Roles of interaction between actuator and nucleotide binding domains of sarco(endo)plasmic reticulum Ca(2+)-ATPase as revealed by single and swap mutational analyses of serine 186 and glutamate 439

Abstract

Roles of hydrogen bonding interaction between Ser(186) of the actuator (A) domain and Glu(439) of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca(2+)-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca(2+)-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wild-type rates. Results showed that the Ser(186)-Glu(439) hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm approximately mm range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser(186) and Glu(439) are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.

FIGURE 1.

Reaction cycle of sarco(endo)plasmic reticulum Ca²⁺-ATPase.

FIGURE 2.

Structure of SERCA1a and formation of Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond between the A and N domains. The coordinates for the structures E1Ca₂·AlF₄⁻·ADP, (the analog for the transition state of the phosphoryl transfer E1PCa₂·ADP^‡, left panel) and E2·MgF₄²⁻ (E2·P_i analog (21), right panel) of Ca²⁺-ATPase were obtained from the Protein Data Bank (PDB accession code 1T5T and 1WPG, respectively (12, 14)). The arrows indicate approximate movements of the A and P domains in the change from E1Ca₂·AlF₄⁻ ·ADP to E2·MgF₄²⁻. Ser¹⁸⁶ and Glu⁴³⁹ are depicted as van der Waals spheres. These two residues form a hydrogen bond in E2·MgF₄²⁻ (see inset). The phosphorylation site Asp³⁵¹, two Ca²⁺ at the transport sites and ADP with AlF₄⁻ at the catalytic site in E1Ca₂·AlF₄⁻·ADP, MgF₄²⁻ bound at the catalytic site in E2·MgF₄²⁻ are depicted. The TGES¹⁸⁴ loop and Val²⁰⁰ loop of the A domain and Tyr¹²² on the top part of M2 are shown. These elements produce three interaction networks between A and P domains and M2 (Tyr¹²²) in E2·MgF₄²⁻ (–26). M1′ and M1-M10 are also indicated.

FIGURE 3.

Ca²⁺-ATPase activities of expressed SERCA1a. The Ca²⁺-ATPase activity of microsomes expressing the wild-type or mutant SERCA1a was determined with 0.1 mm [γ-³²P]ATP as described under “Experimental Procedures.” The activities are divided by the amount of EP formed at steady state (see supplemental Fig. S1), and the turnover rates thus obtained are shown as percentage of that of the wild type (7.29 ± 0.42 s⁻¹ (n = 5)). The values of the mutants presented are the mean ± S.D. (n = 3–5).

FIGURE 4.

Accumulation of ADP-insensitive EP in the presence (A) and absence (B) of K⁺. Microsomes expressing the wild type or mutant were phosphorylated with 10 μm [γ-³²P]ATP at 0 °C for 15 s in 50 μl of a mixture containing 2.5 μg of microsomal protein, 1 μm A23187, 7 mm MgCl₂, 0.05 mm CaCl₂, 50 mm MOPS/Tris (pH 7.0), and 0.1 m KCl (A) or 0.1 m LiCl without KCl (B). The total amount of EP formed was determined by the acid-quenching. For determination of ADP-insensitive EP (E2P), an equal volume of a mixture containing 10 mm ADP, 7 mm MgCl₂, 10 mm EGTA, 50 mm MOPS/Tris (pH 7.0), and 0.1 m KCl (A) or LiCl without KCl (B) was added to the above phosphorylation mixture, and the reaction was quenched at 1 s after the ADP addition. ADP-sensitive EP (E1PCa₂) disappeared entirely within 1 s after the ADP addition. The amount of E2P is shown as percentage of the total amount of EP.

FIGURE 5.

Time course of accumulation of ADP-insensitive EP from E1Ca₂ and ATP. Microsomes expressing the wild type or mutant were phosphorylated with [γ-³²P]ATP at 0 °C for various periods in the presence of 0.1 m LiCl without KCl otherwise as in Fig. 4B. The total amount of EP and the amount of ADP-insensitive EP (E2P) were determined at the indicated time without and with the ADP addition in 0.1 m LiCl (no KCl), otherwise as in Fig. 4B. Solid lines show the least squares fit to a single exponential, and the apparent rates to reach the steady-state E2P level are given in Table 1.

FIGURE 6.

Decay of E1PCa₂ formed from ATP. Microsomes expressing the wild type or mutant were phosphorylated in 0.1 m KCl at 0 °C for 15 s as in Fig. 4A. Phosphorylation was terminated by addition of an equal volume of a buffer containing 8 mm EGTA, 0.1 m KCl, 7 mm MgCl₂, and 50 mm MOPS/Tris (pH 7.0) at 0 °C. The total amount of EP remaining after the EGTA addition was determined at the indicated time. The total amounts of EP obtained at zero time (i.e. immediately before the EGTA addition) are normalized to 100%. Solid lines show the least squares fit to a single exponential, and the decay rates obtained are given in Table 1.

FIGURE 7.

Hydrolysis of E2P formed from P_i without Ca²⁺. Microsomes expressing the wild type or mutant were phosphorylated with 0.1 mm ³²P_i at 25 °C for 10 min in 5 μl of a mixture containing 2 μg of microsomal protein, 20 μm A23187, 1 mm EGTA, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 35% (v/v) Me₂SO. The mixture was then cooled and diluted at 0 °C by 100 μl of a mixture containing 1.05 mm non-radioactive P_i, 105 mm KCl, 15.8 mm EGTA, and 50 mm MOPS/Tris (pH 7.0). At different times after the dilution, the E2P hydrolysis was quenched by acid. The amounts of E2P formed with ³²P_i at zero time are normalized to 100%. Solid lines show the least squares fit to a single exponential, and the rates obtained are given in Table 1.

FIGURE 8.

MgATP dependence of Ca²⁺-ATPase activity. The Ca²⁺-ATPase activity of microsomes expressing the wild type or mutant was determined at various concentrations of [γ-³²P]ATP otherwise as in Fig. 3. Almost all of ATP (more than 97% of total ATP) is in MgATP. The activities of the mutants are presented as a percentage of that of the wild type at 5 mm MgATP.

FIGURE 9.

MgATP dependence of the decay rate of E1PCa₂ formed from ATP. E1PCa₂ was first formed in 50 μl of a microsomes suspension in 10 μm [γ-³²P]ATP, 10 μm Ca²⁺ (0.98 mm CaCl₂ with 1 mm EGTA), and 0.1 m KCl, otherwise as in Fig. 4A. Phosphorylation was terminated by 100 μl of a buffer containing 8 mm EGTA, 1 μm A23187, 0.1 m KCl, 50 mm MOPS/Tris (pH 7.0), and various concentrations of ATP and MgCl₂ (producing MgATP (more than 97% of the total ATP) with 6.2 mm free Mg²⁺). At different times after this MgATP addition, the decay reaction of E1PCa₂ at 0 °C was quenched by acid. The rate of the single exponential decay of E1PCa₂ obtained was plotted versus the MgATP concentration. Solid lines show the least squares fit to the Hill equation, and the parameters V₀ (the rate at the lowest 10 μm MgATP), V_max (the maximum rate), and K_0.5 (MgATP concentration giving the half-maximal change) are given in Table 2.

FIGURE 10.

ATP dependence of the decay rate of E1PCa₂ formed from ATP. The E1PCa₂ decay was followed after addition of various concentrations of ATP and 30 mm EDTA without MgCl₂ (in place of 8 mm EGTA) otherwise as in Fig. 9. The single exponential decay rate of E1PCa₂ obtained was plotted versus the metal-free ATP concentration, and the parameters V₀, V_max, and K_0.5 estimated as in Fig. 9 are given in Table 2.

FIGURE 11.

ATP dependence of the rate of E2P hydrolysis. Microsomes expressing the wild type or mutant were phosphorylated with ³²P_i at 25 °C for 10 min in 4 μl of a mixture containing 1.6 μg of microsomal protein, 0.1 mm ³²P_i, 20 μm A23187, 1 mm EGTA, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 35% (v/v) Me₂SO. The mixture was then diluted at 0 °C by addition of 196 μl of a mixture containing 1.02 mm non-radioactive P_i, 50 mm MOPS/Tris (pH 7.0), 15.3 mm EDTA, and various concentration of ATP. At different times after the dilution, the E2P hydrolysis was quenched by acid. The rate of the single exponential E2P hydrolysis was plotted versus the ATP concentration, and the parameters V₀ (the rate without ATP), V_max (the rate at the highest ATP concentration, 1 mm), and K_0.5 (ATP concentration giving the half-maximal change) are given in Table 2. It should be noted that Mg²⁺ bound at the catalytic site of E2P is occluded (53), and therefore the E2P hydrolysis takes place even after removal of free Mg²⁺.