Assembly of a Tyr¹²² Hydrophobic Cluster in Sarcoplasmic Reticulum Ca²⁺-ATPase Synchronizes Ca²⁺ Affinity Reduction and Release with Phosphoenzyme Isomerization

Background: Ca²⁺ transport by Ca²⁺-ATPase includes phosphoenzyme isomerization with luminal Ca²⁺ release.

Results: Mutation of Leu¹¹⁹, Tyr¹²², or Ile¹⁷⁹ in an interdomain hydrophobic cluster retards release relative to isomerization.

Conclusion: There is a transient Ca²⁺-bound state and affinity reduction during release governed by cluster assembly.

Significance: Ca²⁺ release is a multistep process directed by head domain gathering on transmembrane helix M2.

Abstract

The mechanism whereby events in and around the catalytic site/head of Ca²⁺-ATPase effect Ca²⁺ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca²⁺ by taking advantage of its rapid occlusion upon formation of E1PCa₂ and of stabilization afforded by a high concentration of Ca²⁺. The assay is applicable to minute amounts of Ca²⁺-ATPase expressed in COS-1 cells. It was validated by measuring the Ca²⁺ binding properties of unphosphorylated Ca²⁺-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca²⁺ release process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. In the wild type, Ca²⁺ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa₂ → E2PCa₂) followed by very rapid Ca²⁺ release. In contrast, with alanine mutants of Leu¹¹⁹ and Tyr¹²² on the cytoplasmic part of the second transmembrane helix (M2) and Ile¹⁷⁹ on the A domain, Ca²⁺ release in 10 μm Ca²⁺ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca²⁺. The results suggest that these residues function in Ca²⁺ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca²⁺ release.

Introduction

Sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1a),² a representative member of P-type ion-transporting ATPases, catalyzes Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (for recent reviews, see Refs. 1–3). The enzyme is activated by the binding of two cytoplasmic Ca²⁺ ions at the high-affinity transport sites (E2 to E1Ca₂, steps 1 and 2) and autophosphorylated at Asp³⁵¹ with MgATP to form an ADP-sensitive phosphoenzyme (E1P, step 3), which reacts with ADP to regenerate ATP in the reverse reaction. Upon E1P formation, the two bound Ca²⁺ are occluded in the transport sites (E1PCa₂). The subsequent isomeric transition to the ADP-insensitive E2P form results in rearrangements of the Ca²⁺ binding sites to deocclude Ca²⁺, open the release path, and reduce the affinity, therefore releasing Ca²⁺ into the lumen (steps 4 and 5). Finally, the Asp³⁵¹-acylphosphate in E2P is hydrolyzed to form a Ca²⁺-unbound inactive E2 state (step 6).

FIGURE 1.

Reaction schematic of Ca²⁺-ATPase.

The EP isomerization associated with luminal Ca²⁺ release is a key rate-limiting process and involves a large rotation of the A domain, its association with the P domain and the cytoplasmic part of M2, and an inclination of associated A and P domains and the connected helices M2/M1 and M4/M5 via a steric effect of a M1/M2 V-shaped body (4–20). These motions are coupled to a rearrangement of the transport sites. We have found previously that the hydrophobic association of Leu¹¹⁹/Tyr¹²² on the cytoplasmic part of M2 with the A and P domains (Tyr¹²² hydrophobic cluster) is critical for formation of the Ca²⁺-released E2P ground state structure, with hydrolytic ability at the catalytic site and a properly opened luminal Ca²⁺ release path with reduced affinity at the transport sites (16–18). The Tyr¹²² hydrophobic cluster is formed with residues on the cytoplasmic part of M2 (Tyr¹²²/Leu¹¹⁹), A domain (Ile¹⁷⁹/Leu¹⁸⁰), P domain (Val⁷⁰⁵/Val⁷²⁶), and A/M3 linker (Ile²³²), thereby producing a most compactly organized head in E2P. The postulated E2PCa₂ transient intermediate was successfully trapped in the Ca²⁺-occluded state (before deocclusion) by elongation of the A/M1′ linker with a two- or four-glycine insertion. Consistently in this state, the Tyr¹²² hydrophobic cluster was shown to not yet be fully formed. The finding demonstrates the critical role of the strain in this linker, probably to effect inclination of the A and P domains and connected helices required for deocclusion and Ca²⁺ release (19, 20).

Further understanding of Ca²⁺ binding/release processes in the transport cycle and roles of residues involved, especially the dynamic key process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺, is hampered by an inability to determine and detect the bound but deoccluded Ca²⁺ in minute amounts of expressed wild type and mutants obtained from cultured cells. In this study, we developed a method to determine the bound Ca²⁺ in the non-phosphorylated state as well as in the phosphorylated state of expressed enzymes. We took advantage of the rapid occlusion in E1PCa₂ of bound Ca²⁺, from either E1Ca₂ or E2P species (E2P and, possibly, the transient E2P state with bound Ca²⁺) by addition of a high concentration (10 mm) of Ca²⁺ (plus ATP for E1Ca₂) (21–25), probably trapping the bound Ca²⁺ before possible exchange with the added Ca²⁺. The E1PCa₂ thus formed is very stable, probably because of Ca²⁺ substitution of Mg²⁺ bound at the catalytic Mg²⁺ subsite, as found previously (23, 26–29), therefore withstanding membrane filtration and extensive washing. We then applied this new method to the EP isomerization and Ca²⁺ release kinetic processes in alanine substitution mutants of the Tyr¹²² hydrophobic cluster because they are critical for the Ca²⁺-released E2P ground state structure (16–18). The results indicate the presence of a transient E2P state with bound but deoccluded Ca²⁺ and show that Leu¹¹⁹ and Tyr¹²² on the cytoplasmic part of M2 and Ile¹⁷⁹ on the A domain function via their association to reduce Ca²⁺ affinity during E2P processing and, thereby, to accelerate Ca²⁺ release into the lumen. The detailed analyses suggest that a possibly stepwise assembly of the residues into the Tyr¹²² hydrophobic cluster takes place for proper Ca²⁺ handling (namely, deocclusion, affinity reduction, and release) and, therefore, Ca²⁺ transport coupled with EP processing.

Experimental Procedures

Mutagenesis and Expression

QuikChange^TM site-directed mutagenesis (Stratagene) was utilized for the substitution of residues in rabbit SERCA1a cDNA. The ApaI-KpnI or KpnI-SalI restriction fragment was ligated back into the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector (30). The pMT2 DNA was transfected into COS-1 cells with Lipofectamine and Plus^TM reagent (Invitrogen), and the microsomes were prepared from COS-1 cells as described previously (18, 31).

Determination of EP

Microsomes expressing wild-type or mutant SERCA1a prepared from the COS-1 cells were phosphorylated with [γ-³²P]ATP or ³²P_i under the conditions described in the figure legends. The total amount of EP was determined following the addition of trichloroacetic acid. The amount of E2P was determined by adding an equal volume of a solution containing 2 mm ADP and 5 mm EGTA, followed by the trichloroacetic acid addition 1 s after the ADP addition. The amount of EP was quantified with digital autoradiography after separation by 5% SDS-PAGE at pH 6.0 according to Weber and Osborn (32) and as described previously (33). The amount of EP in expressed SERCA1a was obtained by subtracting the background radioactivity determined in the absence of Ca²⁺. In all mutants and the wild type, the background level was less than 1% of the total amount of EP.

Determination of Bound Ca²⁺

Microsomes were incubated with 0–10 μm ⁴⁵Ca²⁺ at 4 °C in the absence or presence of 1 μm thapsigargin (TG), a highly specific and subnanomolar affinity inhibitor of SERCA that fixes the enzyme in the Ca²⁺-unbound E2 state (34). 50 μl of reaction mixture was spotted on a membrane filter (Millipore, 0.45-μm mixed cellulose membrane HAWP) and washed for 3 s with 1 ml of Ca²⁺ binding assay medium containing 50 mm HEPES/Tris (pH 8.0), 0.1 m KCl, 10 mm CaCl₂, 7 mm MgCl₂, and 0.1 mm ATP. Other experimental conditions are described in detail in the figure legends. The ⁴⁵Ca²⁺ remaining on the filter was quantified by digital autoradiography. The amount of Ca²⁺ specifically bound to the Ca²⁺-ATPase (trapped as occluded in E1PCa₂) was obtained by subtracting the amount of nonspecific Ca²⁺-binding background determined in the presence of TG. We confirmed that all mutants retained TG sensitivity by observing that TG (1 μm used in this study) completely inhibits EP formation from [γ-³²P]ATP in the presence of Ca²⁺, showing its validity for the background determination. The background level of nonspecific Ca²⁺ binding (in the presence of 10 μm Ca²⁺) was ∼100 pmol/mg of microsomal protein, which is 30–50% of total Ca²⁺ binding (see Fig. 4, A and B).

FIGURE 4.

Ca²⁺ concentration dependence of Ca²⁺ binding in non-phosphorylated Ca²⁺-ATPase from COS-1 cells. A and B, microsomes expressing the wild type (A) or mutant L119A (B) were incubated at 4 °C for 20 s with various concentrations of ⁴⁵Ca²⁺ in a mixture containing 20 μg/ml microsomal protein, 50 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, 3 μm A23187, and 10 μm ⁴⁵CaCl₂ with 0–0.09 mm EGTA to give the indicated free Ca²⁺ concentration in the presence (open triangles) or absence (open circles) of 1 μm TG. Then, as described under “Experimental Procedures,” 50 μl of the mixture was spotted on a membrane filter and washed immediately for 3 s with 1 ml of a Ca²⁺ binding assay medium containing 10 mm CaCl₂ and 0.1 mm ATP in 50 mm HEPES/Tris (pH 8.0), 0.1 m KCl, and 7 mm MgCl₂. The values presented are the mean ± S.D. (n = 3–4). The amount of Ca²⁺ specifically bound to the Ca²⁺-ATPase (trapped as occluded Ca²⁺ in E1PCa₂, closed circles) was obtained by subtracting the amount of nonspecific Ca²⁺ binding determined in the presence of TG. The solid lines show the least squares fit to the Hill equation with the fitting parameters K_d and Hill coefficient of 0.22 μm and 1.6 for the wild type and 0.29 μm and 1.6 for the mutant L119A. C, the maximum amount of specific ⁴⁵Ca²⁺ binding in 10 μm ⁴⁵Ca²⁺ was determined as above (Ca) and compared with the maximum amount of EP (EP) determined in the presence of 10 mm Ca²⁺ as in Fig. 2A (i.e. essentially under the same conditions as described for the determination of the catalytic site content in the preparation (45)). It should be mentioned here that, for the comparison, one preparation of microsomes was used throughout for the wild type and the mutant. Actual values of the amount of bound Ca²⁺ and that of EP (pmol/mg of microsomal protein (n = 4)) for the wild type were 287 ± 14 and 136 ± 8, respectively, giving a stoichiometry of Ca/EP = 2.11. Those for the L119A mutant were 95.6 ± 16.2 and 48.6 ± 2.6, respectively, providing a stoichiometry of Ca/EP = 1.97. Note that these maximum Ca²⁺ binding values are slightly different from those in A and B, in which different preparations were used.

Miscellaneous

Protein concentrations were determined according to Lowry et al. (35). Data were analyzed by nonlinear regression using the program Origin (Microcal Software, Inc.). Free Ca²⁺ concentrations were calculated by the Calcon program. Three-dimensional models of SERCA1a were produced by the program VMD (36).

Results

Rapid E1PCa₂ Formation and Its Stabilization

To fix the bound Ca²⁺ in E1Ca₂ in an occluded form in E1PCa₂, it is necessary to rapidly phosphorylate E1Ca₂ to E1PCa₂ before Ca²⁺release and to stabilize E1PCa₂ and prevent its decay during thorough washing. In analysis of the EP isomerization/Ca²⁺ release process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺, the E2P species need to be rapidly converted to a stabilized E1PCa₂ state. The crucial feature for the forward reaction is to add a very high concentration (10 mm) of Ca²⁺ together with ATP. E1PCa₂ is extremely stable in a high concentration of Ca²⁺, probably because of Ca²⁺ replacing catalytic subsite Mg²⁺, as shown previously (23, 26–29). This stabilization prevents possible Ca²⁺ exchange at the transport sites in the reverse conversion in E1PCa₂ ↔ E2P + 2Ca²⁺. Actually, the Mg²⁺ at the catalytic subsite is not “occluded” in E1PCa₂ and exchanges rapidly with Ca²⁺ at such a high concentration (26–28). Also, addition of a high concentration of Ca²⁺ rapidly converts E2P to the stable E1PCa₂ in the reverse reaction (18, 22).

In Fig. 2A, wild-type SERCA1a expressed in microsomes of COS-1 cells was incubated with 10 μm Ca²⁺ to form the E1Ca₂ state in 7 mm Mg²⁺, and then [γ-³²P]ATP plus an excess of EGTA or [γ-³²P]ATP plus 10 mm Ca²⁺ was added in 0.1 m KCl, which accelerates E2P hydrolysis, causing E1PCa₂ accumulation (37). When ATP is added with EGTA, E1PCa₂ forms very rapidly despite the removal of free Ca²⁺ and then decays slowly via the rate-limiting EP isomerization, followed by rapid hydrolysis. The maximum EP level immediately after ATP/EGTA addition, estimated by an extrapolation of the single exponential decay to zero time, is very close to that obtained with ATP plus 10 mm Ca²⁺. E1PCa₂ formation in 10 mm Ca²⁺ is also rapid and reaches a maximum within ∼3 s, albeit slightly slower than that without free Ca²⁺ (i.e. with the substrate MgATP, consistent with previous kinetic studies (21–25)). Importantly, during this 3 s period, ∼90% of E1PCa₂ remains when free Ca²⁺ is removed by EGTA. Therefore, E1PCa₂ formation and resulting Ca²⁺ occlusion by the simultaneous addition of ATP and 10 mm Ca²⁺ is rapid enough to trap almost all ⁴⁵Ca²⁺ originally bound in E1Ca₂ as an occluded E1PCa₂ species before Ca²⁺ release and possible ⁴⁵Ca²⁺-Ca²⁺ exchange (as demonstrated in Fig. 4).

FIGURE 2.

Time course of EP formation and its decay in the wild type. A, microsomes expressing wild-type Ca²⁺-ATPase prepared from COS-1 cells were incubated with 10 μm Ca²⁺ in a mixture containing 20 μg/ml microsomal protein, 20 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, 10 μm CaCl₂, and 3 μm A23187 at 4 °C. Then EP formation was initiated at zero time by mixing with an equal volume of a solution containing 20 μm [γ-³²P]ATP, 0.1 m HEPES/Tris (pH 8.0), 0.1 m KCl, and 7 mm MgCl₂ with 10 mm CaCl₂ (closed circles) or 2 mm EGTA (open circles). B, EP was formed in 20 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, 10 μm CaCl₂, 3 μm A23187, and 10 μm [γ-³²P]ATP at 4 °C for 10 s. Then the reaction was chased at zero time by mixing with an equal volume of a solution containing non-radioactive 0.2 mm ATP and various concentrations of CaCl₂ to give the indicated final Ca²⁺ concentrations (0.01, 1, and 10 mm) or 2 mm EGTA to remove free Ca²⁺ in 0.1 m HEPES/Tris (pH 8.0), 0.1 m KCl, 7 mm MgCl₂, and 3 μm A23187. The amount of EP was normalized to the value at zero time of the chase.

In Fig. 2B, the decay of E1PCa₂ formed with [γ-³²P]ATP in 10 μm Ca²⁺ and 7 mm Mg²⁺ was initiated by a cold (non-radioactive) ATP chase in various concentrations of Ca²⁺. The decay is slowed markedly with increasing Ca²⁺. Actually, E1PCa₂ in 10 mm Ca²⁺ remains almost completely stable during the initial 3 s, a characteristic exploited in the Ca²⁺ binding assays in Figs. 4–10 as a period for membrane filter washing with 10 mm Ca²⁺.

FIGURE 5.

Time courses of EP isomerization and Ca²⁺ release in the wild type. A, for the determination of EP, wild-type Ca²⁺-ATPase was phosphorylated in a mixture containing 20 μg/ml microsomal protein, 20 μm [γ-³²P]ATP, 10 μm CaCl₂, 50 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, and 3 μm A23187. Then the phosphorylation was chased at zero time by mixing with an equal volume of EGTA chase solution containing 1 mm EGTA, 50 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, and 3 μm A23187. The reaction was quenched by acid at the indicated times. Here it should be noted that nearly all the EP was E1P in the presence of KCl (actually more than 95%), as we observed under essentially the same conditions (cf. Fig. 2A in Ref. 42). Therefore, EP decay represents EP isomerization (which is followed by rapid E2P hydrolysis). B, for the Ca²⁺ binding assay, wild-type Ca²⁺-ATPase in microsomes (MS) was incubated for 5 s with 10 μm ⁴⁵Ca²⁺ in 50 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, and 3 μm A23187 and phosphorylated by addition of a small volume of ATP to give 10 μm (ATP) for 20 s. Immediately, the mixture (50 μl) was spotted on the membrane filter (spot), and, at zero time (EGTA), free Ca²⁺ was removed to initiate EP decay by continuous rinsing with the above EGTA chase solution. After various periods (t), the amount of ⁴⁵Ca²⁺ specifically bound to Ca²⁺-ATPase was determined by washing the filter with 1 ml of the Ca²⁺ binding assay medium as in Fig. 4 (wash). The values presented are the mean ± S.D. (n = 3–6). C, the time courses of EP decay (closed circles) and decrease in bound ⁴⁵Ca²⁺ (open circles) determined in A and B are replotted in the same panel. The plots show the mean values in A and B. The solid and broken lines show a single exponential fit in which the rates are 0.23 s⁻¹ for EP decay and 0.22 s⁻¹ for the decrease in bound ⁴⁵Ca²⁺, respectively.

FIGURE 6.

Time courses of Ca²⁺ release and EP isomerization in mutant L119A. A, microsomes (MS) expressing mutant L119A (20 μg/ml microsomal protein) were incubated for 20 s with 10 μm ⁴⁵Ca²⁺ in 50 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, and 3 μm A23187. Then, at zero time (ATP + or −EGTA), phosphorylation was initiated by addition of 10 μm ATP with (open circles) or without 1 mm EGTA (closed circles), the mixture was spotted on the filter, and the amount of ⁴⁵Ca²⁺ specifically bound to the Ca²⁺-ATPase was determined as in Fig. 5B. The values presented are the mean ± S.D. (n = 3–5). The solid lines show a single exponential fit, and the rates of the ⁴⁵Ca²⁺ release thus determined are 0.10 s⁻¹ in an excess of EGTA (open circles) and 0.05 s⁻¹ in the presence of 10 μm ⁴⁵Ca²⁺ (closed circles). B, inset, microsomes expressing the mutant L119A (40 μg/ml microsomal protein) in 10 μm Ca²⁺ were phosphorylated at zero time by mixing with an equal volume of a solution containing 20 μm [γ-³²P]ATP and 10 μm CaCl₂ (circles) or 2 mm EGTA (triangles), and the total amount of EP (closed symbols) and the amount of E2P (open symbols) were determined at the indicated times as in Fig. 5A. Main panel, the fraction of E1P in the total amount of EP was calculated at each time point by subtracting the amount E2P from the total amount of EP (E1P plus E2P). The solid lines show a single exponential fit, and the rates of the decrease in E1P fraction, i.e. the E1P to E2P isomerization, thus determined are 0.108 s⁻¹ in an excess of EGTA (open circles) and 0.089 s⁻¹ in the presence of 10 μm Ca²⁺ (closed circles).

FIGURE 7.

Time courses of Ca²⁺ release and EP isomerization in mutant Y122A. A, Ca²⁺ release from EP in the mutant Y122A was determined as in Fig. 6A. The values presented are the mean ± S.D. (n = 3–5). The solid lines show a single exponential fit, and the rates of the ⁴⁵Ca²⁺ release thus determined are 0.23 s⁻¹ in an excess of EGTA (open circles) and 0.125 s⁻¹ in the presence of 10 μm ⁴⁵Ca²⁺ (closed circles). B, inset, the total amount of EP (closed symbols) and the amount of E2P (open symbols) were determined at the indicated times with the mutant Y122A as described in Fig. 6B. Main panel, the fraction of E1P in the total amount of EP was calculated at each time point by subtracting the amount E2P from the total amount of EP (E1P plus E2P). The solid lines show a single exponential fit, and the rates of the decrease in E1P fraction, i.e. the E1P to E2P isomerization, thus determined are 0.286 s⁻¹ in an excess EGTA (open circles) and 0.280 s⁻¹ in the presence of 10 μm Ca²⁺ (closed circles).

FIGURE 8.

Time courses of EP isomerization in mutants I179A and V705A. The mutants I179A (A) and V705A (B) were phosphorylated with ATP, and the total amount of EP (closed symbols) and the amount of E2P (open symbols) were determined at the indicated time in an excess of EGTA (triangles) or in 10 μm Ca²⁺ (circles) as described in the inset in Fig. 6B. The fraction of E1P in the total amount of EP in the presence of 10 μm Ca²⁺ was calculated at each time point by subtracting the amount E2P from the total amount of EP (E1P plus E2P) and is depicted in Fig. 9B.

FIGURE 9.

Time courses of Ca²⁺ release and EP isomerization in the presence of 10 μm Ca²⁺ in mutants I179A and V705A. A, Ca²⁺ release from EP in the mutants I179A and V705A was determined in the presence of 10 μm ⁴⁵Ca²⁺ as described in Fig. 6A. The values presented are the mean ± S.D. (n = 3–5). The solid lines show a single exponential fit, and the rates of the ⁴⁵Ca²⁺ release thus determined are 0.22 s⁻¹ in I179A (closed triangles) and 0.48 s⁻¹ in V705A (open triangles). B, the fraction of E1P in the total amount of EP was calculated at each time point in the presence of 10 μm Ca²⁺ in Fig. 8 by subtracting the amount E2P from the total amount of EP (E1P plus E2P). The values presented are the mean ± S.D. (n = 3). The solid lines show a single exponential fit, and the rates of the decrease in E1P fraction, i.e. the E1P to E2P isomerization, thus determined are 0.34 s⁻¹ in I179A (closed triangles) and 0.38 s⁻¹ in V705A (open triangles).

FIGURE 10.

E1P fraction and bound Ca²⁺. The amount of bound Ca²⁺ and the E1P fraction in total amount of EP (E1P plus E2P) were determined repeatedly in the presence of an excess 1 mm EGTA (bottom panel) or 10 μm Ca²⁺ (top panel) or at one selected time point during the EP isomerization and Ca²⁺ release time courses (i.e. 2 s after the start of reaction) for the mutants I232A and V726A in comparison with the mutant V705A, as described in Fig. 6. The E1P fraction in the total amount of EP and the amount of bound ⁴⁵Ca²⁺ relative to the maximum ⁴⁵Ca²⁺ binding determined at zero time are shown as indicated (E1P and bound ⁴⁵Ca, respectively). The values presented are the mean ± S.D. (n = 3–4). It should be noted that the Ca²⁺-ATPase is dephosphorylated to the E2 state upon Ca²⁺ removal by an excess of EGTA (bottom panel) and is in all phosphorylated states in the presence of 10 μm Ca²⁺ (top panel, see Figs. 6–8). Therefore, there is no Ca²⁺ bound to non-phosphorylated enzyme under our experimental conditions.

Rapid Luminal Ca²⁺-induced Reverse Conversion E2P + 2Ca²⁺ → E2PCa₂ → E1PCa₂ and Stabilization of E1PCa₂

In Fig. 3, E2P was first formed with P_i and Mg²⁺ in the reverse reaction of hydrolysis, and then, at zero time, its decay was initiated by a large dilution with or without 10 mm Ca²⁺ in the presence of the Ca²⁺ ionophore A23187. When 10 mm Ca²⁺ is added, all E2P becomes stable E1P (as judged from its ADP sensitivity) because of Ca²⁺ binding to the luminally oriented, low-affinity Ca²⁺ transport sites (18, 22), in contrast to the rapid E2P hydrolysis without Ca²⁺ addition. The results show that E2P is very rapidly converted to E1PCa₂ upon addition of 10 mm Ca²⁺, and E1PCa₂ thus formed is stabilized in 10 mm Ca²⁺, in agreement with previous reports (23, 26–29). The results also suggest that E2P with bound Ca²⁺ can be fixed in the stable E1PCa₂ by reverse conversion.

FIGURE 3.

Stabilization of E1PCa₂ formed by reverse conversion from E2P. Wild-type Ca²⁺-ATPase in microsomes was phosphorylated at 25 °C with 0.1 mm ³²P_i in a mixture containing 200 μg/ml microsomal protein, 50 mm MOPS/Tris (pH 7.3), 7 mm MgCl₂, 1 mm EGTA, 30 μm A23187, 7 mm MgCl₂, and 20% (v/v) Me₂SO that strongly favors E2P formation. The mixture was chilled at 4 °C and then, at zero time, diluted with a 19-fold volume of a solution containing 50 mm HEPES/Tris (pH 8.0), 0.105 m KCl, 7 mm MgCl₂, and 10.5 mm CaCl₂ (open and closed circles) or 1 mm EGTA (open triangles) in the absence (open symbols) or presence (closed circles) of 10.5 mm ADP, and the amount of EP was determined at the indicated times.

Ca²⁺ Binding to the Non-phosphorylated Wild Type

Considering all of these advantages of rapid formation and stabilization of E1PCa₂ by the simultaneous addition of high concentrations of Ca²⁺ (10 mm) and ATP, we first determined the bound but non-occluded Ca²⁺ in wild type E1Ca₂ by membrane filtration, as shown in Fig. 4, top panel. Microsomes containing expressed Ca²⁺-ATPase were incubated with various concentrations of ⁴⁵Ca²⁺, spotted on a membrane filter, and exposed to continuous washing with Ca²⁺ binding assay medium containing 0.1 mm ATP and 10 mm non-radioactive Ca²⁺ for 3 s, during which E1PCa₂ is rapidly formed and stabilized and free ⁴⁵Ca²⁺ is washed out. The amount of ⁴⁵Ca²⁺ specifically bound to the Ca²⁺-ATPase was obtained by subtracting the background determined in the presence of TG from that in its absence. The ⁴⁵Ca²⁺ concentration dependence shows a saturation curve with a K_d value and Hill coefficient of 0.22 μm and 1.64, respectively, and a stoichiometry of maximum binding of ∼2 Ca²⁺ per catalytic site (maximum amount of EP, Fig. 4C), in complete agreement with the properties of the high-affinity transport sites established with sarcoplasmic reticulum vesicle Ca²⁺-ATPase (38) (whose values actually observed by this method are a K_d of 0.25 μm, a Hill coefficient of 1.61, and a stoichiometry of ∼2). Therefore, this method clearly fixes the ⁴⁵Ca²⁺ originally bound in E1Ca₂ of expressed SERCA1a in an occluded form as E1PCa₂ before its release and exchange with the non-radioactive Ca²⁺ at 10 mm in the Ca²⁺ binding assay medium. This validates the method for measuring Ca²⁺ binding for small quantities of expressed SERCA1a.

The results also imply that there is virtually no exchange of the bound ⁴⁵Ca²⁺ in E1PCa₂ with the subsequently added 10 mm non-radioactive Ca²⁺. As noted above, this is probably due to stabilization of E1PCa₂ by Ca²⁺ substituting for Mg²⁺ at the catalytic Mg²⁺ subsite (23, 26–29) rather than the Ca²⁺-induced reverse conversion in the equilibrium E1PCa₂ ↔ E2P + 2Ca²⁺, which would result in Ca²⁺ exchange at the transport sites. Notably, also, previous analyses of Ca²⁺ binding/release in non-phosphorylated enzyme E1Ca₂ ↔ E2 + 2Ca²⁺ shows (39–41) that the Ca²⁺ bound at transport site II is exposed to the medium and exchangeable with medium Ca²⁺, whereas Ca²⁺ bound at the deeply located site I is not released (unless site II becomes empty). ATP-induced phosphorylation to E1PCa₂ requires both Ca²⁺ to be bound. Our results indicate that E1PCa₂ formation upon addition of ATP and 10 mm Ca²⁺ is sufficiently fast to avoid such a Ca²⁺ exchange at site II, thereby trapping both of the ⁴⁵Ca²⁺ originally bound in the E1Ca₂ state as E1PCa₂.

Time Course of Ca²⁺ Release and EP Isomerization in the Wild Type

The Ca²⁺ binding assay was applied to analyze the EP isomerization and Ca²⁺ release process in the wild type (Fig. 5). The experiments were performed in the presence of 0.1 m KCl, in which E2P does not accumulate because of its rapid hydrolysis (37), and, therefore, the EP decay reflects rate-limiting E1PCa₂ isomerization to E2P (as shown in our previous report, cf. Fig. 2A in Ref. 42). In Fig. 5B, the expressed wild type was incubated with 10 μm ⁴⁵Ca²⁺ and phosphorylated with ATP to form E1PCa₂, and then an aliquot of the sample was placed on the membrane filter. At zero time, E1PCa₂ decay was initiated by removal of ⁴⁵Ca²⁺ by excess EGTA, and, after the indicated time periods, the sample was washed for 3 s with Ca²⁺ binding assay medium containing 10 mm non-radioactive Ca²⁺. For the determination of EP, [γ-³²P]ATP and non-radioactive Ca²⁺ were used. The decrease in EP (representing EP isomerization) and that in bound Ca²⁺ coincide perfectly with the established mechanism in the wild type. The rate-limiting E1PCa₂ to E2PCa₂ isomerization is followed by the very rapid Ca²⁺ release E2PCa₂ → E2P + 2Ca²⁺. The results obtained by this method are also in complete agreement with those established with sarcoplasmic reticulum vesicles, which contain a large proportion of Ca²⁺-ATPase, by the usual direct Ca²⁺ binding assay (i.e. without washing of the membrane filter) (42). Therefore, the Ca²⁺ release kinetics associated with EP isomerization can be followed by this assay using even small quantities of enzyme expressed in COS-1 cell microsomes.

Ca²⁺ Release in 10 μm Ca²⁺ Is Slower than EP Isomerization in the L119A Mutant

Detailed kinetic measurements of EP isomerization and Ca²⁺ release were made, first with the mutant L119A. We focused on residue Leu¹¹⁹ first because proteinase K-specific cleavage occurs at Leu¹¹⁹ in E2PCa₂ (E2P with occluded Ca²⁺ trapped by elongation of the A/M1′ linker) and its analog E2·BeF₃⁻·Ca₂, but not in the Ca²⁺-released E2P ground state and its analog E2·BeF₃⁻ (7, 19, 20), and Leu¹¹⁹ is critical in the E2P ground state with reduced Ca²⁺ affinity (18). We hypothesized that this residue on the cytoplasmic part of M2, possibly its gathering into the Tyr¹²² hydrophobic cluster, may be involved in Ca²⁺ release during E2P processing and Ca²⁺ release (E2PCa₂ → E2P + 2Ca²⁺). First, we found, as shown in Fig. 4B, that the properties of the Ca²⁺ binding sites of non-phosphorylated L119A (expression level about half of that of the wild type) are a K_d of 0.29 μm, a Hill coefficient of 1.61, and the stoichiometry 2 per one catalytic site (Fig. 4C), essentially the same as those of the wild type (cf. Fig. 4A). After establishing this stoichiometry of Ca²⁺ binding, we compared, in Fig. 6, the rate of Ca²⁺ release with that of EP isomerization.

In Fig. 6A, E1Ca₂ was formed with saturating 10 μm ⁴⁵Ca²⁺ and ionophore A23187, and phosphorylated at zero time to form E1PCa₂ by the addition of 10 μm ATP with and without excess EGTA, and then the sample was spotted on the membrane filter, otherwise as in Fig. 5B. E1PCa₂ is rapidly formed, and, as seen in Fig. 6B, there is almost no change in the total amount of EP during the time period because of the nearly complete block of E2P hydrolysis by the Leu¹¹⁹ mutation, whereas the E2P fraction increases gradually upon EP isomerization. When free ⁴⁵Ca²⁺ is removed by EGTA, ⁴⁵Ca²⁺ release takes place at the same rate as that of E2P formation. On the other hand, in the presence of 10 μm ⁴⁵Ca²⁺, ⁴⁵Ca²⁺ release is slower than E2P formation by ∼2-fold as a result of EP isomerization. The results suggest that, in 10 μm Ca²⁺, there may be a transient intermediate E2P state with bound Ca²⁺ at the transport sites with still a rather high affinity. Therefore, in this L119A mutant, Ca²⁺ affinity reduction at the transport sites likely occurs after EP isomerization (E1PCa₂ → E2PCa₂ at the catalytic site) and release gate opening, causing Ca²⁺ release to be retarded. Note that, for the wild type, this type of kinetic experiment in the presence of 10 μm Ca²⁺ is not feasible because, after Ca²⁺ release, E1Ca₂ is quickly regenerated upon rapid E2P hydrolysis to E2, followed by immediate Ca²⁺ rebinding. The results with the L119A mutant nevertheless suggest that some structural rearrangement takes place at the Leu¹¹⁹ region on the cytoplasmic part of M2, possibly formation of the Tyr¹²² hydrophobic cluster, to effect Ca²⁺ affinity reduction and release.

EP Isomerization and Ca²⁺ Release in other Tyr¹²² Hydrophobic Cluster Mutants

To further explore possible roles in the Ca²⁺ release process of other residues in the Tyr¹²² hydrophobic cluster, i.e. the hydrophobic interactions of the cytoplasmic part of M2 with the A and P domains and A/M3 linker (Leu¹¹⁹/Tyr¹²² (M2), Ile¹⁷⁹/Leu¹⁸⁰ (A), Val⁷⁰⁵/Val⁷²⁶ (P), and Ile²³² (A/M3 linker), see Fig. 11), we performed the same set of detailed kinetic measurements with mutant Y122A on the cytoplasmic part of M2 and I179A and V705A as representative for A and P domain interactions, respectively. It should be mentioned here that the stoichiometry of the maximum ⁴⁵Ca²⁺ binding in Y122A, I179A, and V705A (Ca/EP determined as in Fig. 4C) are 2.03, 1.72, and 2.18, respectively, very similar to those in L119A and the wild type (1.97 and 2.11, respectively, see the legend for Fig. 4). Furthermore, the K_d (μm) and Hill coefficient of the Ca²⁺ binding sites determined with the Ca²⁺ concentration dependence of E1PCa₂ formation from E1Ca₂ are also in essential agreement in these mutants and the wild type, showing respective values 0.31 and 1.93 (Y122A), 0.24 and 1.98 (I179A), 0.35 and 1.90 (L119A), 0.61 and 1.51 (V705A), and 0.29 and 1.91 (wild type) (see Table 1 in Ref. 17).

FIGURE 11.

Structures at Leu¹¹⁹/Tyr¹²² region on the cytoplasmic part of M2 and Tyr¹²² hydrophobic cluster formation in E2·BeF₃⁻ and E2·BeF₃⁻(TG). The structures in E2·BeF₃⁻ (PDB codes 2ZBE (Ref. 12) and 3B9B (Ref. 13)) and in E2·BeF₃⁻(TG) (PDB code 2ZBF (Ref. 12)) are shown as a schematic. The cytoplasmic domains A, P, and N and the cytoplasmic part of M2 are colored yellow, cyan, pink, and purple, respectively. The residues involved in the formation of Tyr¹²² hydrophobic cluster (Leu¹¹⁹/Tyr¹²² on the cytoplasmic part of M2, Ile¹⁷⁹/Leu¹⁸⁰ on the A domain, Val⁷⁰⁵/Val⁷²⁶ on the P domain, and Ile²³² on the A/M3 linker) are shown with van der Waals spheres and are colored green (Leu¹¹⁹/Tyr¹²²), brown (Ile¹⁷⁹/Leu¹⁸⁰), and orange (Val⁷⁰⁵/Val⁷²⁶/Ile²³²).

In Y122A, as in L119A, E2P hydrolysis was nearly completely blocked (17), and the single exponential E2P accumulation in EP isomerization was observed with the same rates in the presence and absence of 10 μm Ca²⁺ (Fig. 7B). The ⁴⁵Ca²⁺ release in 10 μm ⁴⁵Ca²⁺ occurs in a single exponential manner (Fig. 7A) and is slower than E2P formation by more than 2-fold. Therefore, in Y122A, as in L119A, Ca²⁺ release after E2P formation is retarded in 10 μm Ca²⁺.

The ⁴⁵Ca²⁺ release in Y122A upon ⁴⁵Ca²⁺ removal by EGTA exhibits interesting kinetics. Immediately after removal, some portion of the bound ⁴⁵Ca²⁺ (∼35% of the total, Fig, 7A, open circles) disappeared rapidly before formation of E2P (Fig. 7B, inset, closed triangles). The remaining part of the bound Ca²⁺ is released in a single exponential manner at the same rate as that of E2P formation in EP isomerization. This result suggests that the Ca²⁺-occluded structure in E1PCa₂ is destabilized in this mutant so that a fraction of the Ca²⁺ of E1PCa₂ is released rapidly (without affinity reduction because such a rapid release does not occur in 10 μm Ca²⁺). Similarly, we previously observed Ca²⁺ release (escape) from E1PCa₂ without affinity reduction in the wild type in the absence of K⁺ and indicated that the stabilization of E1PCa₂, in this case by the K⁺ binding on its specific site on the P domain, is crucial for stabilizing the Ca²⁺-occluded structure of E1PCa₂ (42). Therefore, similar to the bound K⁺, Tyr¹²² is likely important for the structural stabilization of E1PCa₂ with occluded Ca²⁺.

In the alanine mutants of Ile¹⁷⁹ and Val⁷⁰⁵ that form the Tyr¹²² hydrophobic cluster, EP decay occurs fairly fast because of the faster E2P hydrolysis, as found previously (16, 17) (Fig. 8, see the decay after the Ca²⁺ removal (triangles), although it is still much slower than the wild type (cf. Fig. 5)). Nevertheless, within the initial time period of ∼20 s in the presence of 10 μm Ca²⁺, during which the amount of original EP species remains significant, we were able to compare E2P formation and Ca²⁺ release. We found, with V705A (Fig. 9), that the Ca²⁺ release almost coincides with E2P formation, therefore the Ca²⁺ release from E2P is not retarded by this P domain mutation. For the mutants V726A on the P domain and I232A on the A/M3 linker, we repeatedly determined the amount of E2P and that of bound Ca²⁺ at a time point during the EP isomerization when the amount of E2P fraction reaches ∼40–60% (Fig. 10). In both mutants, the bound Ca²⁺ and the E2P fraction are nearly the same, therefore there is no indication of retardation of Ca²⁺ release from E2P in these mutants as in V705A.

On the other hand, with mutant I179A on the A domain, Ca²⁺ release was obviously slower than the EP isomerization (Fig. 9); therefore, Ca²⁺ release is retarded. A curious observation with this mutant was that apparently all of the ⁴⁵Ca²⁺ is released, although ∼30% of the total amount of EP remains as E1P. This again suggests that the Ca²⁺-occluded structure of E1PCa₂ is partly perturbed in the mutant I179A, in this case causing a slow Ca²⁺ release. The L180A mutant does not accumulate E2P significantly at steady state, as found previously (18). Therefore, it was not possible to compare the kinetics of E2P formation and Ca²⁺ release.

Discussion

We have been able to show that several mutations at an interdomain junction at the cytoplasmic part of transmembrane helix M2 allow a separation of steps during luminal Ca²⁺ release and phosphoenzyme isomerization, steps that, in the wild type, are too fast to distinguish. This has been possible through the development of an assay that specifically measures bound, not occluded, Ca²⁺ during the release process. The difficulties in determining bound Ca²⁺ are to fix it to the protein during washing and to obtain a sufficiently high specific signal over the background, both hurdles especially acute with the small quantity of Ca²⁺-ATPase expressed in COS-1 cell microsomes (∼1% of the total protein). Our assay overcomes such problems by almost instantaneously converting the bound Ca²⁺ to a stable occluded form.

EP Isomerization with Delayed Ca²⁺ Release

In the mutants L119A and Y122A on the cytoplasmic part of M2, Ca²⁺ release in the presence of 10 μm ⁴⁵Ca²⁺ is slower than EP isomerization, whereas they coincide in the absence of Ca²⁺. Retarded release in 10 μm ⁴⁵Ca²⁺ is also observed with the mutant I179A on the A domain. Such results indicate an E2P species with partially exchangeable Ca²⁺ that binds luminal Ca²⁺ with relatively high affinity and slowly proceeds to the Ca²⁺-released form of E2P with affinity reduction. These attributes fit with our previous findings in which we analyzed luminal Ca²⁺-induced reverse conversion of E2P (formed by P_i without Ca²⁺) to E1PCa₂ (18) and found that the E2P of these mutants have luminally facing transport sites with a K_d 120–250 μm for Ca²⁺, which is ∼10 times lower than that of the wild type (K_d 1.48 mm). The kinetic analysis of the reverse conversion further indicated that the Ca²⁺ release path is not fully opened in these mutants compared with the wild type (18). It seems that, in the transient E2P with bound but deoccluded Ca²⁺ in the mutant L119A, Y122A, or I179A, the affinity reduction is less (K_d change from low micromolar to 120–250 μm), allowing Ca²⁺ release at moderate free Ca²⁺ concentrations.

These findings can be described by assuming a transient state *E2P that possesses a high affinity Ca²⁺ site(s) facing the lumen (therefore, Ca²⁺ release and rebinding takes place in the presence of luminal Ca²⁺ as low as 10 μm) and proceeds to the E2P state with affinity reduction, as E1PCa₂ ↔ *E2PCa₂ ↔ E2PCa₂ ↔ E2P + 2Ca²⁺. In the wild type, *E2PCa₂ ↔ E2PCa₂ and subsequent Ca²⁺ (⁴⁵Ca²⁺) release are very rapid; therefore, the *E2P species before the affinity reduction with bound ⁴⁵Ca²⁺ is not trapped by the added non-radioactive ⁴⁰Ca²⁺. The trapping of bound ⁴⁵Ca²⁺ in the *E2P species in the mutants L119A, Y122A, and I179A in the presence of 10 μm ⁴⁵Ca²⁺ upon addition of non-radioactive 10 mm ⁴⁰Ca²⁺ points to a possible sequential Ca²⁺ release/rebinding as E1PCa₂ ↔ *E2PCa₂ ↔ *E2PCa + Ca²⁺ ↔ *E2P + 2Ca²⁺ ↔ E2P + 2Ca²⁺, in which at least one bound ⁴⁵Ca²⁺ is trapped (as *E2P⁴⁵Ca⁴⁰Ca by ⁴⁵Ca²⁺-⁴⁰Ca²⁺ exchange in *E2P) in the reverse conversion to E1PCa₂ by the added 10 mm ⁴⁰Ca²⁺. Such a sequential mechanism fits with the Ca²⁺ release path in E2P being not fully opened in these mutants. However, it is also possible that nonspecific Ca²⁺ binding to luminal polar and negatively charged (gating) residues (43) prevents full ⁴⁵Ca²⁺ release or that Ca²⁺ substitution for Mg²⁺ at the catalytic Mg²⁺ subsite stabilizes *E2P with bound Ca²⁺, as happens in E1PCa₂ (23, 26–29). Whatever the mechanism, we do not mean to imply that all bound ⁴⁵Ca²⁺ in E2P is fixed in the conversion to the occluded E1PCa₂ state but that at least some is.

Structural Rearrangements for Luminal Ca²⁺ Release during E2P Processing

In the intermediate state E2P with occluded Ca²⁺ (E2PCa₂) and its structural E2·BeF₃⁻·Ca₂ analog trapped by elongation of the A/M1′ linker, the Leu¹¹⁹-specific site is cleaved by proteinase K and, therefore, sterically exposed, which is in contrast to the complete resistance in the Ca²⁺-released E2P ground state and its analog E2·BeF₃⁻, formed from E2 in the absence of Ca²⁺ and in its TG-bound state E2·BeF₃⁻(TG) (7, 19, 20). Evidently, some structural rearrangement takes place at the flexible Leu¹¹⁹ and Tyr¹²² region on the cytoplasmic part of M2 and Ile¹⁷⁹ region on the A domain to effect Ca²⁺ deocclusion and release. Tyr¹²²/Leu¹¹⁹ on the cytoplasmic part of M2 forms a hydrophobic interaction network (Tyr¹²² hydrophobic cluster) with five other residues on the A and P domains and the A/M3 linker (Ile¹⁷⁹/Leu¹⁸⁰, Val⁷⁰⁵/Val⁷²⁶, and Ile²³²) in E2P (Fig. 11), and Ile¹⁷⁹ is positioned most closely to Leu¹¹⁹/Tyr¹²² on the cytoplasmic part of M2 (Fig. 11, 2ZBE and 3B9B). This cluster formation has been found previously (17, 18) to be critical for the E2P ground state structure with potential hydrolytic activity at the catalytic site and luminally opened transport sites with reduced Ca²⁺ affinity. Therefore, the assembly of Tyr¹²²/Leu¹¹⁹ on the cytoplasmic part of M2 with the other residues and formation of the Tyr¹²² hydrophobic cluster is almost certainly accomplished during E2P processing to the E2P ground state (E2PCa₂ → E2P + 2Ca²⁺) (Fig. 12, schematic).

FIGURE 12.

Schematic for E2P processing and Ca²⁺ handling. Top panel, structures of E1Ca₂·AlF₄⁻·ADP (a structural analog for the transition state in phosphorylation, E1PCa₂·ADP^‡, PDB code 1T5T (Ref. 10)), E2·BeF₃⁻ (PDB code 2ZBE (Ref. 12)), E2·BeF₃⁻(TG) (PDB code 2ZBF (Ref. 12)), and E2·AlF₄⁻(TG) ((a structural analog for the transition state in hydrolysis, E2P^‡, PDB code 1XP5 (Ref. 11)) are shown as a schematic. In these structures, the residues involved in the formation of the Tyr¹²² hydrophobic cluster (Y122-HC) are depicted with van der Waals spheres and are colored as in Fig. 10 (see the residue numbers in Fig. 10). The cytoplasmic part of M2 and the TGES¹⁸⁴ loop are colored in purple and blue, respectively, and the A, P, and N domains are colored in yellow, cyan, and pink, respectively. The open or closed state of the Ca²⁺ path (luminal gate) and the assembling state of the Tyr¹²² hydrophobic cluster are indicated above the structures. Bottom panel, schematic of the structural changes for Tyr¹²² hydrophobic cluster formation and for the property of the Ca²⁺ transport sites and release gate during E2P processing with Ca²⁺ release (E2PCa₂ to E2P). In this model, the main body of Ca²⁺-ATPase is shown in red, orange, and yellow to indicate the open high-affinity, open low-affinity, and closed states, respectively, for the property of the Ca²⁺ transport sites and release gate. The green semicircle indicates the part of Tyr¹²² hydrophobic cluster formed in E2PCa₂ upon EP isomerization (E1PCa₂ → E2PCa₂) and composed of Ile¹⁷⁹/Leu¹⁸⁰ on the A domain, Val⁷⁰⁵/Val⁷²⁶ on the P domain, and Ile²³² on the A/M3 linker. The pink structure indicates the cytoplasmic part of M2, including Leu¹¹⁹ and Tyr¹²². During E2P processing, Tyr¹²²/Leu¹¹⁹ gathers to Ile¹⁷⁹ and then to the assembled five residues, completing the Tyr¹²²-hydrophobic cluster, which is coupled with affinity reduction. Mutation of Leu¹¹⁹, Tyr¹²², or Ile¹⁷⁹ probably destabilizes the hydrophobic cluster, thereby retarding affinity reduction. Also note that our previous detailed kinetic study has revealed (18) that the E2P ground state structure has a closed luminal gate and that the Tyr¹²² hydrophobic cluster is tightly fixed (as described under “Discussion”), which is depicted here with a yellow body.

However, in the crystal structure E2·BeF₃⁻, Leu¹¹⁹/Tyr¹²² are not yet associated with the other five clustered residues despite being very close to Ile¹⁷⁹, which is already gathered with the other four hydrophobic residues (Leu¹⁸⁰, Val⁷⁰⁵/Val⁷²⁶, and Ile²³²) (12, 13), whereas all seven residues are assembled in the thapsigargin-fixed structure E2·BeF₃⁻(TG) (Fig. 11) as well as in E2·AlF₄⁻(TG), E2·MgF₄²⁻(TG), and E2(TG) and E2 (6, 9, 12, 13, 15).

Therefore, at first glance, the presently available E2·BeF₃⁻ crystal structures seem to not fit with the clustering having a critical function in the E2P ground state. However, it is possible that interaction of the cytoplasmic part of M2 Leu¹¹⁹/Tyr¹²² with the A domain Ile¹⁷⁹ is the final process in assembling the Tyr¹²² hydrophobic cluster, and, for some reason, it is not seen in the analog crystal structures. The cytoplasmic part of M2 in the E2P ground state appears rather flexible, judging from the lack of interactions here in the crystal structures, and the absence of Ca²⁺ at the transport sites may have something to do with this. We can hypothesize that even mild perturbations, such as detergent solubilization, as well as mutations here could keep the residues apart. In fact, TG binding rearranges the helices in this state to produce a tightly closed gate.

It is also of interest to note that, in our detailed analyses of the Ca²⁺ dependences of the luminal Ca²⁺-induced reverse conversion kinetics E2P + 2Ca²⁺ → E1PCa₂ and its analog E2·BeF₃⁻ + 2Ca²⁺ → E1Ca₂·BeF₃⁻, the K_0.5 of luminal Ca²⁺ was found to be 1.5 mm for E2P (18) and 0.4 mm for E2·BeF₃⁻ (14). Therefore, if this is not a kinetic effect, then the affinity in E2·BeF₃⁻ is somewhat higher than that for E2P even though E2·BeF₃⁻ seemingly possesses all of the characteristics of the E2P ground state; i.e. a hydrophobic catalytic site, the same cytoplasmic domain organization, luminally open low-affinity transport sites, and the same intrinsic tryptophan fluorescence level that reflects arrangement of the transmembrane helices. It is definitely an E2P ground state structural analog (7), and yet there may be subtle differences that may arise from a slight difference in the active site between the covalently bound phosphate at the catalytic aspartate (Asp³⁵¹) and the E2·BeF₃⁻ directly ligated with this aspartate. Alternatively, the crystallization process in detergent may impose a difference, possibly by selecting one of the putative flexible/fluctuating structures, somewhat between an “open high-affinity” state and an “open low-affinity” state. Again, we assert that the E2P ground state does, in fact, have a fully assembled hydrophobic cluster that completes the Ca²⁺ release process, otherwise it is difficult to explain the mutational effects.

We summarized our findings in a schematic in Fig. 12, which depicts that, after EP isomerization (E1PCa₂ → E2PCa₂ and loss of ADP sensitivity), the transport sites proceed from a state of open high affinity to one of open low affinity for Ca²⁺ release. The rearrangement on the cytoplasmic part of M2 (Tyr¹²²/Leu¹¹⁹) and its assembly into a hydrophobic cluster are accomplished in this affinity reduction process by gathering of Tyr¹²²/Leu¹¹⁹ on the cytoplasmic part of M2 with the Ile¹⁷⁹/Leu¹⁸⁰ region on the A domain that is already associated with the P domain during the EP isomerization E1PCa₂ → E2PCa₂. In the mutants L119A/Y122A and I179A, the Tyr¹²² hydrophobic cluster is destabilized, and formation of the open low-affinity state is retarded.

It is not clear why the mutations of residues Val⁷⁰⁵/Val⁷²⁶ on the P domain and Ile²³² on the A/M3 linker in the hydrophobic cluster do not retard Ca²⁺ release, but it is probably because there are several interactions between the A and P domains, and a single conservative substitution here is not enough to disrupt A-P domain association. Also, these two domains are already associated in E2PCa₂, and mild perturbations may be without effect on the later affinity reduction and release. There are large changes during EP isomerization. The A domain rotates and docks on the P domain, allowing Ile¹⁷⁹/Leu¹⁸⁰ on the A domain to cluster with Val⁷⁰⁵/Val⁷²⁶ on the P domain and Ile²³² on the A/M3 linker associated with the P domain. In E2PCa₂, the interactions at the Val²⁰⁰ loop on the A domain with polar residues on the P domain (19, 44) and those at the TGES¹⁸⁴ loop on the A domain with the catalytic site on the P domain (which causes the loss of ADP sensitivity) are already produced. To effect affinity reduction and Ca²⁺ release, the associated A and P domains move together, allowing the cytoplasmic part of M2-A domain interaction at Tyr¹²²/Leu¹¹⁹ and Ile¹⁷⁹ to form and complete the critical Tyr¹²² hydrophobic cluster.

Regarding a possible “closed” state in the E2P ground state (Fig. 12), we have found previously, with detailed kinetics in the wild type (18), that the rate of luminal Ca²⁺-induced reverse conversion E2P + 2Ca²⁺ → E1PCa₂ increases linearly with an increase in the luminal Ca²⁺ concentration and is not saturated even at 3 mm. This suggests that luminal Ca²⁺ access to the open low-affinity transport sites in E2P is rate-limiting, and, therefore, the gate in E2P may actually be closed, and the rate-limiting Ca²⁺ access may reflect opening of the closed state to an open low-affinity state. We have also found that this reverse conversion is retarded by alanine mutation of each of the seven residues in the Tyr¹²² hydrophobic cluster, and the extent of the retardation was almost the same in all seven mutants (Figs. 8 and 9 and supplemental Fig. 3 in Ref. 18). Therefore, all residues in the cluster seem important for reverse opening.

This finding (18) and the mutation-induced retardation of the forward Ca²⁺ release process found here can be accounted for by a destabilization of the open low-affinity state and transition states in the forward and reverse processes by the cluster mutations. Furthermore, the fact that all seven residues in the cluster influence the closed state implies that the change from the open low-affinity state to a tightly fixed closed state requires further rearrangements, which then permits advance to the transition state for hydrolysis (E2-P^‡ mimicked by E2·AlF₄⁻), in which the gate becomes tightly closed (7).

P-type ATPases possess a common molecular structure with N, P, and A domains connected to transmembrane helices, including the long M2 helix critical for gating (1–3), and almost certainly all of them utilize a common mechanism for ion pumping. We predict that ion release to the trans side of the membrane will entail rearrangements at the cytoplasmic part of M2 and engagement of the A and P domains. A stepwise affinity reduction and release on this trans side during phosphoenzyme isomerization may be discernable even in some wild-type pumps of other P-type ATPases.

Author Contributions

K. Y. and H. S. conceived and coordinated the study and wrote the paper. K. Y. designed, performed, and analyzed the experiments. T. D. provided critical discussions and technical advice. S. D. provided technical assistance and contributed to the preparation of the figures and manuscript. All authors reviewed the results and approved the final version of the manuscript.