Roles of Interaction between Actuator and Nucleotide Binding Domains of Sarco(endo)plasmic Reticulum Ca2+-ATPase as Revealed by Single and Swap Mutational Analyses of Serine 186 and Glutamate 439

Roles of hydrogen bonding interaction between Ser¹⁸⁶ of the actuator (A) domain and Glu⁴³⁹ of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca²⁺-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 Ca²⁺-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 Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm～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 Ser¹⁸⁶ and Glu⁴³⁹ 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.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a)² is a representative member of P-type ion-transporting ATPases and catalyzes Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). In the catalytic cycle, the enzyme is activated by binding of two Ca²⁺ ions at the transport sites (E2 to E1Ca₂, steps 1–2) and then autophosphorylated at Asp³⁵¹ with MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3), which can react with ADP to regenerate ATP. Upon formation of this EP, the bound Ca²⁺ ions are occluded in the transport sites (E1PCa₂). The subsequent isomeric transition to ADP-insensitive form (E2P) results in a change in the orientation of the Ca²⁺ binding sites and reduction of their affinity, and thus Ca²⁺ release into lumen (steps 4 and 5). Finally, the hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca²⁺-unbound form (E2, step 6). E2P can also be formed from P_i in the presence of Mg²⁺ and the absence of Ca²⁺ by reversal of its hydrolysis.Open in a separate windowFIGURE 1.Reaction cycle of sarco(endo)plasmic reticulum Ca²⁺-ATPase.The cytoplasmic three domains N, A, and P largely move and change their organization states during the Ca²⁺ transport cycle (10–22). These changes are linked with the rearrangements in the transmembrane helices. In the EP isomerization (loss of ADP sensitivity) and Ca²⁺ release, the A domain largely rotates (by ～110° parallel to membrane plane), intrudes into the space between the N and P domains, and the P domain largely inclines toward the A domain. Thus in E2P, these domains produce the most compactly organized state (see Fig. 2 for the change E1Ca₂·AlF₄^?·ADP →E2·MgF₄^2? as the model for the overall process E1PCa₂·ADP^? → E2·P_i).Open in a separate windowFIGURE 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·MgF₄^2? (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·MgF₄^2?. Ser¹⁸⁶ and Glu⁴³⁹ are depicted as van der Waals spheres. These two residues form a hydrogen bond in E2·MgF₄^2? (see inset). The phosphorylation site Asp³⁵¹, two Ca²⁺ at the transport sites and ADP with AlF₄^? at the catalytic site in E1Ca₂·AlF₄^?·ADP, MgF₄^2? bound at the catalytic site in E2·MgF₄^2? 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·MgF₄^2? (23–26). M1′ and M1-M10 are also indicated.We have found that the interactions between the A and P domains at the Val²⁰⁰-loop (Asp¹⁹⁶-Asp²⁰³) with the residues of the P domain (Arg⁶⁷⁸/Glu⁶⁸⁰/Arg⁶⁵⁶/Asp⁶⁶⁰) (23) and at the Tyr¹²² hydrophobic cluster (24–26) (see Fig. 2) play critical roles for Ca²⁺ deocclusion/release in E2PCa₂ → E2P + 2Ca²⁺ after the loss of ADP sensitivity (E1PCa₂ to E2PCa₂ isomerization). The proper length of the A/M1′ linker is critical for inducing the inclining motion of the A and P domains for the Ca²⁺ deocclusion and release from E2PCa₂ (27, 28). The importance of the interdomain interaction between Arg⁶⁷⁸ (P) and Asp²⁰³ (A) in stabilizing the E2P and E2 intermediates and its influence on modulatory ATP activation were pointed out by the mutation R678A (29). Regarding the N domain, the importance of Glu⁴³⁹ in the EP isomerization and E2P hydrolysis was previously noted by its alanine substitution, and possible importance of its interaction with Ser¹⁸⁶ on the A domain has been suggested since Glu⁴³⁹ forms a hydrogen bond with Ser¹⁸⁶ in the E2P analog structures (29) (see Fig. 2). The Darier disease-causing mutations of Ser¹⁸⁶ of SERCA2b, S186P and S186F also alter the kinetics of the EP processing and its importance as the residue in the immediate vicinity of TGES¹⁸⁴ has been pointed out (30, 31). Notably also, Glu⁴³⁹ is situated near the adenine binding pocket and its importance in the ATP binding and ATP-induced structural change have been shown (32, 33). In the structure E2(TG)AMPPCP (E2·ATP), Glu⁴³⁹ interacts with the modulatory ATP binding via Mg²⁺, and is involved in the acceleration of the Ca²⁺-ATPase cycle (16).Considering these critical findings on each of Glu⁴³⁹ and Ser¹⁸⁶, it is crucial to reveal the role of the Ser¹⁸⁶-Glu⁴³⁹ hydrogen-bonding interaction between the A and N domains in the EP processing and its ATP modulation (i.e. regulatory ATP-induced acceleration). We therefore made a series of mutants on both Ser¹⁸⁶ and Glu⁴³⁹ including the swap substitution mutant, S186A, E439A, S186A/E439A, S186E, E439S, S186E/E439S, and explored their kinetic properties. Results showed that the Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond is critical for the stabilization of the E2P ground state structure, and possibly functioning as to make the E2P resident time long enough for Ca²⁺ release (E2PCa₂ → E2P + 2Ca²⁺) thus to avoid its hydrolysis without Ca²⁺ release. Results also revealed that the side-chain configurations of Ser¹⁸⁶ and Glu⁴³⁹ are fixed by their hydrogen bond, thereby conferring the proper modulatory ATP binding to occur at the cellular ATP level to accelerate the rate-limiting EP isomerization.