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-m
m∼m
m 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.Sarcoplasmic reticulum Ca
2+-ATPase (SERCA1a)
2 is a representative member of P-type ion-transporting ATPases and catalyzes Ca
2+ transport coupled with ATP hydrolysis () (
1–
9). In the catalytic cycle, the enzyme is activated by binding of two Ca
2+ ions at the transport sites (
E2 to
E1Ca
2, steps 1–2) and then autophosphorylated at Asp
351 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 Ca
2+ ions are occluded in the transport sites (
E1PCa
2). The subsequent isomeric transition to ADP-insensitive form (
E2P) results in a change in the orientation of the Ca
2+ binding sites and reduction of their affinity, and thus Ca
2+ release into lumen (steps 4 and 5). Finally, the hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca
2+-unbound form (
E2, step 6).
E2P can also be formed from P
i in the presence of Mg
2+ and the absence of Ca
2+ by reversal of its hydrolysis.
Open in a separate windowReaction cycle of sarco(endo)plasmic reticulum Ca
2+-ATPase.The cytoplasmic three domains N, A, and P largely move and change their organization states during the Ca
2+ transport cycle (
10–
22). These changes are linked with the rearrangements in the transmembrane helices. In the
EP isomerization (loss of ADP sensitivity) and Ca
2+ 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 for the change
E1Ca
2·AlF
4−·ADP →
E2·MgF
42− as the model for the overall process
E1PCa
2·ADP
‡ →
E2·P
i).
Open in a separate windowStructure of SERCA1a and formation of Ser
186-Glu
439 hydrogen bond between the A and N domains. The coordinates for the structures
E1Ca
2·AlF
4−·ADP, (the analog for the transition state of the phosphoryl transfer
E1PCa
2·ADP
‡,
left panel) and
E2·MgF
42− (
E2·P
i analog (
21),
right panel) of Ca
2+-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
E1Ca
2·AlF
4− ·ADP to
E2·MgF
42−. Ser
186 and Glu
439 are depicted as van der Waals spheres. These two residues form a hydrogen bond in
E2·MgF
42− (see
inset). The phosphorylation site Asp
351, two Ca
2+ at the transport sites and ADP with AlF
4− at the catalytic site in
E1Ca
2·AlF
4−·ADP, MgF
42− bound at the catalytic site in
E2·MgF
42− are depicted. The TGES
184 loop and Val
200 loop of the A domain and Tyr
122 on the
top part of M2 are shown. These elements produce three interaction networks between A and P domains and M2 (Tyr
122) in
E2·MgF
42− (
23–
26). M1′ and M1-M10 are also indicated.We have found that the interactions between the A and P domains at the Val
200-loop (Asp
196-Asp
203) with the residues of the P domain (Arg
678/Glu
680/Arg
656/Asp
660) (
23) and at the Tyr
122 hydrophobic cluster (
24–
26) (see ) play critical roles for Ca
2+ deocclusion/release in
E2PCa
2 →
E2P + 2Ca
2+ after the loss of ADP sensitivity (
E1PCa
2 to
E2PCa
2 isomerization). The proper length of the A/M1′ linker is critical for inducing the inclining motion of the A and P domains for the Ca
2+ deocclusion and release from
E2PCa
2 (
27,
28). The importance of the interdomain interaction between Arg
678 (P) and Asp
203 (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 Glu
439 in the
EP isomerization and
E2P hydrolysis was previously noted by its alanine substitution, and possible importance of its interaction with Ser
186 on the A domain has been suggested since Glu
439 forms a hydrogen bond with Ser
186 in the
E2P analog structures (
29) (see ). The Darier disease-causing mutations of Ser
186 of SERCA2b, S186P and S186F also alter the kinetics of the
EP processing and its importance as the residue in the immediate vicinity of TGES
184 has been pointed out (
30,
31). Notably also, Glu
439 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), Glu
439 interacts with the modulatory ATP binding via Mg
2+, and is involved in the acceleration of the Ca
2+-ATPase cycle (
16).Considering these critical findings on each of Glu
439 and Ser
186, it is crucial to reveal the role of the Ser
186-Glu
439 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 Ser
186 and Glu
439 including the swap substitution mutant, S186A, E439A, S186A/E439A, S186E, E439S, S186E/E439S, and explored their kinetic properties. Results showed that the Ser
186-Glu
439 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 Ca
2+ release (
E2PCa
2 →
E2P + 2Ca
2+) thus to avoid its hydrolysis without Ca
2+ release. Results also revealed that the side-chain configurations of Ser
186 and Glu
439 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.
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