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

Abstract

The mechanism whereby events in and around the catalytic site/head of Ca(2+)-ATPase effect Ca(2+) release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca(2+) by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca(2+). The assay is applicable to minute amounts of Ca(2+)-ATPase expressed in COS-1 cells. It was validated by measuring the Ca(2+) binding properties of unphosphorylated Ca(2+)-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca(2+) release process E1PCa2 → E2PCa2 → E2P + 2Ca(2+). In the wild type, Ca(2+) release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa2 → E2PCa2) followed by very rapid Ca(2+) release. In contrast, with alanine mutants of Leu(119) and Tyr(122) on the cytoplasmic part of the second transmembrane helix (M2) and Ile(179) on the A domain, Ca(2+) release in 10 μm Ca(2+) lags EP isomerization, indicating the presence of a transient E2P state with bound Ca(2+). The results suggest that these residues function in Ca(2+) 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 Ca(2+) release.

Keywords: calcium ATPase; calcium transport; enzyme kinetics; enzyme structure; site-directed mutagenesis.

FIGURE 1.

Reaction schematic of Ca²⁺-ATPase.

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.

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.

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.

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.

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).

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.