major Intermediates and Western Blot Analysis—Major intermediates and its stable analogs of the Ca²⁺-ATPase were produced and subjected to the structural analysis by limited proteolysis with trypsin and proteinase K (PrtK) as described in Fig. 12 legend. The digests were separated by 10.5 or 7.5% SDS-PAGE, according to Laemmli (31), and blotted onto a polyvinylidene fluoride membrane and then incubated with IIH11 monoclonal antibody to the rabbit SERCA1a (Affinity Bioreagents), which recognizes an epitope between Ala¹⁹⁹-Arg⁵⁰⁵. After incubation with secondary antibody (goat anti-mouse IgG-horseradish peroxidase-conjugated), the bound proteins were probed using an enhanced chemiluminescence-linked detection system (ECL Plus, GE Healthcare).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Ca2+-ATPase activities of expressed SERCA1a. The Ca2+-ATPase activity of expressed SERCA1a was determined as described under "Experimental Procedures." The activities are represented as percentage of that of the wild type (1.2 ± 0.1 µmol/min/mg of SERCA1a protein (n = 6)). The values presented are the mean ± S.D. (n = 3-4). 1Gi-46/47 to 6Gi-46/47 represent the mutants with a one to six glycine(s) insertion between Gly46 and Lys47. Likewise, 1Gi-42/43 and 4Gi-42/43 are for the mutants with a one and four glycine(s) insertion between Pro42 and Ala43, respectively; 4Ai-22/23 and 4Ai-53/54 are for the mutants with a four alanine insertion between Thr22 and Gly23 and between Val53 and Ile54, respectively. In the mutant 4950525354S, Leu49/Trp50/Leu52/Val53/Ile54 on M1' are all substituted by serine. In the mutant 5155565859A, Glu51/Glu55/Gln56/Glu58/Asp59 on M1' are all substituted by alanine.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ca²⁺-ATPase Activity—The specific Ca²⁺-ATPase activity of the expressed SERCA1a mutants was determined at saturating 50 µM Ca²⁺ and 25 °C and compared with that of the wild type (Fig. 3). Insertion of one glycine between Gly⁴⁶ and Lys⁴⁷ (1Gi-46/47) and between Pro⁴² and Ala⁴³ (1Gi-42/43) within the A/M1 linker slowed the ATPase activity by 50%. Insertion of two or more glycines at each of these sites abolished the activity almost completely (2Gi-46/47 to 6Gi-46/47 and 4Gi-42/43). Thus, the elongation of the linker by the glycine insertion at the two different positions within the A/M1 linker exhibited the same inhibitory effects on the ATPase, indicating the importance of the correct length of this loop in the function.³

We then examined the possible effects of the 4-amino acidinsertion in the C- and N-terminal regions of the A/M1 linker: between Val⁵³ and Ile⁵⁴ on the helix M1' and between Thr²² and Gly²³ in the immediate vicinity of the Thr²⁵-Tyr³⁶ helix (see Fig. 2b). In the mutants 4Ai-53/54 and 4Ai-22/23, alanines were inserted (for M1', we intended to minimize possible disruption of the helical structure). These mutants exhibited the high ATPase activity (Fig. 3) and the Ca²⁺ transport coupled with the ATP hydrolysis (data not shown). Thus the insertions at the adjacent regions of the A/M1 linker did not inhibit the activity. We inserted amino acids also in the immediate N-terminal region of the A/M1 linker, for example at His³²-Leu³³ and at Gly³⁷-His³⁸; however, the protein expression levels of these mutants were extremely low (less than 10% of the wild type); therefore, their functional analysis was not possible.

We also investigated the possible importance of amphipathic property of the helix M1' (Trp⁵⁰-Glu⁵⁸ in E1·AlF_x·ADP or Leu⁴⁹-Gln⁵⁶ in ), which is directly connected with the A/M1 linker and formed by kinking of M1. M1' lies on the membrane surface, having hydrophobic residues aligned on the membrane side (Leu⁴⁹/Trp⁵⁰/Val⁵³/Ile⁵⁴) and the polar residues (Glu⁵¹/Glu⁵⁵/Gln⁵⁶/Glu⁵⁸) on the cytoplasmic side (Fig. 2b). Therefore, the hydrophobic interactions of M1' with the membrane core and/or its hydrophilic interactions at the membrane surface may possibly be important for function (13). Previously the mutations of the single residues on the M1' region were found to have almost no or only a slight effect on the activity (23, 34). In this study, we therefore introduced the extensive nonconservative substitutions as follows: the serine substitution of all Leu⁴⁹/Trp⁵⁰/Leu⁵²/Val⁵³/Ile⁵⁴ (4950525354S) and the alanine substitution of all Glu⁵¹/Glu⁵⁵/Gln⁵⁶/Glu⁵⁸/Asp⁵⁹ (5155565859A). The mutant 4950525354S exhibited the markedly reduced ATPase activity (17% that of the wild type), whereas the mutant 5155565859A exhibited a fairly high activity (70% that of the wild type). The results indicate that the hydrophobic interaction of M1' with the membrane core may be important.

EP Formation from ATP and the E2-E1Ca₂ Transition—The amount of EP formed from ATP at saturating 50 µM Ca²⁺ was determined at steady state and 0 °C with 10 µM ATP under the conditions otherwise the same as those for the ATPase assay (open bars in Fig. 4). All the mutants formed EP with the amounts comparable with that of the wild type (3.31 ± 0.14 nmol/mg of the SERCA1a protein (n = 4)) except that the mutant 4950525354S exhibited a somewhat reduced amount. The Ca²⁺ affinity of the mutants in the E2to E1Ca₂ transition was estimated by the Ca²⁺ dependence of the EP formation from ATP and was found to be nearly the same as that of the wild type (see the Ca²⁺ affinity and the Hill coefficient in Table 1). The mutant 4Ai-53/54 among those examined showed a slightly reduced affinity. We further found that the first-order rate constants of the E2to E1Ca₂ transition (steps 1 and 2) in the examined mutants were nearly the same as in the wild type (Table 1). In this experiment, the enzyme was first preincubated in the absence of Ca²⁺ at pH 6, where the equilibrium between E1 and E2 is most shifted to E2 (35), and then the phosphorylation was initiated by the simultaneous addition of saturating Ca²⁺ and ATP. When ATP was added to the enzyme preincubated with Ca²⁺, otherwise as above, the EP formation was much faster in the mutants as well as in the wild type, and therefore the rates obtained above actually reflect the rate-limiting E2to E1Ca₂ transition.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Total amount of EP formed from ATP at steady state and the amount of ADP-insensitive EP. Microsomes expressing the wild-type or mutant SERCA1a were phosphorylated with [-32P]ATP at 0 °C for 1 min in 50 µl of a mixture containing 1 µg of microsomal protein, 10 µM [-32P]ATP, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, 0.55 mM CaCl2, 0.5 mM EGTA, and 50 mM MOPS/Tris (pH 7.0). The total amount of EP formed (open bars) was determined by the acid quenching as described under "Experimental Procedures." For determination of ADP-insensitive EP (closed bars), an equal volume of a mixture (50 µl) containing 4 mM ADP, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, 10 mM EGTA, and 50 mM MOPS/Tris (pH 7.0) was added to the above phosphorylation mixture, and the reaction was quenched at 1 s after the ADP addition. ADP-sensitive EP (E1P) disappeared entirely within 1 s after the ADP addition.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Time course of accumulation of ADP-insensitive EP. Microsomes expressing the wild-type or mutant SERCA1a were phosphorylated with [-32P]ATP at 0 °C for various periods as indicated on the abscissa in 50 µl of a mixture containing 1 µg of microsomal protein, 10 µM [-32P]ATP, 1 µM A23187, 7 mM MgCl2, 0.55 mM CaCl2, 0.5 mM EGTA, and 50 mM MOPS/Tris (pH 7.0) in the presence of 0.1 M LiCl without added KCl (A and B) or 0.1 M KCl without LiCl (C and D). The reaction was quenched by acid, and the total amount of EP (A and C) was determined. For determination of ADP-insensitive EP (B and D), an equal volume (50 µl) of a mixture containing 4mM ADP, 1 µM A23187, 7 mM MgCl2, 10 mM EGTA, 50 mM MOPS/Tris (pH 7.0), and 0.1 M LiCl without KCl (B) or 0.1 M KCl without LiCl (D) was added to the above phosphorylation mixture at the indicated time. At 1 s after the ADP addition, the reaction was quenched by acid. Solid lines show the least squares fit to a single exponential, and the apparent rates to reach the steady-state level are given in Table 1. The maximum values of the total amount of EP obtained at infinite time in the fitting are normalized to 100% (A and C), and the amounts of the ADP-insensitive EP are shown as percentages of the maximum value of the total amount of EP (B and D).

It should be noted that the mutant 4950525354S exhibited the almost exclusive accumulation of ADP-insensitive EP both in the absence (Fig. 5B) and presence (Fig. 4) of K⁺ at steady state. In this regard, the removal of the hydrophobic property of M1' by the serine substitutions (removal of the likely hydrophobic interaction with the membrane core) caused the same consequence as that of the elongation of the A/M1 linker. Besides, the observed slow EP formation from E1Ca₂ and ATP in this mutant 4950525354S (Fig. 5A) suggests the importance of the hydrophobic property of this region in rapid structural changes for ATP binding and phosphorylation. The mutants 5155565859A on M1' and 4Ai-22/23 and 4Ai-53/54 adjacent to the A/M1 linker exhibited the extent and rate of the accumulation of ADP-insensitive EP almost the same as those of the wild type (see Table 1 for the rates, and Figs. 4 and 5B for the extent).

Decay of EP Formed from ATP and Ca²⁺—The decay of EP formed from ATP and Ca²⁺ was determined at 0 °C in the presence of K⁺ and is shown with the representative mutants in Fig. 6. The decay time courses were fitted well with a single exponential (Fig. 6A), and the rates were summarized in Table 1. The fraction of the ADP-insensitive EP (E2P) remaining in the decay course was also determined (Fig. 6B). In the wild type, the EP remaining was exclusively the ADP-sensitive EP (E1P), and this is consistent with the well known rate-limiting E1P to E2P transition in the ATPase cycle (36, 37). The EP decay was slightly slowed in the single glycine-insertion mutants 1Gi-46/47 (Fig. 6A) and 1Gi-42/43 (see Table 1), being consistent with the slight reduction in the ATPase activity in these mutants (cf. Fig. 3). The EP decay was almost completely blocked in the mutants with two or more glycine insertions in the A/M1 linker, 2Gi-46/47 to 4Gi-46/47 (Fig. 6A) and 4Gi-42/43 (see Table 1). This is consistent with the complete loss of the ATPase activity in these mutants. EP present at the start and course of the decay reaction was exclusively the ADP-insensitive one (E2P) in the mutants 3Gi-46/47 and 4Gi-46/47, and 50% in the mutant 2Gi-46/47 (Fig. 6B, as also shown in Figs. 4 and 5D). In the mutants with the elongated A/M1 linker with the two or more glycine insertions, these results show that the E1P-E2P isomerization was strongly shifted toward the ADP-insensitive EP (E2P) and that the decay of the ADP-insensitive EP was blocked. In the mutant 4950525354S on M1', the EP decay was markedly slowed (Fig. 6A), and the EP remaining was almost exclusively the ADP-insensitive EP during the decay reaction (data not shown, but see Fig. 4). Thus in this mutant, the decay of the ADP-insensitive EP was markedly retarded. The mutants 5155565859A (Fig. 6A), 4Ai-22/23, and 4Ai-53/54 (Table 1) showed the rapid EP decay as the wild type, being consistent with their high ATPase activities.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Decay of EP formed from ATP and the fraction of the ADP-insensitive EP during the decay. Microsomes expressing the wild-type or mutant SERCA1a were phosphorylated at 0 °C for 1 min in 50 µl of a mixture containing 1 µg of microsomal protein, 10 µM [-32P]ATP, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, 0.55 mM CaCl2, 0.5 mM EGTA, and 50 mM MOPS/Tris (pH 7.0). Phosphorylation was terminated by addition of an equal volume (50 µl) of a buffer containing 10 mM EGTA, 0.1 M KCl, 7 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0) at 0 °C. The total amount of EP remaining after the EGTA addition was determined by the acid quenching at the indicated time (A). The total amounts of EP obtained at zero time (i.e. immediately before the EGTA addition) are normalized to 100%. Solid lines show the least squares fit to a single exponential, and the decay rates thus obtained are given in Table 1. For determination of ADP-insensitive EP (B), an equal volume (100 µl) of a mixture containing 4 mM ADP, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, 10 mM EGTA, and 50 mM MOPS/Tris (pH 7.0) was added to the above EGTA-added phosphorylation mixture at the indicated time. At 1 s after the ADP addition, the reaction was quenched by acid. The amount of the ADP-insensitive EP at each of the indicated times thus determined is shown as percentage of the total amount of EP determined at zero time.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Hydrolysis of E2P formed from Pi without Ca2+. Microsomes expressing the wild-type or mutant SERCA1a were phosphorylated with 32Pi at 25 °C for 10 min in 5 µl of a mixture containing 1 µg of microsomal protein, 0.1 mM 32Pi, 1 µM A23187, 1 mM EGTA, 10 mM MgCl2, 50 mM MOPS/Tris (pH 7.0), and 35% (v/v) Me2SO. The mixture was then cooled and diluted at 0 °C by addition of 95 µl of a mixture containing 2.1 mM nonradioactive Pi, 105 mM KCl, 1 mM EGTA, 10 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0). At different times after the dilution, the E2P hydrolysis was quenched by acid. The amounts of E2P formed with 32Pi at zero time are normalized to 100%. Solid lines show the least squares fit to a single exponential, and the rates thus obtained are given in Table 1.

Ca²⁺ Occlusion in Stable E2P—The observed block of the decay of ADP-insensitive EP (E2P) formed from ATP with Ca²⁺ (Fig. 6) and the rapid hydrolysis of E2P formed from P_i without Ca²⁺ (Fig. 7) in the mutants indicate that there may be a kinetic limit for the decay of E2P formed with Ca²⁺ before the hydrolysis of E2P without bound Ca²⁺. This limiting step is possibly the Ca²⁺-releasing step from E2PCa₂; E2PCa₂ E2P + 2Ca²⁺ and E2PCa₂ may be stabilized and accumulated in the mutants. In Fig. 8, this possibility was directly examined by the determination of ⁴⁵Ca²⁺ occlusion in E2P accumulated from ATP and ⁴⁵Ca²⁺. With the representative mutants 4Gi-46/47 (Fig. 8, A and C) and 2Gi-46/47 (Fig. 8, B and C), EP was first formed from ATP and ⁴⁵Ca²⁺ at steady state in the absence or presence of the Ca²⁺ ionophore A23187 [GenBank] , and then the EP decay was initiated by the addition of excess EGTA. The amount of occluded ⁴⁵Ca²⁺ was determined at the indicated time by membrane filtration with an extensive washing with a solution containing EGTA and A23187. [GenBank] The total amount of EP and the fraction of ADP-insensitive EP (E2P) was determined by the use of [-³²P]ATP and nonradioactive Ca²⁺.

View larger version (15K): [in this window] [in a new window]   FIGURE 8. Occlusion of Ca2+ in EP formed from ATP and Ca2+. A, microsomes expressing the mutant 4Gi-46/47 were phosphorylated for 10 s with ATP and 45Ca2+ at 25 °C in 50 µl of a mixture containing 2 µg of microsomal protein, 1 µM ATP, 10 µM 45CaCl2, 0.1 M KCl, 7 mM MgCl2, and 30 mM MOPS/Tris (pH 7.0) in the presence () or absence () of 1 µM A23187. Then a small volume of EGTA was added to give 2 mM to terminate the phosphorylation. At the indicated time after this EGTA addition, the mixture was diluted at 25 °C by 2 ml of a washing solution containing 5 µM A23187, 0.1 M KCl, 7 mM MgCl2, 2 mM EGTA, and 50 mM MOPS/Tris (pH 7.0) and immediately filtered through a 0.45-µm nitrocellulose membrane filter. The samples on the filter were rapidly washed four times by 2 ml of the above washing solution at 25 °C. The amount of 45Ca2+ specifically bound to the expressed SERCA1a, i.e. the occluded Ca2+ in SERCA1a, was determined after this extensive washing by subtracting the amount of nonspecific Ca2+ binding, which was determined by including 1 µM TG in the above phosphorylation mixture, otherwise as described under "Experimental Procedures." The values are shown as "nmol per mg of SERCA1a applied on the filter" (, ). By using [-32P]ATP and nonradioactive CaCl2, the total amount of EP present in the above EGTA-added phosphorylation mixture was determined at the indicated time by acid quenching (, ). EP present was found to be exclusively the ADP-insensitive one (E2P) as also demonstrated with this mutant in Figs. 4, 5 and 6. The amounts of EP were also determined without the acid quenching but with the membrane filtration method as the above determination of the occluded 45Ca2+. The amounts of EP thus determined by the two methods actually agreed well with each other, and therefore the acid-quenching method was employed throughout the experiments in A and B for convenience. B, microsomes expressing the mutant 2Gi-46/47 were phosphorylated with ATP and 45Ca2+ in the presence of 1 µM A23187 at 10 °C otherwise as described in A. The phosphorylation was terminated by the EGTA addition as in A, and at the indicated time after this EGTA addition, the mixture was diluted at 10 °C by 2 ml of a washing solution containing 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, 2 mM EGTA, and 50 mM MOPS/Tris (pH 7.0) in the presence () or absence () of 2 mM ADP. The sample was immediately filtered through the membrane filter, washed four times by 2 ml of the washing solution with () and without () ADP at 10 °C, and the amount of bound 45Ca2+ was determined. By using [-32P]ATP and nonradioactive CaCl2, the total amount of EP () and the amount of ADP-insensitive EP () present in the above EGTA-added phosphorylation mixture were determined at the indicated time. C, stoichiometry of Ca2+ occluded in EP was calculated at each time point in the EP decay in A and B. The mutants 4Gi-46/47 and 2Gi-46/47 and the conditions employed are indicated with different symbols in the figure.

In Fig. 8, B and C, the experiments were performed with the mutant 2Gi-46/47 in the presence of A23187 [GenBank] at 10 °C. At this temperature, the EP decay was most conveniently followed in this mutant. The phosphorylated samples were filtered at the indicated time with and without addition of ADP immediately before the filtration. Approximately 50% of the total amount of EP was ADP-insensitive EP (E2P) throughout the decay reaction (compare open and closed squares in Fig. 8B, also see Fig. 6B). The total amount of EP, the amount of ADP-insensitive EP, and the amounts of occluded Ca²⁺ determined without and with the ADP addition decreased very slowly and concomitantly (Fig. 8B). Thus, as plotted in Fig. 8C, the stoichiometry of the occluded Ca²⁺ was always found to be two in the total amount of EP (E1P plus E2P) and in the amount of ADP-insensitive EP (E2P) throughout the decay reaction. The results show that two Ca²⁺ ions are occluded in both the ADP-sensitive EP, i.e. E1PCa₂, and the ADP-insensitive EP, i.e. E2PCa₂, in the mutant 2Gi-46/47. The results are consistent with the view that the E1PCa₂-E2PCa₂ equilibrium was largely shifted to E2PCa₂, and the Ca²⁺ deocclusion from E2PCa₂ was blocked in this mutant. It should be mentioned for the mutant 4950525354S that, as indicated by the kinetic analyses in Figs. 4, 5, 6 and 7, E2PCa₂ is probably accumulated in this mutant from ATP and Ca²⁺ as in the mutant 4Gi-46/47. The Ca²⁺-binding experiments were not possible, however, with this mutant because of its very low protein expression level (only 15-20% that of 4Gi-46/47 and 2Gi-46/47 or wild type).

View larger version (14K): [in this window] [in a new window]   FIGURE 9. Formation of stable E2P in the reverse reaction by addition of a high concentration of Ca2+ to E2P formed from Pi without Ca2+. Microsomes expressing the wild-type or mutant SERCA1a were phosphorylated with 32Pi at 25 °C for 10 min in 2.5 µl of a mixture containing 1 µg of microsomal protein, 0.1 mM 32Pi, 1 µM A23187, 1 mM EGTA, 10 mM MgCl2, 50 mM MOPS/Tris (pH 7.0), and 35% (v/v) Me2SO. The mixture was cooled and diluted at 0 °C with 22.5 µl of the "Ca solution" containing 22.3 mM CaCl2 (to give 20 mM Ca2+), 1 µM A23187, 111 mM KCl, 7 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0) and incubated for 1 min. This mixture was then further diluted 10-fold at 0 °C by the addition of 225 µl of the "EGTA solution" containing 44.4 mM EGTA, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0). At different times after this Ca2+-removal, the EP decay was quenched by acid, and the amounts of EP remaining were determined (A). The amounts of EP obtained at zero time (i.e. immediately before the addition of the EGTA solution) are normalized to 100%. It should be noted that the amount of E2P formed from Pi (at zero time) was comparable with that of EP formed from ATP and Ca2+ shown in Fig. 4 in the wild type and in each of the mutants (e.g. in the wild type, E2P formed from Pi was 3.45 ± 0.22 nmol/mg SERCA1a protein (n = 4) and EP formed from ATP was 3.31 ± 0.14 nmol/mg of the SERCA1a protein (n = 4)). B, for determination of the ADP-insensitive EP, an equal volume (250 µl) of a mixture containing 4 mM ADP, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, 10 mM EGTA, and 50 mM MOPS/Tris (pH 7.0) was added to the above EGTA-diluted phosphorylation mixture at the indicated time. At 1 s after this addition, the reaction was quenched by acid. The amount of the ADP-insensitive EP determined at each of the indicated time is shown as percentage of the total amount of EP determined at zero time.

With the representative mutant 4Gi-46/47, we then actually determined the ⁴⁵Ca²⁺ occlusion in the stable E2P formed from E2P with the subsequently added 3 mM ⁴⁵Ca²⁺ (rather than 20 mM because of the experimental limitation), otherwise as above.⁴ We found that the amounts of stable E2P formed and the occluded Ca²⁺ were 1.65 ± 0.13 and 3.33 ± 0.25 nmol/mg of expressed SERCA1a protein (n = 4), respectively, and thus the stoichiometry of the occluded Ca²⁺ in E2P was 2.02. The results demonstrated that two Ca²⁺ ions are occluded in the stable E2PCa₂, which is formed in the reverse reaction as in the forward reaction. The results also indicated that the reverse transition from E2PCa₂ to E1PCa₂ was dramatically retarded (blocked) in the mutants 3Gi-46/47 and 4Gi-46/47.

View larger version (16K): [in this window] [in a new window]   FIGURE 10. Accessibility of luminal Ca2+ in E2P formed from Pi without Ca2+. Microsomes expressing the wild type (A) or the mutant 2Gi-46/47 (B) were phosphorylated with 32Pi in 2.5 µl of a mixture as described in Fig. 9. The mixture was then cooled and diluted 100-fold at 0 °C with 247.5 µl of a solution containing various concentrations of CaCl2 in 1 µM A23187, 101 mM KCl, 1 mM EGTA, 7 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0) to give the final Ca2+ concentrations as indicated with different symbols. The amount of EP remaining at the indicated time after this Ca2+ addition was determined and shown as percentage of the amount of EP at zero time, which was determined immediately before the Ca2+ addition. The EP decay occurred in two phases. The first and rapid phase completed within a few seconds corresponding to the hydrolysis of E2P without bound Ca2+ (see Fig. 7). Essentially the same results were observed in the mutants 3Gi-46/47 and 4Gi-46/47 (data not shown) as in 2Gi-46/47. C, content of EP in the slow and second phase at each Ca2+ concentration was obtained by extrapolating to the zero time and plotted versus the Ca2+ concentration. K0.5 values for the Ca2+ activation and Hill coefficients obtained by fitting to the Hill equation (solid lines) were 1.4 mM and 1.5 (wild type), 1.3 mM and 1.5 (1Gi-46/47), 1.2 mM and 1.4 (2Gi-46/47), 1.0 mM and 1.2 (3Gi-46/47), 0.9 mM and 1.4 (4Gi-46/47), as summarized in Table 1.

In the mutants 2Gi-46/47 (Fig. 10B) and 3Gi-46/47 and 4Gi-46/47 (data not shown), the EP decay in the second phase was extremely slow, and actually almost no decay occurred during the period of observation. The decay of EP in these mutants was extremely slow even after removal of Ca²⁺ as shown in Fig. 9. The EP remaining in this slow phase was almost exclusively the ADP-insensitive one (E2P) in the mutants 3Gi-46/47 and 4Gi-46/47 even at the highest Ca²⁺ concentration 20 mM and 50% in the mutant 2Gi-46/47 at 20 mM Ca²⁺ (see Fig. 9).

The content of EP in the second and slow phase was obtained by extrapolating to the zero time and plotted versus the Ca²⁺ concentrations (Fig. 10C). The content increased with the increase in the Ca²⁺ concentration and was nearly saturated at 10 mM Ca²⁺. K_0.5 was estimated to be 1.4 mM in the wild type and similarly 0.9-1.3 mM in the mutants in Fig. 10 and other mutants as well (see Table 1). These values are actually consistent with the high Ca²⁺ concentrations required for the Ca²⁺-induced reverse reaction from E2P determined previously with SR Ca²⁺-ATPase as the access of luminal Ca²⁺ to the transport sites of E2P (41-44). The results indicate that in these mutants with the elongated A/M1 linker, the luminal Ca²⁺ can access and bind to the transport sites of E2P as in the wild type, and the mutants produce the stable ADP-insensitive EP, i.e. E2PCa₂.

In this context, it should be noted that the Ca²⁺-dependent increase in the stable EP in Fig. 10C reflects mostly the relative values between the Ca²⁺-dependent increasing rate of the formation of the stable EP versus the rate of the hydrolysis of the Ca²⁺-unbound E2P. Namely, the curve reflects mostly the relative rates between E2P + 2Ca²⁺ E2PCa₂ (or further to E1PCa₂) versus E2P + H₂O E2 + P_i, rather than the relative rates between the reverse and forward reactions in E2P + 2Ca²⁺ E2PCa₂ (or E1PCa₂) (i.e. the Ca²⁺ affinity). This is because the curve in Fig. 10C is the plot of the amount of EP stably remaining after the hydrolysis of the Ca²⁺-unbound E2P, and because the amount of the remaining EP is dependent on the rate of its formation relative to the rate of the E2P hydrolysis. Furthermore, the decay of the remaining EP in the second phase was extremely slow in the wild type as well as in the mutants, and thus virtually negligible as compared with its rapid formation and the rapid hydrolysis of the Ca²⁺-unbound E2P both in the wild type and mutants.⁵ Importantly, the E2P hydrolysis rate was found to be essentially the same between the wild type and the mutants (Fig. 7). Therefore, the very similar Ca²⁺ dependence curves of the wild type and mutants in Fig. 10C indicate that the rates of the formation of the stable EP upon the luminal Ca²⁺ binding to E2P are very similar between them. Thus we concluded that E2P formed from P_i without Ca²⁺ in the mutants possesses the luminally opened Ca²⁺ release pathway as in the wild type.

View larger version (14K): [in this window] [in a new window]   FIGURE 11. Thapsigargin accelerates the decay of E2PCa2 accumulated with the mutant. , , microsomes expressing the mutant 4Gi-46/47 were phosphorylated with [-32P]ATP and Ca2+ at 25 °C in 25 µl of the mixture otherwise as described in Fig. 6 legend. , , microsomes expressing the mutant 4Gi-46/47 were phosphorylated with 32Pi in 2.5 µl of a mixture, and then diluted at 25 °C with 22.5 µl of the "Ca solution" containing 22.3 mM CaCl2, 1 µM A23187, 111 mM KCl, 7 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0), and incubated for 1 min, otherwise as described in Fig. 9 legend. Both of the above phosphorylation mixtures were then diluted 10-fold at 25 °C by addition of 225 µl of the EGTA solution containing 44.4 mM EGTA, 1 µM A23187, 0.1 M KCl, 7 mM MgCl2, and 50 mM MOPS/Tris (pH 7.0) in the absence (, ) or presence (, ) of 1 µM TG. At different times after this dilution, the amount of EP remaining was determined by the acid quenching.

Structure of E2PCa₂ Revealed by Proteolytic Analysis as an Intermediate State between E1PCa₂ and E2P—In the transport cycle, the cytoplasmic three domains, N, P, and A largely move and change their organization states (7, 9-17). These changes are definitely monitored as the changes in the resistance of the specific cleavage sites against trypsin and PrtK (15-17). As one of most notable examples, the tryptic T2 site Arg¹⁹⁸ on the outermost Val²⁰⁰ loop (Asp¹⁹⁶-Asp²⁰³) of the A domain is rapidly cleaved in E1PCa₂, by contrast, it is completely resistant in E2P (15-17). This is because the A domain largely rotates parallel to the membrane plane by 110°, and the Val²⁰⁰ loop, including Arg¹⁹⁸, associates with the P domain by forming an ionic interaction network and thus blocks sterically against the tryptic attack, as seen in E1·AlF_x·ADP (or ) (see Refs. 11-14) (see Figs. 2 and 13).

View larger version (83K): [in this window] [in a new window]   FIGURE 12. Structural analysis of major intermediates and E2PCa2 by limited proteolysis. The major