Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca²⁺-ATPase with Occluded Ca²⁺ by Beryllium Fluoride

Abstract

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca²⁺-ATPase E1PCa₂·Mg, a complex of E1Ca₂·BeF_x, was successfully developed by addition of beryllium fluoride and Mg²⁺ to the Ca²⁺-bound state, E1Ca₂. In E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, two Ca²⁺ are occluded at high affinity transport sites, its formation required Mg²⁺ binding at the catalytic site, and ADP decomposed it to E1Ca₂, as in E1PCa₂·Mg. Organization of cytoplasmic domains in E1Ca₂·BeF_x was revealed to be intermediate between those in E1Ca₂·AlF₄⁻ ADP (transition state of E1PCa₂ formation) and E2·BeF₃⁻·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca₂·BeF_x in contrast to no superfluorescence of TNP-AMP bound to E1Ca₂·AlF_x. E1Ca₂·BeF_x with bound TNP-AMP slowly decayed to E1Ca₂, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF₃⁻, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca₂·BeF_x mimics E1PCa₂·Mg, and between those of E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻. E1Ca₂·BeF_x at low 50–100 μm Ca²⁺ was converted slowly to E2·BeF₃⁻ releasing Ca²⁺, mimicking E1PCa₂·Mg → E2P·Mg + 2Ca²⁺. Ca²⁺ replacement of Mg²⁺ at the catalytic site at approximately millimolar high Ca²⁺ decomposed E1Ca₂·BeF_x to E1Ca₂. Notably, E1Ca₂·BeF_x was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca²⁺ with 15 mm Mg²⁺. Also, stable E1Ca₂·BeF_x was produced from E2·BeF₃⁻ at 0.7 mm lumenal Ca²⁺ by binding two Ca²⁺ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca²⁺ → E1PCa₂·Mg.

Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a),² a representative member of the P-type ion transporting ATPases, catalyze Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). The enzyme forms phosphorylated intermediates from ATP or P_i in the presence of Mg²⁺ (10–13). In the transport cycle, the enzyme is first activated by cooperative binding of two Ca²⁺ ions at high affinity transport sites (E2 to E1Ca₂, steps 1–2) (14) and autophosphorylated at Asp³⁵¹ with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which reacts with ADP to regenerate ATP in the reverse reaction. Upon this E1P formation, the two bound Ca²⁺ are occluded in the transport sites (E1PCa₂). Subsequent isomeric transition to the ADP-insensitive form (E2PCa₂), i.e. loss of ADP sensitivity at the catalytic site, results in rearrangement of the Ca²⁺ binding sites to deocclude Ca²⁺, reduce the affinity, and open the lumenal gate, thus releasing Ca²⁺ into the lumen (E2P, steps 4–5). Finally Asp³⁵¹-acylphosphate in E2P is hydrolyzed to form the Ca²⁺-unbound inactive E2 state (steps 6 and 7). Mg²⁺ bound at the catalytic site is required as a physiological catalytic cofactor in phosphorylation and dephosphorylation and thus for the transport cycle. The cycle is totally reversible, e.g. E2P can be formed from P_i in the presence of Mg²⁺ and absence of Ca²⁺, and subsequent Ca²⁺ binding at lumenally oriented low affinity transport sites of E2P reverses the Ca²⁺-releasing step and produces E1PCa₂, which is then decomposed to E1Ca₂ by ADP.

FIGURE 1.

Ca²⁺ transport cycle of Ca²⁺-ATPase.

Various intermediate structural states in the transport cycle were fixed as their structural analogs produced by appropriate ligands such as AMP-PCP (non-hydrolyzable ATP analog) or metal fluoride compounds (phosphate analogs), and their crystal structures were solved at the atomic level (15–22). The three cytoplasmic domains, N, P, and A, largely move and change their organization state during the transport cycle, and the changes are coupled with changes in the transport sites. Most remarkably, in the change from E1Ca₂·AlF₄⁻·ADP (the transition state for E1PCa₂ formation, E1PCa₂·ADP·Mg^‡) to E2·BeF₃⁻ (the ground state E2P·Mg) (23–25), the A domain largely rotates by more than 90° approximately parallel to the membrane plane and associates with the P domain, thereby destroying the Ca²⁺ binding sites, and opening the lumenal gate, thus releasing Ca²⁺ into the lumen (see Fig. 2). E1PCa₂·Ca·AMP-PN formed by CaAMP-PNP without Mg²⁺ is nearly the same as E1Ca₂·AlF₄⁻·ADP and E1Ca₂·CaAMP-PCP in their crystal structures (17, 18, 22).

FIGURE 2.

Structure of SERCA1a and its change during processing of phosphorylated intermediate. E1Ca₂·AlF₄⁻·ADP (the transition state analog for phosphorylation E1PCa₂·ADP·Mg^‡) and E2·BeF₃⁻ (the ground state E2P analog (25)) were obtained from the Protein Data Bank (PDB accession code 1T5T (17) and 2ZBE (21), respectively). Cytoplasmic domains N (nucleotide binding), P (phosphorylation), and A (actuator), and 10 transmembrane helices (M1–M10) are indicated. The arrows on the domains, M1′ and M2 (Tyr¹²²) in E1Ca₂·AlF₄⁻·ADP, indicate their approximate motions predicted for E1PCa₂·ADP·Mg^‡ → E2P·Mg. The phosphorylation site Asp³⁵¹, TGES¹⁸⁴ of the A domain, Arg¹⁹⁸ (tryptic T2 site) on the Val²⁰⁰ loop (DPR¹⁹⁸AV²⁰⁰NQD) of the A domain, and Thr²⁴² (proteinase K site) on the A/M3-linker are shown. Seven hydrophobic residues gather in the E2P state to form the Tyr¹²²-hydrophobic cluster (Y122-HC); Tyr¹²²/Leu¹¹⁹ on the top part of M2, Ile¹⁷⁹/Leu¹⁸⁰/Ile²³² of the A domain, and Val⁷⁰⁵/Val⁷²⁶ of the P domain. The overall structure of E1Ca₂·AlF₄⁻·ADP is virtually the same as those of E1Ca₂·CaAMP-PCP and E1PCa₂·Ca·AMP-PN (17, 18, 22).

Despite these atomic structures, yet unsolved is the structure of E1PCa₂·Mg, the genuine physiological intermediate E1PCa₂ with bound Mg²⁺ at the catalytic site without the nucleotide. Its stable structural analog has yet to be developed. E1PCa₂·Mg is the major intermediate accumulating almost exclusively at steady state under physiological conditions. Its rate-limiting isomerization results in Ca²⁺ deocclusion/release producing E2P·Mg as a key event for Ca²⁺ transport. In E1Ca₂·CaAMP-PCP, E1Ca₂·AlF₄⁻·ADP, and E1PCa₂·Ca·AMP-PN, the N and P domains are cross-linked and strongly stabilized by the bound nucleotide and/or Ca²⁺ at the catalytic site, thus they are crystallized (17, 18, 22). Kinetically, E1PCa₂·Ca formed with CaATP is markedly stabilized due to Ca²⁺ binding at the catalytic Mg²⁺ site, and its isomerization to E2P is strongly retarded in contrast to E1PCa₂·Mg (26, 27). Thus, the bound Ca²⁺ at the catalytic Mg²⁺ site likely produces a significantly different structural state from that with bound Mg²⁺.

Therefore, it is now essential to develop a genuine E1PCa₂·Mg analog without bound nucleotide and thereby gain further insight into the structural mechanism in the Ca²⁺ transport process. It is also crucial to further clarify the structural importance of Mg²⁺ as the physiological catalytic cation. In this study, we successfully developed the complex E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, as the E1PCa₂·Mg analog by adding beryllium fluoride (BeF_x) to the E1Ca₂ state without any nucleotides. For its formation, Mg²⁺ binding at the catalytic site was required and Ca²⁺ substitution for Mg²⁺ was absolutely unfavorable, revealing a likely structural reason for its preference as the physiological cofactor. In E1Ca₂·BeF₃⁻, two Ca²⁺ ions bound at the high affinity transport sites are occluded. It was also produced from E2·BeF₃⁻ by lumenal Ca²⁺ binding at the lumenally oriented low affinity transport sites, mimicking E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. All properties of the newly developed E1Ca₂·BeF₃⁻ fulfilled the requirements as the E1PCa₂·Mg analog, and hence we were able to uncover the hitherto unknown nature of E1PCa₂·Mg as well as structural events occurring in the phosphorylation and isomerization processes. Also, we successfully found the conditions that perfectly stabilize the E1Ca₂·BeF₃⁻ complex.

EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles and Treatment with BeF_x, AlF_x, and AlF_x·ADP

SR vesicles were prepared from rabbit skeletal muscle as described (28). The content of the phosphorylation site in the vesicles determined according to Barrabin et al. (29) was 4.49 ± 0.22 nmol/mg of vesicle protein (n = 5). The Ca²⁺-dependent ATPase activity determined at 25 °C in a mixture containing 5 μg/ml microsomal protein, 1 mm ATP, 1.7 μm A23187, 7 mm MgCl₂, 0.1 m KCl, 50 mm MOPS/Tris (pH 7.0), and 0.6 mm CaCl₂ with 0.5 mm EGTA (or 2 mm EGTA without added CaCl₂) was 1.87 ± 0.14 μmol/min/mg of vesicle protein (n = 3). The Ca²⁺-ATPase was purified from the vesicles by deoxycholate as described (30, 31). The E1Ca₂ state ATPase was incubated with fluoride compounds, 2 mm potassium fluoride and 100 μm BeSO₄ or AlCl₃, at 25 °C for 30 min in the presence of 0.1 mm Ca²⁺, 15 mm MgCl₂, 0.1 m KCl, 30 mm MOPS/Tris buffer (pH 7.0), unless stated otherwise. E1Ca₂· AlF₄⁻·ADP was formed by including 50 μm ADP in the above AlF_x incubation mixture as described (32). E2·BeF₃⁻, E2·AlF₄⁻, and E2·MgF₄²⁻ were produced as described (23–25).

Determination of EP

EP formation was performed with 3 μm [γ-³²P]ATP in 100 (or 50) μm Ca²⁺ at 0 °C for 3 s, and terminated by trichloroacetic acid containing carrier P_i. The amount of EP formed was determined as described previously (28). The background level determined with excess 5 mm EGTA was less than 0.5% of the phosphorylation sites.

Ca²⁺ Binding and Occlusion

⁴⁵Ca²⁺ binding and occlusion at the transport sites was determined at 25 °C with 2 ml of the SR vesicle mixture (0.2 mg/ml protein) with a 0.45-μm nitrocellulose membrane filter (Millipore) as described (31). In some cases, the vesicles on the filter were washed for 10 s by perfusion with 2 ml of a washing solution containing 5 mm EGTA. The amount of Ca²⁺ bound at the transport sites was obtained by subtracting the nonspecific Ca²⁺ binding level determined as described in the figure legends.

Proteolysis

SR vesicles (0.45 mg/ml protein) were treated at 25 °C with trypsin (0.3 mg/ml) or proteinase K (0.1 mg/ml) as described (23, 24) and as noted in the figure legends. The samples were subjected to Laemmli SDS-PAGE (33) and densitometric analyses of the Coomassie Brilliant Blue R-250-stained gels (23, 24).

Fluorescence Measurements

The TNP-AMP fluorescence and intrinsic tryptophan fluorescence of Ca²⁺-ATPase (0.06 mg/ml protein) were measured on a RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) with excitation and emission wavelengths 408 and 540 nm for TNP-AMP (with band widths 5 and 10 nm), and 290 and 338.4 nm for tryptophan (with bandwidth 1.5 and 5 nm), unless otherwise described (28).

Miscellaneous

Trypsin (l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) and proteinase K were obtained from Sigma. TNP-AMP was synthesized according to Hiratsuka (34). Protein concentrations were determined by the method of Lowry et al. (35) with bovine serum albumin as a standard. Free Ca²⁺ concentrations were calculated by the Calcon program. Data were analyzed by nonlinear regression using the program Origin (Microcal Software, Inc., Northampton, MA). Three-dimensional models of the enzyme were reproduced by the program VMD (36).

RESULTS

Formation of E1PCa₂ Analogs by Fluoride Compounds

The Ca²⁺-ATPase of SR vesicles in 100 μm Ca²⁺ and 15 mm Mg²⁺ was incubated with beryllium fluoride (BeF_x) or aluminum fluoride (AlF_x) for 30 min at 25 °C. The ability of EP formation from ATP was almost completely lost (actually within 1 min) (Fig. 3), showing the stable complex formation with BeF_x and AlF_x. No inhibition occurred with Mg²⁺ and fluoride without beryllium and aluminum (E1Ca₂+(MgF_x)). Thus, MgF_x (MgF₄²⁻) was not able to produce a complex with E1Ca₂, in contrast to E2·MgF₄²⁻ formation from E2, the E2·P_i product analog in E2P hydrolysis (19, 25). This finding agrees with the in-line phosphorylation of E1Ca₂ to E1PCa₂ (37), in which there is no state with non-covalently bound P_i.

FIGURE 3.

Inhibition of EP formation by binding of BeF_x and AlF_x to E1Ca₂. The E1Ca₂ state ATPase of SR vesicles in 0.1 or 5 mm free Ca²⁺ was treated for 30 min at 25 °C with fluoride compounds in the presence of the indicated concentrations of the ligands. Subsequently, the samples were cooled to 0 °C and phosphorylated for 3 s by 3 μm [γ-³²P]ATP. The amount of EP formed was shown as the percentage of the control value obtained without F⁻, Be²⁺, and Al³⁺. The EP formation was not inhibited when the incubations were performed with Be²⁺ or Al³⁺ but without F⁻. The error bars show the S. D. of five independent experiments.

The binding of Mg²⁺ at the catalytic site as a physiological cation is nevertheless required for EP formation. Actually in Fig. 3, Mg²⁺ was required for complex formation with BeF_x in 100 μm Ca²⁺. The apparent Mg²⁺ affinity (K_0.5 of 5 mm, supplemental Fig. S1) was consistent with that of the catalytic site in phosphorylation from ATP or P_i (e.g. Refs. 11, 12, and 38–41). The BeF_x-induced inhibition also occurred with Mn²⁺ with apparent affinity (K_0.5 of 0.6 mm) significantly higher than that of Mg²⁺, as found in E1PCa₂ formation and ATP hydrolysis with Mn²⁺ (40, 42).

When Ca²⁺ over millimolar amounts was added in place of Mg²⁺, the EP formation was not inhibited by BeF_x (BeF_x(5 mm Ca)). Therefore, Ca²⁺ substitution probably at the catalytic Mg²⁺ site abolished complex formation with BeF_x. Although CaATP as a substrate and Ca²⁺ bound at the catalytic Mg²⁺ site are able to function for E1PCa₂ formation (26, 27), Ca²⁺ bound at the catalytic site likely produces different structure from the Mg²⁺ bound structure (as in fact found, see below). The complex formation of E1Ca₂ by AlF_x in 100 μm Ca²⁺ also required Mg²⁺ or Mn²⁺ with somewhat higher apparent affinities than those for BeF_x-induced complex formation, and was abolished by Ca²⁺ binding at the catalytic site.

Ca²⁺ Binding and Occlusion at Transport Sites

In Fig. 4, Ca²⁺ binding and occlusion in formation of the Ca²⁺-ATPase complexes with fluoride compounds were determined in 15 mm Mg²⁺ with and without a 10-s EGTA filter washing. In all the cases without washing, Ca²⁺ was bound to the Ca²⁺-ATPase with high affinities; K_0.5 at sub-micromolar to millimolar ranges, Hill coefficient ∼2, and maximum levels of 9–10 nmol/mg of protein, i.e. the stoichiometry of two Ca²⁺ per phosphorylation site (inset at 50 μm Ca²⁺). Therefore, E1Ca₂·BeF_x and E1Ca₂·AlF₄⁻·ADP/E1Ca₂·AlF_x were produced by cooperative binding of two Ca²⁺ ions at high affinity transport sites. This finding agrees with the property of the sites for Ca²⁺ binding and the resulting enzyme activation for phosphorylation by ATP as nicely demonstrated for the first time by Inesi et al. (14). Upon the EGTA washing of E1Ca₂ that is complexed with BeF_x, the two bound Ca²⁺ were not removed, and therefore occluded in the complex as “E1Ca₂·BeF_x.” The two Ca²⁺ are occluded also in E1Ca₂·AlF₄⁻·ADP and less strongly in E1Ca₂·AlF_x.

FIGURE 4.

Ca²⁺ binding and occlusion at transport sites. The E1Ca₂ state ATPase of SR vesicles in various concentrations of ⁴⁵Ca²⁺ and 15 mm MgCl₂ was incubated with BeF_x, AlF_x, and AlF_x·ADP or without these compounds (E1Ca₂) for 30 min at 25 °C. The amounts of bound (upper panel) and occluded (lower panel) ⁴⁵Ca²⁺ were determined without and with the perfusion of the membrane filter with a 5 mm EGTA-containing washing solution (without CaCl₂ and fluoride compounds otherwise as the above incubation solution). The nonspecific Ca²⁺ binding was determined by including 10 μm thapsigargin before the addition of fluoride compounds, and subtracted. When ADP was used for E1Ca₂·AlF₄⁻·ADP, 5 μm A23187 was included to avoid Ca²⁺ accumulation in the vesicles by ATP produced from ADP due to adenylate kinase in the vesicles. In the inset, the stoichiometries of bound Ca²⁺ (open bars) and occluded Ca²⁺ (closed bars) to the phosphorylation site (P-site) were determined at saturating 50 μm ⁴⁵Ca²⁺. Solid lines in the upper panel show the least squares fit to the Hill equation. K_0.5 of Ca²⁺ and Hill coefficients obtained were 1.3 μm and 1.7 (E1Ca₂), 4.3 μm and 1.7 (E1Ca₂·BeF_x), 2.3 μm and 1.7 (E1Ca₂·AlF_x), and 0.4 μm and 1.4 (E1Ca₂·AlF₄⁻·ADP). In the lower panel, the values for E1Ca₂·BeF_x and E1Ca₂·AlF₄⁻·ADP are essentially not altered by EGTA washing (4.7 μm and 1.9, and 0.4 μm and 1.9, respectively).

Note that the Ca²⁺ affinity became 2–3-fold lower for BeF_x and AlF_x. This may be because the Ca²⁺-free E2 state produces E2·BeF₃⁻ and E2·AlF₄⁻ (25), and therefore competes with Ca²⁺ binding for formation of E1Ca₂·BeF_x and E1Ca₂·AlF_x. On the other hand, the observed ∼3-fold Ca²⁺-affinity increase in formation of E1Ca₂·AlF₄⁻·ADP is probably brought about by the fact that ADP together with AlF_x strongly stabilizes the cross-linked N-P domains (17, 18), which is unfavorable for formation of the Ca²⁺-free E2 and E2·AlF₄⁻, because for these structures the A domain should rotate into the opened space between the N and P domains and associate with them (19, 23, 24).

Cytoplasmic Structure in E1Ca₂·BeF_x Is Intermediate between Those in E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻

Proteolytic analysis was made to reveal the organization state of the cytoplasmic domains in the newly developed E1PCa₂·Mg analog E1Ca₂·BeF_x, and to compare with E1Ca₂·AlF₄⁻·ADP/E1Ca₂·AlF_x and E2·BeF₃⁻ (E2P·Mg) (see the typical cleavage in supplemental Fig. S2). The initial rate of the “A1” appearance upon cleavage at the T2 site (Arg¹⁹⁸ on the Val²⁰⁰ loop of the A domain) in E1Ca₂·BeF_x was substantially slower than the rapid cleavage of E1Ca₂·AlF₄⁻·ADP and E1Ca₂·AlF_x as well as E1Ca₂ (Table 1). The slowed T2 cleavage was also observed when E1Ca₂·BeF_x was formed with 3 mm Mn²⁺ in place of 15 mm Mg²⁺ (data not shown). Also important was the slow but definitely occurring T2 cleavage in E1Ca₂·BeF_x, in sharp contrast to its complete resistance in E2·BeF₃⁻. Therefore A-P domain organization at the Val²⁰⁰ loop in E1Ca₂·BeF_x is intermediate between those in E1Ca₂·AlF₄⁻·ADP/E1Ca₂·AlF_x and E2·BeF₃⁻.

TABLE 1.

Summary of fluorescence changes and proteolysis rates

Maximal TNP-AMP fluorescence intensity at saturating 4 μm TNP-AMP is given as % value of that of E2·BeF₃⁻ without A23187. Tryptophan fluorescence change upon complex formation with the ligand from E1Ca₂ (or from other state when indicated in parentheses) is shown as % value of the intensity of E1Ca₂ (see also supplemental Fig. S3E). The cleavage rate at the T2 site (Arg¹⁹⁸) with trypsin and the digestion rate of the 110-kDa ATPase chain with proteinase K were obtained by the detailed time course analysis in the initial 1 (T2) and 30 min (proteinase K), and shown as % values of those determined with E1Ca₂ in 0.1 mm Ca²⁺. Some of these experiments were done at 0.05 mm Ca²⁺ instead of 0.1 mm, but the results were virtually the same and therefore are represented with 0.1 mm Ca²⁺ for simplicity. It should also be mentioned that the digestion rates in E1Ca₂ were not altered by 5 or 0.7 mm Ca²⁺ or by A23187 (being 97–101% of the rates of E1Ca₂ in 0.1 mm Ca²⁺ without A23187), and that the ligand-free E2 state was also rapidly digested by trypsin and proteinase K, and TG binding to E2 and A23187 did not alter essentially the rapid cleavage rates (Refs. 23–25). The other Ca²⁺-ATPase complexes were produced under the same buffer conditions as those for E1Ca₂·BeF_x formation, otherwise as follows and noted below: E1Ca₂·MgAMP-PCP by 5 mm MgAMP-PCP; E1Ca₂·CaAMP-PCP by 5 mm CaAMP-PCP; E1PCa₂·Ca·AMP-PN by 5 mm CaAMP-PNP; E1PCa₂·Ca by 5 mm CaATP. E1Ca₂·BeF_x + 5 mm Ca²⁺, the E1Ca₂·BeF_x complex was formed in 15 mm Mg²⁺ and 100 μm Ca²⁺ and then incubated with the subsequently added 5 mm Ca²⁺ for 3 h; E1Ca₂ + 5 mm Ca²⁺ + BeF_x, the E1Ca₂ state ATPase was incubated with BeF_x for 10 min in the presence of 5 mm Ca²⁺ without Mg²⁺.

ATPase state (→ consequent state, if altered)	Ca2+ (mm)/Mg2+ (mm)	Relative TNP-AMP fluorescence intensity, see Fig. 5A and supplemental Fig. S5	Change in tryptophan fluorescence from E1Ca2 (or from another state), supplemental Fig. S3 ↑ increase, ↓ decrease	Relative digestion rate
				Trypsin (T2), supplemental Fig. S2	Proteinase K, supplemental Fig. S2
	mm	%	% of E1Ca2 level	%
E1Ca2	0.1/15	7	(3.27 ↑ from E2)	100	100
E1Ca2·MgAMP-PCP	0.1/15		0	64	25
E1Ca2·CaAMP-PCP	5/0		0.80 ↑	61	9
E1Ca2·AlF4−·ADP	0.1/15		0.84 ↑	65	3
E1PCa2·Ca·AMP-PN	5/0		0.77 ↑	65	4
E1Ca2·AlFx	0.1/15	7	0	70	10
E1PCa2·Ca	5/0		0.89 ↓	80	6
E1PCa2·Mg	0.1/15		1.18 ↓
E1Ca2·BeFx	0.1/15	75	1.27 ↓	35	13
E1Ca2·BeFx + 5 mm Ca2+ (→ partially E1Ca2)	0.1/15 + 5 mm Ca2+	30		70	32
E1Ca2 + 5 mm Ca2+ + BeFx	5/0	16	0	116	90
E2·BeF3−	0/15	100	(0.66 ↑ from E2)	0	0
E1Ca2·BeFx + TG (→ E2·BeF3−(TG))	0.1/15	64	(5.35 ↓ from E1Ca2·BeFx)	0	0
E2·BeF3− + TG (→ E2·BeF3−(TG))	0/15	65	(4.62 ↓ from E2·BeF3−)	0	0
E1Ca2·BeFx + A23187 (→ E2·BeF3−)	0.1/15	100		10	0
E2·BeF3− + A23187	0/15	100		0	0
E1PCa2·Mg (0.7 mm Ca2+)	0.7/15		1.19 ↓
E1Ca2·BeFx (0.7 mm Ca2+)	0.7/15	75	1.27 ↓	39	15
E1Ca2·BeFx (0.7 mm Ca2+ + A23187)	0.7/15	75		32	8
E2·BeF3− + 0.7 mm Ca2+	0/15 + 0.7 mm Ca2+	100	(0 from E2·BeF3−)	0	2
E2·BeF3− + 0.7 mm Ca2+ + A23187 (→ E1Ca2·BeF3−)	0/15 + 0.7 mm Ca2+	75		38	16

All complexes were almost completely resistant to proteinase K at the major site of Thr²⁴² on the A/M3-linker that produces the “p83” fragment. Therefore, in E1Ca₂·BeF_x, the A domain is rotated perpendicular to the membrane plane from its position in E1Ca₂ thereby causing the A/M3-linker strain, as in E1Ca₂·CaAMP-PCP, E1Ca₂·AlF₄⁻·ADP (18, 19, 24), and E1Ca₂·AlF_x.

These analyses revealed that in the change E1Ca₂·AlF₄⁻· ADP → E1Ca₂·BeF_x (i.e. upon the ADP release from the transition state), the A domain moves, i.e. probably rotates to some extent parallel to the membrane plane likely due to the A/M3-linker strain, and thereby Arg¹⁹⁸ on the Val²⁰⁰ loop comes close to the P domain. In the subsequent change, E1Ca₂·BeF_x → E2·BeF₃⁻, the A domain rotates further (by the A/M3-linker strain as predicted to be motive force (18, 19, 43, 44)) and produces its tight association with the P domain at the Val²⁰⁰ loop, mimicking E1PCa₂·Mg → E2P·Mg + 2Ca²⁺.

Ca²⁺ Ligation at the Catalytic Mg²⁺ Site

The proteolysis further revealed that E1Ca₂·BeF_x was not produced from E1Ca₂ in 5 mm Ca²⁺ without Mg²⁺ (E1Ca₂ + 5 mm Ca²⁺ + BeF_x in Table 1), and that E1Ca₂·BeF_x produced in 15 mm Mg²⁺ and 50–100 μm Ca²⁺ was decomposed to the E1Ca₂ by 5 mm Ca²⁺ (E1Ca₂·BeF_x + 5 mm Ca²⁺), as shown by the rapid cleavage rates at the T2 and proteinase K sites. In E1PCa₂·Ca and E1Ca₂· CaAMP-PCP formed in 5 mm Ca²⁺ without Mg²⁺ (Table 1),³ the T2 site was also rapidly cleaved, in contrast to its substantially slowed cleavage in E1Ca₂·BeF_x formed with Mg²⁺. Thus, for organization of the cytoplasmic domains at the T2 site (Arg¹⁹⁸), E1Ca₂·CaAMP-PCP and E1PCa₂·Ca are very similar to E1Ca₂·AlF₄⁻·ADP, but differ from E1Ca₂·BeF_x. The close similarity between E1Ca₂·CaAMP-PCP and E1Ca₂·AlF₄⁻·ADP is in agreement with their nearly same atomic structures and previous observations (17, 18, 45). Also notably, structure E1PCa₂·Ca·AMP-PN formed by CaAMP-PNP in 10 mm Ca²⁺ without Mg²⁺ (22) is almost identical with those of E1Ca₂·CaAMP-PCP and E1Ca₂·AlF₄⁻·ADP (see also Table 1).

In E1Ca₂·CaAMP-PCP and E1PCa₂·Ca·AMP-PN, the N-P domain cross-linked state is stabilized by Ca²⁺ bound at catalytic Mg²⁺ site I (Asp³⁵¹/Thr³⁵³/Asp⁷⁰³ and the phosphate) and by the nucleotide to be nearly identical to the state stabilized with AlF₄⁻ plus ADP in E1Ca₂·AlF₄⁻·ADP (17, 18, 22). The results on E1PCa₂·Ca further indicated that such an N-P domain closed state is stabilized solely by site I Ca²⁺ ligation without the nucleotide. The stabilization of this state in E1PCa₂·Ca is consistent with its markedly retarded isomerization to E2P (27), because isomerization requires the A domain rotation into the space between the N and P domains. In E1Ca₂·BeF_x formed with Mg²⁺ at the catalytic site (site I), such a Ca²⁺ ligation effect is obviously not present. Therefore, the N and P domains are probably more easily separated from each other, and the A domain can rotate into the space between the N and P domains to some extent thus resulting in partial T2 resistance (but not yet as completely as in E2·BeF₃⁻). As the cause of the Ca²⁺-induced E1Ca₂·BeF_x to E1Ca₂ decomposition, Ca²⁺ replacement of Mg²⁺ at site I altered the domain organization state and made the BeF_x ligation unfavorable (see “Discussion”).

TNP-AMP Superfluorescence

TNP-AMP binds to the nucleotide binding site with an extremely high affinity (46–48), and in the E2P ground state and its analog E2·BeF₃⁻, the bound TNP-AMP develops its extremely high fluorescence “superfluorescence” (25), which reflects a strongly hydrophobic atmosphere around Asp³⁵¹ (49, 50). On the other hand, it has been controversial whether E1PCa₂ develops TNP-AMP superfluorescence, mostly because its tight binding to the nucleotide binding site prevents phosphorylation to form E1PCa₂·Mg, so the TNP-AMP·E1PCa₂·Mg complex is not formed in significant amounts. Nakamoto and Inesi (47), nevertheless, predicted the development of superfluorescence in E1PCa₂.

Here, with E1Ca₂·BeF_x as the E1PCa₂·Mg analog, we examined the superfluorescence without ATP. In Fig. 5, A and B, we first formed E1Ca₂·BeF_x in 15 mm Mg²⁺ and 50 μm Ca²⁺, then TNP-AMP was added to give a saturating level of 4 μm. E1Ca₂·BeF_x rapidly developed superfluorescence, and then the fluorescence decreased slowly (much more slowly and extensively at 4 °C).⁴ The proteolysis after the loss of superfluorescence revealed that E1Ca₂·BeF_x was decomposed to E1Ca₂ (data not shown). Thus, E1Ca₂·BeF_x develops superfluorescence, and the TNP-AMP binding per se causes its decomposition to E1Ca₂, in sharp contrast to the completely stable E2·BeF₃⁻ even after TNP-AMP binding. The maximum superfluorescence level of E1Ca₂·BeF_x was slightly lower than that of E2·BeF₃⁻ (Fig. 5, A-C), which is the same as that of E2P·Mg formed from P_i (25). The results clearly revealed that the atmosphere around Asp³⁵¹ in E1Ca₂·BeF_x is strongly hydrophobic, similar to E2·BeF₃⁻, although the cytoplasmic domain organization in E1Ca₂·BeF_x distinctly differs from and did not yet reach the most compactly organized state in E2·BeF₃⁻.

FIGURE 5.

TNP-AMP superfluorescence of E1Ca₂·BeF_x and E2·BeF₃⁻. E1Ca₂·BeF_x and E1Ca₂·AlF_x were produced in 50 μm Ca²⁺ and 15 mm MgCl₂, then at 25 (A) and 4 °C (B), a small volume of TNP-AMP was added to give saturating 4 μm, and the TNP-AMP fluorescence was followed. The fluorescence of E2·BeF₃⁻ produced with BeF_x in the absence of Ca²⁺, the E1Ca₂ and E2 states without the fluoride compounds, and without SR vesicles (no SRV) were also followed. C, the TNP-AMP fluorescence intensities were measured at various concentrations of TNP-AMP at 25 °C, otherwise as described for A. For E1Ca₂·BeF_x, the maximum level of transient superfluorescence was determined by extrapolating its decrease to the time of TNP-AMP addition. In the lower panel in C, the low fluorescence was replotted on the expanded scale. The maximum fluorescence intensities at saturating 4 μm TNP-AMP were obtained by subtracting the background level without TNP-AMP and the level of 4 μm TNP-AMP without the SR vesicles, and given as the relative values in Table 1.

E1Ca₂·AlF_x as well as E2 and E1Ca₂ did not develop superfluorescence despite high affinity TNP-AMP binding. Therefore the catalytic site in E1Ca₂·AlF_x is hydrophilic and differs critically from the strongly hydrophobic site in E1Ca₂·BeF_x. Note also that E2·AlF₄⁻ (E2-P^‡) and E2·MgF₄²⁻ (E2·P_i) do not develop TNP-AMP superfluorescence (25). The superfluorescence therefore develops solely with Ca²⁺-ATPase complexed with BeF_x; E1Ca₂·BeF_x and E2·BeF₃⁻.

The superfluorescence development of E1Ca₂·BeF_x in the presence of 15 mm Mg²⁺ and 50 μm Ca²⁺ was rapidly diminished with increasing Ca²⁺ over millimolar concentrations of Ca²⁺ (see Fig. 8 and Table 1). Also, inclusion of 5 mm Ca²⁺ without Mg²⁺ in the E1Ca₂·BeF_x formation mixture abolished the superfluorescence (Table 1). The results agree with the above findings that E1Ca₂·BeF_x is not produced from and decomposed to E1Ca₂ by Ca²⁺ ligation at the catalytic Mg²⁺ site (site I).

FIGURE 8.

Stability of E1Ca₂·BeF_x in various Ca²⁺ concentrations. A, E1Ca₂·BeF_x was first produced in SR vesicles in 0.1 mm Ca²⁺ and 15 mm MgCl₂ with BeF_x. Subsequently, Ca²⁺ was changed to 0.1 (unchanged), 0.4, 0.7, 1.1, 3.1, and 10.1 mm, and the incubations continued at 25 °C for 72 h. At the indicated periods, the superfluorescence with 4 μm TNP-AMP was examined at 25 °C, and the maximum levels obtained as described in the legend to Fig. 6 are shown. E2·BeF₃⁻ formed without Ca²⁺, E1Ca₂ in 0.1–10 mm Ca²⁺, and E2 without Ca²⁺ were also incubated. B, in the presence of 0.7 mm Ca²⁺, E1Ca₂ in SR vesicles was first incubated with and without 1.2 μm A23187 (A23), then BeF_x was added (E1Ca₂·BeF_x). E1Ca₂·BeF_x (DOC-E1Ca₂·BeF_x) was also produced from E1Ca₂ in 0.7 mm Ca²⁺ of the Ca²⁺-ATPase purified and delipidated from SR vesicles by deoxycholate (DOC) treatment (30). The incubation was continued for 12 days, otherwise as indicated in A. E2·BeF₃⁻ (E2·BeF₃⁻, DOC-E2·BeF₃⁻) without Ca²⁺ and E1Ca₂ in 0.7 mm Ca²⁺ (E1Ca₂, DOC-E1Ca₂) and E2 without Ca²⁺ (E2, DOC-E2) were also incubated. C–G, the fluorescence traces upon TNP-AMP addition were shown for the representative samples with incubation periods and Ca²⁺ concentration (mm). Note that at 0.7 mm Ca²⁺, both with and without A23187, the development of the E1Ca₂·BeF_x characteristic transient superfluorescence remained perfectly the same for 12 days. By contrast, the transient superfluorescence was converted to the stable and higher superfluorescence characteristic of E2·BeF₃⁻ at 0.1 and 0.4 mm Ca²⁺, and it was markedly reduced by 3 and 10 mm Ca²⁺ due to decomposition to E1Ca₂.

Transmembrane Domain Structure

The 12 tryptophan residues among 13 in the Ca²⁺-ATPase are located at the transmembrane region. The tryptophan fluorescence changes in fact reflect the transmembrane domain structural changes, i.e. rearrangements of the transmembrane helices upon Ca²⁺ binding to the high affinity transport sites and during the transport cycle (28, 51, 52) as found originally by Dupont and Leigh (53). As summarized in Table 1 with typical fluorescence traces in supplemental Fig. S3, the fluorescence changes were determined at 4 °C upon formation of the E1PCa₂ analogs by the addition of fluoride compounds to E1Ca₂ in 15 mm Mg²⁺ and 100 μm Ca²⁺. E1Ca₂·BeF_x formation decreased fluorescence by 1.3% very similar to the decrease in E1PCa₂·Mg formation from E1Ca₂ by MgATP, i.e. in E1Ca₂·MgATP → E1PCa₂·Mg (52). In contrast, E1Ca₂· AlF_x formation did not cause any change. The E1Ca₂·AlF₄⁻·ADP formation increased the fluorescence by 0.8%. (F⁻ alone and ADP alone did not cause any change, except the dilution (F⁻) and absorption of excitation light (ADP).) Thus the transmembrane structure of E1Ca₂·BeF_x mimics that of E1PCa₂· Mg, but those of E1Ca₂·AlF₄⁻·ADP and E1Ca₂·AlF_x differ substantially although the Ca²⁺ ions are occluded at the transport sites (or less strongly in E1Ca₂·AlF_x, Fig. 4). This observation is consistent with the finding in proteolysis and TNP-AMP superfluorescence that organization of the cytoplasmic domains and structure at the catalytic site in E1Ca₂·BeF_x substantially differ from those in E1Ca₂·AlF₄⁻·ADP and E1Ca₂·AlF_x. It is concluded that the transmembrane structure with the occluded Ca²⁺ adopts not simply one state, but changes with the change in the cytoplasmic region during phosphoryl transfer and ADP release (see the diagram of tryptophan fluorescence change in supplemental Fig. S3E (with Ref. 54)).

Upon formation of E1Ca₂· CaAMP-PCP and E1PCa₂·Ca·AMP-PN by CaAMP-PCP and CaAMP-PNP, respectively, the fluorescence increased by 0.8% equal to that upon E1Ca₂·AlF₄⁻·ADP formation (Table 1), in agreement with their essentially identical structures with occluded Ca²⁺ (17, 18, 22, 45). By contrast, the fluorescence did not change upon formation of the E1Ca₂·MgAMP-PCP, which is the Ca²⁺-unoccluded state (28, 45), and in rapid equilibrium with E1Ca₂.

Upon the exclusive accumulation of E1PCa₂·Ca by CaATP without Mg²⁺, tryptophan fluorescence decreased by 0.9%, being slightly less than that by formation of E1PCa₂·Mg and E1Ca₂·BeF_x (Table 1). Thus in the overall structure, E1PCa₂·Ca may be between E1Ca₂·CaAMP-PCP and E1PCa₂·Mg (E1Ca₂·BeF_x), and closer to the latter state. Although Ca²⁺ ligation at catalytic Mg²⁺ site I in E1PCa₂·Ca favors the N-P domain closed state, similar to E1Ca₂·CaAMP-PCP, the absence of the N-P domain cross-linking nucleotide in E1PCa₂·Ca likely altered the overall structure slightly.

Upon formation of E2·BeF₃⁻ from E2 by BeF_x and Mg²⁺ without Ca²⁺, the fluorescence increased by 0.7%, mimicking the change upon E2P·Mg formation from E2 with P_i and Mg²⁺, and reflecting the opening of the lumenal gate from the closed state (25). As a consequence, the fluorescence of E1Ca₂·BeF_x was definitely higher by ∼1.3% than that of E2·BeF₃⁻, showing their distinct difference in the transmembrane structure. In agreement, the previous kinetic analysis have shown (28) that tryptophan fluorescence decreases by ∼1% in the isomerization/Ca²⁺ release, E1PCa₂·Mg → E2P·Mg + 2Ca²⁺, reflecting the transmembrane structural change from the Ca²⁺-occluded state to the Ca²⁺-released and lumenally opened state.

Upon the addition of thapsigargin (TG) to E1Ca₂·BeF_x and E2·BeF₃⁻, tryptophan fluorescence decreased rapidly by 5.4 and 4.6%, respectively, and reached the level of E2·BeF₃⁻ with bound TG (E2·BeF₃⁻(TG), see Table 1). TNP-AMP superfluorescence (supplemental Fig. S5, A and B) and proteolysis (Table 1) also demonstrated that E1Ca₂·BeF_x was converted by TG to E2·BeF₃⁻(TG). Importantly, as described under supplemental Fig. S4, two Ca²⁺ occluded in E1Ca₂·BeF_x are most likely released into the lumen by the TG-induced structural perturbation and trapped in the lumen by the bound TG, as TG fixes the lumenal gate in the closed state and suppresses Ca²⁺ leakage (16, 55).

E1Ca₃·BeF_x Is ADP-sensitive

In Fig. 6, two ⁴⁵Ca²⁺ occluded in E1Ca₂·BeF_x were rapidly removed by washing with 1 mm ADP, whereas the occluded ⁴⁵Ca²⁺ remained completely without ADP. Thus ADP caused the loss of Ca²⁺ occlusion. In agreement, ADP binding to E1Ca₂·BeF_x increased tryptophan fluorescence to the E1Ca₂ level, and resulted in the tryptic T2 site cleavage as E1Ca₂ with bound ADP (data not shown). By contrast, ADP binding to E2·BeF₃⁻ did not alter its structure (data not shown). The ADP-induced decomposition of E1Ca₂·BeF_x to E1Ca₂ was also demonstrated with the ADP-induced loss of TNP-AMP superfluorescence, in contrast to normal superfluorescence development in E2·BeF₃⁻ after ADP incubation (data not shown). Thus E1Ca₂·BeF_x is ADP-sensitive as E1PCa₂·Mg, and E2·BeF₃⁻ is ADP-insensitive as E2P·Mg.

FIGURE 6.

ADP causes the loss of Ca²⁺ occlusion in E1Ca₂·BeF_x. E1Ca₂·BeF_x or E1Ca₂ were produced in 50 μm ⁴⁵Ca²⁺ and 15 mm Mg²⁺ as described in the legend to Fig. 4, then subjected to membrane filtration without and with perfusion for 20 s by the same buffer containing 50 μm non-radioactive Ca²⁺ (in place of ⁴⁵Ca²⁺) with and without 1 mm ADP. The amounts of ⁴⁵Ca²⁺ specifically bound and occluded in the Ca²⁺-ATPase were determined by subtracting the nonspecific background level (2.03 ± 0.04 nmol/mg) obtained with the above perfusion without ADP of the E1Ca₂ state. The error bars show the S. D. of three independent experiments.

Conversion of E1Ca₂·BeF_x to E2·BeF₃⁻ at 50 μm Ca²⁺

In Fig. 7, E1Ca₂·BeF_x was first formed in SR vesicles with BeF_x at 25 °C in 50 μm Ca²⁺ and 15 mm Mg²⁺, then further incubated at 25 and 4 °C in the presence of these ligands. The amount of bound and occluded Ca²⁺ was lost slowly (t½ = ∼2 h at 25 °C and ∼7 h at 4 °C). TNP-AMP superfluorescence (Fig. 8) and tryptic and proteinase K proteolyses (data not shown) revealed that E1Ca₂·BeF_x turned to E2·BeF₃⁻ with Ca²⁺ loss. Thus E1Ca₂· BeF_x proceeded its spontaneous slow conversion to E2·BeF₃⁻, as the autoisomerization of E1PCa₂·Mg to E2P·Mg. The Ca²⁺ ions released into the lumen may leak out during such long periods. In E1Ca₂·AlF_x and E1Ca₂·AlF₄⁻·ADP, the amount of bound (occluded) Ca²⁺ was not decreased during the above 10-h incubation at 25 °C (data not shown). The proteolysis showed that these complexes were not converted to the Ca²⁺-released forms, E2·AlF₄⁻ (E2·AlF_x) with and without ADP (data not shown). The results indicate that the product state E1PCa₂·Mg in the phosphoryl transfer acquires the structure ready for autoisomerization to E2P·Mg releasing Ca²⁺, whereas the transition state structure is not yet fully prepared for autoisomerization to the Ca²⁺-released E2P form. Interestingly, as described in supplemental Figs. S4 and S5 (with Refs. 56 and 57), the conversion E1Ca₂·BeF_x → E2·BeF₃⁻ was markedly accelerated by the transmembrane structural perturbation with hydrophobic reagents such as A23187, lasalocid, and C₁₂E₈, as well as TG. In contrast, E1Ca₂·AlF_x and E1Ca₂·AlF₄⁻·ADP were resistant against these reagents.

FIGURE 7.

Conversion of E1Ca₂·BeF_x to E2·BeF₃⁻ with Ca²⁺ release. The complex E1Ca₂·BeF_x was produced at 25 °C in 50 μm ⁴⁵Ca²⁺ and 15 mm MgCl₂ as described in the legend to Fig. 4. The complex was then further incubated in the presence of these ligands at 25 and 4 °C for various periods, and the bound ⁴⁵Ca²⁺ was determined with and without EGTA washing as described in the legend to Fig. 4A. In the control, the bound ⁴⁵Ca²⁺ in E1Ca₂ without BeF_x was determined.

E1Ca₂·BeF_x Is Perfectly Stabilized at 0.7 mm Ca²⁺

As found here, Ca²⁺ binding at high affinity transport sites in E1Ca₂ is obligatorily required for E1Ca₂·BeF_x formation, whereas millimolar high Ca²⁺ (Ca²⁺ ligation at the catalytic Mg²⁺ site I) decomposes this complex to E1Ca₂. Furthermore, E1Ca₂·BeF_x at 50 μm Ca²⁺ is spontaneously and slowly converted to E2·BeF_x releasing Ca²⁺, and the conversion is markedly accelerated by transmembrane perturbation with hydrophobic reagents such as C₁₂E₈ and A23187 (see supplemental materials). The results showed that E1Ca₂·BeF_x as the E1PCa₂·Mg analog possesses the structure prepared for its isomerization to E2·BeF₃⁻ with Ca²⁺ release as E1PCa₂·Mg → E2P·Mg + 2Ca²⁺. On the other hand, it is essential for crystallographic studies to find conditions to perfectly stabilize the E1Ca₂·BeF_x complex. In Fig. 8A, E1Ca₂·BeF_x was first formed in 0.1 mm Ca²⁺ and 15 mm Mg²⁺, then further incubated with various concentrations of Ca²⁺ with and without A23187. The structural state was monitored by TNP-AMP superfluorescence (Fig. 8), proteolysis, and tryptophan fluorescence (see Table 1 for representative data). Then we successfully found that Ca²⁺ at a very narrow concentration range, 0.7 mm, perfectly stabilizes E1Ca₂·BeF_x and maintains this complex for at least 12 days at 25 °C (and 4 °C) even in the presence of A23187. The ⁴⁵Ca²⁺ binding measurements on E1Ca₂·BeF_x in 0.7 mm ⁴⁵Ca²⁺ demonstrated that two Ca²⁺ ions are bound and occluded in this complex (Fig. 9A).

FIGURE 9.

Two Ca²⁺ are bound in E1Ca₂·BeF_x formed from E1Ca₂ and from E2·BeF₃⁻ at 0.7 mm Ca²⁺. A, SR vesicles in 0.7 mm ⁴⁵Ca²⁺ were incubated with BeF_x in the absence or presence of 5 μm A23187, otherwise as described in the legend to Fig. 4. The amount of bound Ca²⁺ was obtained with subtraction of the background level (10.1 ± 0.5 nmol/mg (n = 6)) determined by EGTA washing the vesicles incubated without BeF_x and A23187. The occluded Ca²⁺ was determined by EGTA washing in the absence of A23187 and by subtraction of the background level. (Here the determination of occluded Ca²⁺ in A23187 by EGTA washing was not feasible, because in the absence of (or even in 0.1 mm) Ca²⁺, A23187 converts E1Ca₂·BeF_x very rapidly to E2·BeF₃⁻ releasing Ca²⁺ (supplemental Figs. 4 and 5). B, E2·BeF₃⁻ was first produced in SR vesicles without A23187 and Ca²⁺. Subsequently, the samples were diluted 10 times with the buffer containing ⁴⁵CaCl₂ and BeF_x with and without 5 μm A23187, to give 0.7 mm ⁴⁵Ca²⁺ and the same buffer conditions as in A. At 15 s after dilution, the amount of bound ⁴⁵Ca²⁺ was determined without EGTA washing and by subtracting the nonspecific Ca²⁺ binding (1.0 ± 0.1 nmol/mg (n = 6)) determined by EGTA washing the sample incubated without BeF_x and A23187.

The perfectly stable E1Ca₂·BeF_x was produced from E1Ca₂ even in the presence of A23187 if 0.7 mm Ca²⁺ was included before BeF_x addition (Fig. 8, B and F, and Table 1). Also, E1Ca₂·BeF_x was successfully produced with the Ca²⁺-ATPase purified from SR vesicles by delipidation with deoxycholate (30); in this case again, by including 0.7 mm Ca²⁺ before BeF_x addition. E1Ca₂·BeF_x thus produced with the purified and delipidated Ca²⁺-ATPase was perfectly stable at least for 12 days at 4 and 25 °C in 0.7 mm Ca²⁺ (Fig. 8, B and G, at 25 °C).

E1Ca₂·BeF₃⁻ Is Produced from E2·BeF₃⁻ by Lumenal Ca²⁺ Binding

We successfully found also that E1Ca₂·BeF_x (E1Ca₂·BeF₃⁻) can be produced from E2·BeF₃⁻ by lumenal Ca²⁺ binding, as mimicking the lumenal Ca²⁺-induced reverse transition, E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. In Fig. 10, we added various concentrations of Ca²⁺ to E2·BeF₃⁻ formed in SR vesicles in 15 mm Mg²⁺ without Ca²⁺ in the presence and absence of A23187, then at 10 s after Ca²⁺ addition the structural state was examined by TNP-AMP superfluorescence. With increasing Ca²⁺ to 0.7 mm in the presence of A23187, the stable superfluorescence of E2·BeF₃⁻ was converted to the transient and slightly lower superfluorescence characteristic of E1Ca₂·BeF_x with K_0.5 of 0.4 mm Ca²⁺ and a Hill coefficient of 4 (Fig. 10, A and D). A further Ca²⁺ increase in the millimolar range caused the marked loss of superfluorescence with K_0.5 of 1.7 mm and a Hill coefficient of 1 (Fig. 10, B and D). The proteolysis also clearly showed that E2·BeF₃⁻ was converted to E1Ca₂·BeF_x by 0.7 mm Ca²⁺ in A23187 (Table 1), and this complex was further decomposed to E1Ca₂ by 10 mm Ca²⁺ (data not shown). In Fig. 9B, two ⁴⁵Ca²⁺ were shown to be bound producing E1Ca₂·BeF_x, when 0.7 mm ⁴⁵Ca²⁺ was added to E2·BeF₃⁻ in the presence of A23187. In contrast, in the absence of A23187, E2·BeF₃⁻ was neither converted to E1Ca₂·BeF_x nor decomposed to E1Ca₂ even at 10 mm Ca²⁺ (Figs. 9B and 10, C and E, and Table 1 (proteolysis at 0.7 mm Ca²⁺)).

FIGURE 10.

Formation of E1Ca₂·BeF₃⁻ from E2·BeF₃⁻ by lumenal Ca²⁺ binding. E2·BeF₃⁻ was first produced in SR vesicles with BeF_x in the presence and absence of 1.2 μm A23187 at 25 °C in 0.5 mm EGTA, 15 mm MgCl₂, 0.1 m KCl, and 30 mm MOPS/Tris (pH 7.0). Then a small volume of CaCl₂ was added to give various Ca²⁺ concentrations as indicated. At 10 s after this Ca²⁺ addition, TNP-AMP fluorescence was monitored with 4 μm TNP-AMP. In A and B, the maximum intensity in the presence of A23187 was plotted in semi-log scale at 0–0.9 mm Ca²⁺ (A) and 0.7–15 mm Ca²⁺ (B). Note also the different scales in the ordinates. The inset in A is the linear plot at 0–0.9 mm Ca²⁺ to clearly show the saturation of the first phase of the Ca²⁺-dependent change, i.e. the formation of E1Ca₂·BeF₃⁻ from E2·BeF₃⁻. In C, the maximum superfluorescence intensity in the absence of A23187 was plotted at 0–15 mm Ca²⁺. In D and E, the traces of superfluorescence development upon TNP-AMP addition in the presence of A23187 (D) or its absence (E) were shown at the representative Ca²⁺ concentrations (0, 0.7 or 1.0, and 10 mm). Solid lines in A and B show the least squares fit to the Hill equation. Apparent Ca²⁺ affinity (K_0.5) and Hill coefficients at 0–0.9 mm Ca²⁺ are 0.43 mm and 3.8 with the intensity decrease from 170 to 142, and those at Ca²⁺ over 0.7 mm are 1.7 mm and 1.0 with a further intensity decrease to 36.

The results demonstrated that E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, was produced from E2·BeF₃⁻ by the lumenal Ca²⁺ binding at the lumenally oriented low affinity transport sites, and further that Ca²⁺ substitution of Mg²⁺ at the catalytic site in E1Ca₂·BeF₃⁻ produced from E2·BeF₃⁻ caused its decomposition to E1Ca₂, therefore as the change E2·BeF₃⁻ + 2Ca²⁺ → E1Ca₂·BeF₃⁻ → E1Ca₂. Note that Mg²⁺ bound at the catalytic site in E2P·Mg is occluded, whereas it is not and therefore is exchangeable in E1PCa₂·Mg (42). Thus, these two distinctly different states of the ligated Mg²⁺ at the catalytic site (site I) in E2P·Mg and E1PCa₂·Mg are obviously mimicked here by the respective analogs E2·BeF₃⁻ and E1Ca₂·BeF₃⁻. The perfect stabilization of E1Ca₂·BeF₃⁻ achieved by 0.7 mm Ca²⁺ (Fig. 10) obviously involves lumenal Ca²⁺ binding and prevention of the Ca²⁺ release into the lumen. The stabilization by 0.7 mm Ca²⁺ in the absence of A23187 is probably due to Ca²⁺ moved passively into the vesicle lumen during the long incubation periods. All these findings show that the forward and reverse transition, E1PCa₂·Mg ↔ E2P·Mg + 2Ca²⁺, is mimicked by the forward and reverse conversion, E1Ca₂·BeF₃⁻ ↔ E2·BeF₃⁻ + 2Ca²⁺.

It is of interest to note the Hill coefficient of 4 in the lumenal Ca²⁺-induced reverse conversion, E2·BeF₃⁻ + 2Ca²⁺ → E1Ca₂·BeF₃⁻ at 0.1–0.7 mm Ca²⁺ in Fig. 10A. This might be indicative of the existence of lumenal Ca²⁺ access sites in addition to transport sites and their possible cooperative involvement in lumenal Ca²⁺ access to the transport sites. In fact, two such sites besides the two transport sites have been suggested by the kinetics and protein-chemical study on the lumenal loops (58, 59).

DISCUSSION

Formation of E1Ca₂·BeF₃⁻

As a structural analog of the physiological intermediate E1PCa₂·Mg, the E1Ca₂·BeF_x complex was successfully produced by BeF_x binding to the E1Ca₂ state Ca²⁺-ATPase and from E2·BeF₃⁻ by lumenal Ca²⁺ binding to the lumenally oriented low affinity transport sites. All the revealed properties of E1Ca₂·BeF_x met the requirements for the E1PCa₂·Mg analog; i.e. two Ca²⁺ occluded at the transport sites, Mg²⁺ bound (but not occluded) at the catalytic site, the ADP-released but still ADP-sensitive state, and its isomerization to the ADP-insensitive Ca²⁺-released state E2P·Mg (E2·BeF₃⁻) and reversal by lumenal Ca²⁺ binding, E1PCa₂·Mg ↔ E2P·Mg + 2Ca²⁺.

Furthermore, the coordination chemistry of beryllium fluoride, actually BeF₃⁻, fulfills the requirement of E1Ca₂·BeF_x as the E1PCa₂·Mg analog. In chemistry, beryllium fluoride compounds are known to adopt tetrahedral geometry with the Be-F 1.55-Å bond length, thereby making them strictly isomorphous to the tetrahedral phosphate group (60). Moreover, because of the high charge density due to the small size, beryllium is able to coordinate the aspartate-oxygen in addition to F⁻. The -O-BeF₃⁻ thus produced with Asp³⁵¹-oxygen in fact possesses the tetrahedral geometry superimposable with the covalently bound phosphate at the aspartate, as actually seen in E2·BeF₃⁻, the E2P·Mg ground-state analog (21, 22, 25). MgF₄²⁻ also possesses the tetrahedral geometry, but magnesium is not able to be coordinated directly with the Asp³⁵¹-oxygen, as seen in E2·MgF₄²⁻, the E2·P_i analog. AlF₄⁻ in E1Ca₂·AlF₄⁻·ADP and E2·AlF₄⁻ (17, 18, 20) (or AlF₃ in some cases in the haloacid dehalogenase superfamily (61)) possesses planar geometry, in which Asp³⁵¹-oxygen and ADP β-phosphate or the hydrolytic water (E2·AlF₄⁻) coordinate the aluminum at apical positions producing the bipyramidal structure superimposable to the penta-coordinated phosphorus in the transition state of in-line phosphoryl transfer E1PCa₂·ADP·Mg^‡ and acylphosphate hydrolysis E2-P·Mg^‡. Thus all chemical properties of the P_i analogs agree with the conclusion that E1Ca₂·BeF_x is the analog for E1PCa₂·Mg, and BeF_x is most probably BeF₃⁻, i.e. E1Ca₂·BeF₃⁻.

Here note that the replacement of phosphate with BeF₃⁻ produces stabilization of the E1PCa₂·Mg structure with the same geometry of BeF₃⁻ as phosphate, and therefore probably with the same binding residues for them within the catalytic site. The E1Ca₂·BeF₃⁻ stability is likely brought about by the specific chemical nature of fluoride. Namely, it possesses a significantly higher electronegativity than oxygen (actually the highest among all atoms) and a small size, therefore producing stronger BeF₃⁻ binding in the catalytic site and fixing the intermediate structure.

Structure of E1Ca₂·BeF₃⁻

Then with the newly developed E1Ca₂·BeF₃⁻, we explored its structural properties and uncovered the hitherto unknown nature of the physiological intermediate E1PCa₂·Mg and structural changes during the phosphoryl transfer/ADP release and subsequent EP isomerization/Ca²⁺ release. The observed proteinase K resistance of Thr²⁴² on the A/M3-linker revealed that, in E1PCa₂·Mg (E1Ca₂·BeF₃⁻) the A domain is already rotated perpendicular to the membrane plane from the position in E1Ca₂, thereby bringing up its junction with the A/M3-linker and imposing a strain on this linker, similarly to E1Ca₂·AlF₄⁻·ADP and E1Ca₂·CaAMP-PCP (17, 18). As described for the E1Ca₂·CaAMP-PCP structure (18), the A/M3-linker strain, i.e. the A domain perpendicular rotation is brought about by bending the P domain due to binding of the phosphate moiety and Ca²⁺ at the catalytic site (Mg²⁺-site I, Asp³⁵¹/Thr³⁵³/Asp⁷⁰³) on the P domain (see Figs. 4 and 5 in Ref. 18).⁵ Our results revealed that such a strained state is achieved even without the N-P domain cross-linking nucleotide but solely with BeF₃⁻ and Mg²⁺ binding at the catalytic site, and therefore remains in E1PCa₂·Mg after ADP release.

The strain of the A/M3-linker thus imposed has been predicted with the atomic structure (18, 19) to function as a motive force for the A domain rotation parallel to the membrane in the E1P to E2P isomerization. The partial resistance at T2 site Arg¹⁹⁸ in E1Ca₂·BeF₃⁻ (as compared with the rapid cleavage in E1Ca₂·CaAMP-PCP/E1Ca₂·AlF₄⁻·ADP and E1Ca₂·AlF_x) further indicated that in E1PCa₂·Mg, the A domain is already likely rotated parallel to the membrane to some extent from the position in E1Ca₂·MgATP*/E1PCa₂·ADP·Mg^‡. Thus, the A/M3-linker strain is likely functioning for this partial A domain rotation during the phosphoryl transfer/ADP release to produce E1PCa₂·Mg, and further for the large and complete rotation to achieve the tight A-P domain association at Arg¹⁹⁸ on the Val²⁰⁰ loop in the Ca²⁺-released state E2P·Mg (E2·BeF₃⁻). The A-P domain interaction at the Val²⁰⁰ loop is actually critical for formation of the proper Ca²⁺-released structure, E2P·Mg and its analog E2·BeF₃⁻ (25, 62, 63).

Here, it is of interest to note that residues Asp³⁵¹, Thr³⁵³, and Asp⁷⁰³ ligating Mg²⁺ and phosphate will come more proximate to each other during E1PCa₂·ADP·Mg^‡ → E1PCa₂·Mg + ADP (E1Ca₂·AlF₄⁻·ADP/E1Ca₂·AlF_x → E1Ca₂·BeF_x). As a consequence, a further P domain bending and more strain for the A/M3-linker will likely be induced by this coordination-chemical change, thereby contributing to inducing the A domain rotations during E1PCa₂·Mg formation and subsequent isomerization to E2PCa₂·Mg (besides the release of the N-P domain cross-linking nucleotide ADP). In any case, our results show that E1PCa₂·Mg (E1Ca₂·BeF₃⁻) as the product of the phosphorylation reaction acquires the structure ready for isomerization and Ca²⁺ deocclusion/release (i.e. ready for the large A domain rotation to produce E2P·Mg (E2·BeF₃⁻)), whereas the transition state structure in the phosphorylation (E1Ca₂·AlF₄⁻·ADP and E1Ca₂·AlF_x) is not yet fully prepared. Note again that the E1PCa₂·Mg structure before such motions for its isomerization to E2P·Mg is stabilized with replacement of phosphate with BeF₃⁻ in E1Ca₂·BeF₃⁻.

Important also, we found that the Ca²⁺-occluded transmembrane structure adopts not simply one state, but will proceed through changes during the phosphoryl transfer and ADP release to form E1PCa₂·Mg (see supplemental Fig. S3E). The structural change is probably coupled with the above described motions of the P and A domains (more bending and rotation) during this process. In the subsequent Ca²⁺ deocclusion/release in E1PCa₂·Mg → E2P·Mg + 2Ca²⁺, the transmembrane structure changes further (52), which was also clearly mimicked here in the change E1Ca₂·BeF₃⁻ → E2·BeF₃⁻. Thus, the structures of the transmembrane domain as well as the cytoplasmic domains in E1Ca₂·BeF₃⁻ (E1PCa₂·Mg) are intermediate between those of E1Ca₂·AlF₄⁻·ADP (E1PCa₂·ADP·Mg^‡) and E2·BeF₃⁻ (E2P·Mg).

Mg²⁺ as Physiological Catalytic Cation

Important questions regarding the E1Ca₂·BeF₃⁻ structure are why Ca²⁺ coordination at the catalytic Mg²⁺ site (site I, Asp³⁵¹/Thr³⁵³/Asp⁷⁰³) is absolutely unfavorable for E1Ca₂·BeF₃⁻ formation, and why the Mg²⁺-coordinated structure E1Ca₂·BeF₃⁻ differs from Ca²⁺-coordinated E1PCa₂·Ca, E1Ca₂·CaAMP-PCP, and E1PCa₂·Ca·AMP-PN structures as well as from E1Ca₂· AlF₄⁻·ADP/E1Ca₂·AlF_x, especially in A domain positioning. These questions may be relevant to the questions of why forward isomerization of E1PCa₂·Ca to E2P is markedly retarded in contrast to E1PCa₂·Mg and thus why Mg²⁺ is preferred as the catalytic cation. In stringent coordination chemistry, the coordination distance of Mg²⁺ is shorter than Ca²⁺, typically by 0.2 Å (e.g. 2.1 versus 2.3 Å (64, 65)). As a consequence, in the case of E1Ca₂·CaAMP-PCP, the distance between the γ-phosphate and Asp³⁵¹-oxygen becomes 3.24 Å, being greater by 0.3 Å than that predicted in E1Ca₂·MgAMP-PCP. Therefore MgAMP-PCP (MgATP) binding would result in steric clash, and E1Ca₂·CaAMP-PCP is more stable than E1Ca₂·MgAMP-PCP, and therefore has less tendency to decompose to E1Ca₂ (also in the forward direction to the EP formation and its decay in the case of E1Ca₂·CaATP) (45). In E1Ca₂·BeF₃⁻ formed here with Mg²⁺, the direct coordination between Asp³⁵¹ and the beryllium and their proximate positioning would probably favor the closely positioned ligand residues (Thr³⁵³/Asp⁷⁰³/Asp³⁵¹) for BeF₃⁻ and Mg²⁺ but not for Ca²⁺. Therefore Ca²⁺ substitution of Mg²⁺ probably disrupted the precise geometry and decomposed the E1Ca₂·BeF₃⁻ complex. Also, a possible difference in the coordination number might be involved; Mg²⁺ prefers definitely six, whereas Ca²⁺ can accommodate seven or eight ligands (65–69).

Furthermore, the difference in A domain positioning between the Mg²⁺-coordinated state E1Ca₂·BeF₃⁻ and the Ca²⁺-coordinated states may be reasonably understood by the consequence of the stringent coordination chemistry. Namely, because the shorter coordination distance of Mg²⁺, P domain bending, and the resulting A domain rotation perpendicular to the membrane will be greater in the Mg²⁺-coordinated state. Therefore the strain of the A/M3-linker and A domain rotation parallel to the membrane will be more in the Mg²⁺ state E1Ca₂·BeF_x. In this context, it is also reasonable that E1PCa₂·Mg is more rapidly isomerized to E2P with less energy barrier for the large A domain rotation, in contrast to the retarded isomerization in E1PCa₂·Ca that is stabilized by the likely conformational inadequacy. Here note that the cause of the E1PCa₂·Ca stabilization is obviously different from that of E1PCa₂·Mg stabilization produced by replacement of phosphate with BeF₃⁻ (see the above discussion for E1Ca₂·BeF₃⁻ stabilization).

Previously it was documented (45, 64, 70) that destabilization of the non-covalent complex E1Ca₂·MgATP by Mg²⁺ (as found with MgAMP-PCP versus CaAMP-PCP) together with stabilization of the transition state by Mg²⁺ (as found with E1Ca₂·AlF₄⁻·ADP bound Mg²⁺ at both sites I and II) leads to a decrease of the activation energy and a rapid phosphoryl transfer. As another critical reason for Mg²⁺ preference for catalysis, we predict here by exploring the property of the E1Ca₂·BeF_x that the Mg²⁺ bound at the catalytic site produces the proper E1PCa₂ structure, which is ready for rapid transition to E2P in this rate-limiting process of the transport cycle.

Hydrophobic Catalytic Site in E1Ca₂·BeF₃⁻

The microenvironment around Asp³⁵¹ in E1PCa₂·Mg was further predicted by TNP-AMP superfluorescence in E1Ca₂·BeF₃⁻ to be strongly hydrophobic and thus a closed state, and this will become even more in the change E1PCa₂·Mg → E2P·Mg + 2Ca²⁺ (E1Ca₂·BeF₃⁻ → E2·BeF₃⁻). The observed distinct difference between E1Ca₂·BeF₃⁻ and E2·BeF₃⁻ (transient versus stable and slightly higher superfluorescence) is probably ascribed to the distinct difference in the organization state of cytoplasmic domains. The superfluorescence, nevertheless, developed solely in the Ca²⁺-ATPase complexed with beryllium fluoride, E1Ca₂·BeF₃⁻ and E2·BeF₃⁻ (E2P·Mg), but no development in E1Ca₂·AlF_x and in E2·AlF₄⁻ and E2·MgF₄²⁻ (25). Therefore, the hydrophobic closed catalytic site is accomplished by the direct coordination and close proximity of the beryllium with Asp³⁵¹-oxygen and by the specific coordination of the tetrahedral -O-BeF₃⁻, i.e. Asp³⁵¹-acylphosphate within the catalytic site. This is obviously not the case in AlF₄⁻ (the penta-coordinated phosphorus of the transition states) and MgF₄²⁻ (non-covalently bound P_i), thus in these states, the catalytic site is more accessible to nonspecific water molecules.

Whether E1PCa₂·Mg develops the superfluorescence had been controversial. In addition to the obvious problem of TNP-AMP competition against ATP for phosphorylation, the observed TNP-AMP-induced decomposition of E1Ca₂·BeF₃⁻ further revealed that the E1PCa₂·Mg structure may be similarly disrupted rapidly by TNP-AMP binding, therefore making it virtually impossible to examine the superfluorescence development in E1PCa₂·Mg. The TNP-AMP-induced E1Ca₂·BeF₃⁻ decomposition might have occurred by means of a similar structural change as the ADP-induced one, i.e. disruption of the cytoplasmic domain organization and possible BeF₃⁻ release. The most important conclusion here is that the hydrophobic and closed property of the phosphorylated catalytic site both in E1PCa₂·Mg and E2P·Mg may be requisite to avoid a possible attack of nonspecific water molecules on the Asp³⁵¹-acylphosphate thus accomplishing Ca²⁺ release into the lumen and energy coupling.

Formation of E1Ca₂·BeF₃⁻ from E2·BeF₃⁻ and Perfect Stabilization of E1Ca₂·BeF₃⁻

E1Ca₂·BeF₃⁻ was produced also from E2·BeF₃⁻ by binding two lumenal Ca²⁺ to the lumenally oriented low affinity transport sites at 0.7 mm Ca²⁺ and 15 mm Mg²⁺, as mimicking the reverse transition E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. At the critical concentration of 0.7 mm Ca²⁺ in 15 mm Mg²⁺, E1Ca₂·BeF₃⁻ is perfectly stabilized without decomposition to E1Ca₂ or conversion to E2·BeF₃⁻. The perfect E1Ca₂·BeF₃⁻ stabilization is obviously achieved by preventing Ca²⁺ release into the lumen and by avoiding the absolutely unfavorable Ca²⁺ substitution of Mg²⁺ in site I at the most appropriately balanced concentrations of Ca²⁺ and Mg²⁺. As noted in the last paragraph under “Results,” stabilization of E1Ca₂·BeF₃⁻ might possibly involve lumenal Ca²⁺ access at the putative lumenal gating sites besides the transport sites. If this is the case, the gate-opening and Ca²⁺ release into the lumen takes place when the lumenal Ca²⁺ is low enough to avoid the possible lumenal Ca²⁺ access to the gate.

Integrated Picture of EP Processing

Recently, we successfully identified and trapped for the first time the intermediate state E2PCa₂·Mg, ADP-insensitive EP with two Ca²⁺ occluded at transport sites, by elongating the A/M1′-linker (71), and revealed that the proper length of this linker is critical for inducing structural changes for Ca²⁺ deocclusion and release from E2PCa₂·Mg. This dependence on the length of the linker is probably because the length controls the extent of strain between the A domain and M1′, which causes motions of the cytoplasmic A and P domains thereby transmitting the structural signal to the transmembrane transport sites. In trapped E2PCa₂·Mg, the A domain is already largely rotated, and A-P domain associations at Val²⁰⁰ and TGES¹⁸⁴ loops are already produced, although the interaction network is not produced properly at the Tyr¹²²-hydrophobic cluster (71), which is critical for Ca²⁺ deocclusion/release and E2P hydrolysis (72–74). In the Ca²⁺-released E2P·Mg, this cluster is formed from seven residues of the A (Ile¹⁷⁹/Leu¹⁸⁰/Ile²³²) and P (Val⁷⁰⁵/Val⁷²⁶) domains and the top part of M2 (Leu¹¹⁹/Tyr¹²²) (see Fig. 2).

The results indicated that the successive structural changes take place as follows: in E1PCa₂·Mg → E2PCa₂·Mg, the A domain rotates largely (further from the position in E1PCa₂·Mg) into the space between the N and P domains and docks onto the Asp³⁵¹-acylphosphate of the P domain, thereby causing loss of ADP sensitivity and also the strain of the A/M1′-linker (because the A domain is brought above the P domain). The strain thus imposed will cause inclinations of the A and P domains and the connected M2 and M4/M5 thereby rearranging the helices to destroy Ca²⁺ sites and open the lumenal gate thus to release Ca²⁺. Upon these motions, the Tyr¹²²-hydrophobic cluster is produced from the inclined A and P domains and M2. Hence, interactions at this cluster and at the Val²⁰⁰ loop stabilize the Ca²⁺-released structure E2P·Mg, and also produce the catalytic site for the acylphosphate hydrolysis to occur after Ca²⁺ release, ensuring energy coupling (63, 72–74). Atomic level structural studies of E1Ca₂·BeF₃⁻ as E1PCa₂·Mg and the trapped intermediate state E2PCa₂·Mg will contribute to further understanding of EP processing, Ca²⁺ handling, and E2P hydrolysis.