Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca²⁺-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating

Abstract

Ca²⁺ transport by sarcoplasmic reticulum Ca²⁺-ATPase involves ATP-dependent phosphorylation of a catalytic aspartic acid residue. The key process, luminal Ca²⁺ release occurs upon phosphoenzyme isomerization, abbreviated as E1PCa₂ (reactive to ADP regenerating ATP and with two occluded Ca²⁺ at transport sites) → E2P (insensitive to ADP and after Ca²⁺ release). The isomerization involves gathering of cytoplasmic actuator and phosphorylation domains with second transmembrane helix (M2), and is epitomized by protection of a Leu¹¹⁹-proteinase K (prtK) cleavage site on M2. Ca²⁺ binding to the luminal transport sites of E2P, producing E2PCa₂ before Ca²⁺-release exposes the prtK-site. Here we explore E2P structure to further elucidate luminal gating mechanism and effect of membrane perturbation. We find that ground state E2P becomes cleavable at Leu¹¹⁹ in a non-solubilizing concentration of detergent C₁₂E₈ at pH 7.4, indicating a shift towards a more E2PCa₂-like state. Cleavage is accelerated by Mg²⁺ binding to luminal transport sites and blocked by their protonation at pH 6.0. Results indicate that possible disruption of phospholipid-protein interactions strongly favors an E2P species with looser head domain interactions at M2 and responsive to specific ligand binding at the transport sites, likely an early flexible intermediate in the development towards ground state E2P.

Introduction

Sarco(endo)plasmic reticulum (SR) Ca²⁺-ATPase (expressed in adult fast-twitch skeletal muscle, SERCA1a), a representative member of P-type ion transporting ATPases, catalyzes Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (for recent reviews, see Refs 1, 2, 3). The enzyme consists of three large cytoplasmic domains, Nucleotide binding (N), Phosphorylation (P), and Actuator (A), and ten transmembrane helices (M1~M10) (Figs 1 and 2). Ca²⁺ transport requires communication between the catalytic site on the cytoplasmic domains and the transport sites in the transmembrane helices via coupled structural changes, i.e. cytoplasmic domain motions and rearrangements of transmembrane helices. The enzyme is activated by the binding of two cytoplasmic Ca²⁺ ions at the high affinity transport sites composed of residues located on M4, M5, M6, and M8 (E2 to E1Ca₂ in Fig. 1). Then it is auto-phosphorylated at the catalytic residue Asp³⁵¹ with ATP to form an ADP-sensitive phosphoenzyme (E1P), which is capable of reacting 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 involves a large rotation of the A domain to associate with the P domain, thereby rearranging the Ca²⁺ binding sites to deocclude Ca²⁺, open the release path (luminal gate), and reduce the affinity, thus allowing Ca²⁺ release into the lumen. As a consequence, the catalytic site in E2P is prepared for subsequent aspartyl phosphate hydrolysis by tightening of associated A and P domains. In the first step towards hydrolysis, progressing from the ground state to the transition state, namely E2P + H₂O → E2~P^‡, the transport sites are protonated and the luminal gate closes tightly, preventing luminal Ca²⁺ access and driving the process forward^4,5. The cytoplasmic part of the second transmembrane helix, M2, plays a crucial role in coupling A-domain motion and tilting of the P domain during the rearrangements of transport sites^4,6,7,8,9.

Figure 1: Reaction sequence of Ca²⁺-ATPase.

The sequence is shown with intermediates and transition states (E1~PCa₂ADP^‡ and E2~P^‡). Stable structural analog for each state developed with phosphate analogs BeF₃⁻, AlF₄⁻ and MgF₄^{2− 4,6,17,19} is shown with gray-highlight. In the crystal structures E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻ (PDB code: 2ZBD⁸ and 2ZBE⁸, respectively), the cytoplasmic domains A (yellow), P (cyan), and N (pink), M1~M10, occluded two Ca²⁺, and membrane position are indicated. Arrows on the domains in E1Ca₂·AlF₄⁻·ADP indicate their approximate motions to the E2·BeF₃⁻ structure to show changes in E1PCa₂ → E2P+ 2Ca²⁺ as an available model.

Figure 2: Crystal structures E2·BeF₃⁻ and E2·BeF₃⁻(TG).

Structures E2·BeF₃⁻ with bound Mg²⁺ at the transport sites (formed at pH 7.0 and 50 mM Mg²⁺), E2·BeF₃⁻ with most probably protonated transport sites (formed at pH 5.7), and E2·BeF₃⁻(TG) (PDB code: 3B9B¹¹, 2ZBE⁸, 2ZBF⁸, respectively) are shown as a cartoon model. The cytoplasmic region indicated by the red broken line on the whole molecule of E2·BeF₃⁻ with bound Mg²⁺ is enlarged in the three top panels. In the three bottom panels, the view of transport sites from the luminal side as indicated by a large green arrow is shown. The A, P, and N domains and cytoplasmic part of M2 are yellow, cyan, pink, and purple, respectively. The Mg²⁺ and water molecules at the Ca²⁺ binding sites (transport sites) and Na⁺ bound at the K⁺ (Na⁺) site on the P domain are green, red, and blue spheres, respectively. The seven residues involved in the formation of Tyr¹²²-hydrophobic cluster, Y122-HC (Leu¹¹⁹/Tyr¹²² on 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 colored green (Leu¹¹⁹/Tyr¹²²), brown (Ile¹⁷⁹/Leu¹⁸⁰), and orange (Val⁷⁰⁵/Val⁷²⁶/Ile²³²). The BeF₃⁻ coordinated in the catalytic site behind the residues involved in Y122-HC is shown by a space-filling model (cyan for beryllium and purple for fluoride) and Asp³⁵¹ (the auto-phosphorylation site) is shown in a ball-stick model in the panels (note that they are obscured by Y122-HC in E2·BeF₃⁻(TG)). The Mg²⁺ bound to the catalytic site is not depicted as it is also hidden by Y122-HC. The TGES¹⁸⁴ loop and Val²⁰⁰ loop (Lys¹⁸⁹-Lys²⁰⁵) are colored by a red loop and a blue loop, respectively in all panels. The prtK-cleavage sites at Leu¹¹⁹ and Thr²⁴² and the trypsin-cleavage sites at Arg¹⁹⁸ and Arg⁵⁰⁵ are indicated (backbone carbon).

The E2P ground state, transition state (E2~P^‡), and product complex (E2·P_i) in the E2P hydrolysis process are mimicked by the stable structural analogs E2·BeF₃⁻, E2·AlF₄⁻, and E2·MgF₄²⁻, respectively, as produced with the respective phosphate analogs for different configurational states⁴. Their crystal structures, without or with the potent inhibitor thapsigargin (TG), have been solved at atomic level^7,8,10,11 following purification of the protein using a non-ionic detergent octaethylene glycol monododecyl ether (C₁₂E₈). Commensurate with the structural changes mentioned, the crystal structures are subtly different although the overall molecular structure of the compactly organized cytoplasmic A, P, and N domains with tightly bound BeF₃⁻ and occluded Mg²⁺ at the catalytic site and the arrangement of transmembrane helices are similar. Namely, the E2·BeF₃⁻ crystal produced at pH 7.0 in 50 mM Mg²⁺ has wide open transport sites (luminal gate open) with one bound Mg²⁺¹¹ and that at pH 5.7, where the transport sites are protonated and Mg²⁺ is absent, the luminal access pathway is less open⁸ (Fig. 2). The structures with bound TG at a cavity surrounded by M3, M5, and M7, namely E2·BeF₃⁻(TG), and those of E2·AlF₄⁻(TG) and E2·MgF₄²⁻(TG), are different again, and the luminal gate is tightly closed. The closure is associated with formation of hydrophobic interaction network, the Tyr¹²²-hydrophobic cluster (Y122-HC) by Leu¹¹⁹/Tyr¹²² on the cytoplasmic part of M2 and five residues of the gathered A and P domains (Ile¹⁷⁹/Leu¹⁸⁰ (A), Val⁷⁰⁵/Val⁷²⁶ (P)) and A/M3-linker (Ile²³² on the loop connecting the A domain with M3). Significantly, in the E2·BeF₃⁻ crystals without TG, where the gate is open, the side chains of Leu¹¹⁹/Tyr¹²² are close but pointing away from the other gathered five residues, indicative of weaker domain interactions here (Fig. 2).

Extensive mutation and kinetic studies have demonstrated^12,13,14,15 that all seven residues involved in Y122-HC including Leu¹¹⁹/Tyr¹²² are crucial for opening the gate, reducing Ca²⁺ affinity, and allowing rapid Ca²⁺-release (E2PCa₂ → E2P + 2Ca²⁺), and for subsequent gate-closure and the formation of a catalytic site with hydrolytic ability. Investigation of the structural changes during these events has been aided by proteolytic digestion patterns, including a prtK site at Leu^{119 4,16,17}. The site is exposed in the unphosphorylated E2 form but protected in E2·BeF₃⁻, E2·AlF₄⁻, and E2·MgF₄²⁻ as well as in the TG-bound forms of these analogs. Thus susceptibility to prtK attack or otherwise seems a good indicator of the state of the gathering of the head domains on M2. Significantly, E2PCa₂ an early E2P species, is uniquely susceptible to attack, an indication of a loose arrangement of head domains on M2 prior to progression to ground state E2P^4,18,19.

Unexpectedly, we now find that low, non-solubilizing concentrations of C₁₂E₈ render the prtK site at Leu¹¹⁹ in E2P (E2·BeF₃⁻) susceptible to attack. It is as though the detergent has released constraints at the transmembrane helices to favor a state closer to that on Ca²⁺ binding to the luminal sites, namely E2PCa₂. The phenomenon uncovers a hitherto undescribed intermediate just prior to ground state E2P, stabilized by detergent that is uniquely susceptible to diverse ligand binding and cross-protein conformational changes. It shows that phospholipid-protein interactions directly participate the conformational changes associated with luminal gating events and expedite Ca²⁺ release.

Results

PrtK-cleavage of Leu¹¹⁹-site in E2·BeF₃⁻ with C₁₂E₈ at pH 7.4

In Fig. 3a, prtK-proteolysis of E2·BeF₃⁻ is performed at pH 7.4 in 0.1 M K⁺ without and with a non-solubilizing low concentration of C₁₂E₈. In the absence of C₁₂E₈, E2·BeF₃⁻ is completely resistant to prtK both without and with A23187 as found previously⁴. In the presence of C₁₂E₈, a 95-kDa fragment (p95) is produced by specific prtK-cleavage at the Leu¹¹⁹-site without any other cleavages. Cleavage is accelerated by 30 mM Mg²⁺, but no cleavage occurs in the absence of C₁₂E₈ even at 30 mM Mg²⁺. In Fig. 3d, the Mg²⁺ concentration dependence of the specific prtK-cleavage rate at the Leu¹¹⁹-site is determined in C₁₂E₈ and different monovalent cations (K⁺, Na⁺, and Li⁺) at 0.1 M. The rate increases with increasing Mg²⁺ concentration – binding to a low affinity site favors exposure. The cleavage is faster in Na⁺ and K⁺ as compared with that in Li⁺ or in the absence of monovalent cation, thus K⁺ or Na⁺ binding at the K⁺ site on the P domain^20,21 increases prtK attack at Leu¹¹⁹.

Figure 3: Effects of C₁₂E₈ and various factors on proteolysis of E2·BeF₃⁻, E2·AlF₄⁻, and E2·MgF₄²⁻.

The proteolysis was performed for various times with prtK and trypsin as indicated with E2·BeF₃⁻ (a,e), E2·AlF₄⁻ (b,f), and E2·MgF₄²⁻ (c,f) of SR vesicles in the presence or absence of 0.15 mg/ml C₁₂E₈ or 15 μM A23187 in 50 mM MOPS/Tris pH 7.4 (a–c) or MES/Tris pH 6.0 (e,f), 0.1 M KCl, 1 mM EGTA, and 0 or 30 mM MgCl₂ without or with 4 μM TG (“TG”), as indicated. The “E2·TG” state of SR vesicles un-treated with the metal fluoride was subjected to the proteolysis as a control. In (d), the rate of prtK digestion of 110 kDa-ATPase chain in C₁₂E₈ at pH 7.4 was determined at various concentrations of MgCl₂ in 0.1 M KCl, NaCl, or LiCl or in the absence of these salts, otherwise as in (a) and as described under “METHODS”. The fragments indicated on the right of a panel are p95 produced by the prtK-cleavage at the Leu¹¹⁹-site on M2, p81/p83 produced by the prtK-cleavage at the Thr²⁴²-site on A/M3-linker (p83) and Ala⁷⁴⁶ on M5 (p81)^16,39, and the tryptic A1 fragment produced by cleavage at the Arg¹⁹⁸-site on the A fragment (N-terminal half), which is formed very rapidly together with the B fragment (C-terminal half) by cleavage at Arg⁵⁰⁵-site⁴⁰.

E2·BeF₃⁻ cleavage in C₁₂E₈ is inhibited by thapsigargin (TG), which binds tightly to a cavity surrounded by M3, M5, and M7, fixing the arrangement of transmembrane helices with a tightly closed luminal gate^22,23 (Fig. 3a “C₁₂E₈+ TG” for E2·BeF₃⁻). On the other hand, in the BeF₃⁻-free state with bound TG (“E2·TG”) with Mg²⁺ as well as without Mg²⁺, the 110-kDa ATPase chain is very rapidly cleaved producing p95 and p81/p83 fragments by cleavages at Leu¹¹⁹ and at Thr²⁴² (p83) and Ala⁷⁴⁶ (p81), respectively, in agreement with previous findings¹⁶.

Tryptic T2 (Arg¹⁹⁸)-site in E2·BeF₃⁻ is completely resistant in C₁₂E₈

The association of the Val²⁰⁰ loop (Lys¹⁸⁹-Lys²⁰⁵) on the A domain with the P domain by ionic interactions is crucial for E2P structure formation and occurs as a consequence of the A domain’s large rotation during the E1PCa₂ → E2P isomeric transition^6,7,24. With the changes, the Arg¹⁹⁸-tryptic T2 site in this loop becomes completely resistant to tryptic attack^4,6. In Fig. 3a, the trypsin proteolysis was performed as described above with prtK. In the BeF₃⁻-free state with bound TG as a control (“E2·TG”) in which the A and P domains are not fixed, the Arg¹⁹⁸-site is cleaved producing the A1 and A2 fragments (the A2 fragment is not seen because it is at the gel front) as found previously⁶. In E2·BeF₃⁻, the A1 and A2 fragments are not produced regardless of the presence of C₁₂E₈ and 30 mM Mg²⁺, thus the Arg¹⁹⁸-site is completely resistant, consistent with association of the A and P domains by an ionic network as seen in the E2·BeF₃⁻ crystal structures^8,11.

E2·BeF₃⁻ in C₁₂E₈ is completely resistant to prtK at pH 6.0

In Fig. 3e, prtK-proteolysis was performed at pH 6.0 otherwise as in Fig. 3a. At this pH the luminal transport sites are expected to be protonated. No cleavage of the 110 kDa-ATPase chain occurred even in C₁₂E₈ and 30 mM Mg²⁺. The tryptic Arg¹⁹⁸-site was also completely resistant at pH 6.0 as at pH 7.4 without and with C₁₂E₈ and 30 mM Mg²⁺.

E2·AlF₄⁻ and E2·MgF₄²⁻ are completely resistant to prtK even in C₁₂E₈ at pH 7.4 and 6.0

E2·AlF₄⁻, the analog for the transition state E2~P^‡ is completely resistant to prtK at pH 7.4 and 6.0 even in the presence of C₁₂E₈ both without and with 30 mM Mg²⁺ (Fig. 3b,f). The Arg¹⁹⁸-site is also protected from trypsin in all these conditions. E2·MgF₄²⁻, the analog for the product complex (E2·P_i) is completely resistant to prtK and to trypsin in all these conditions as E2·AlF₄⁻ (Fig. 3c,f).

Hydrophobic nature of the nucleotide/catalytic site revealed by TNP-AMP superfluorescence

TNP-AMP binds to the ATP binding site with a very high affinity and develops an extremely high “superfluorescence” in the E2P ground state and its analog E2·BeF₃^−4,25. The TNP moiety binds at the adenine position in the N domain and the superfluorescence can be ascribed to a favorable TNP moiety Phe⁴⁸⁷ interaction and site-occlusion that excludes non-specific water and increases hydrophobicity by the contribution of Arg¹⁷⁴ on the A domain at the A-N interface on the TNP binding pocket²⁶. The superfluorescence is completely lost during E2P + H₂O → E2~P^‡, as demonstrated with the change E2·BeF₃⁻ → E2·AlF₄^− 4, probably through TNP-Phe⁴⁸⁷ mal-alignment and water influx here. In Fig. 4, the superfluorescence development in E2·BeF₃⁻ upon the TNP-AMP binding at saturating 4 μM was examined without and with C₁₂E₈ at pH 7.4 and 6.0 and various concentrations of Mg²⁺ in 0.1 M K⁺ or Li⁺. There was almost no effect of C₁₂E₈ on superfluorescence development. Specific K⁺ binding on the P domain^20,21 also had virtually no effect (compare the data in K⁺ with those in Li⁺). Increasing Mg²⁺ concentration up to 60 mM caused only slight decrease. The results show that the catalytic/nucleotide site, starting from the E2P ground state, is not affected by C₁₂E₈, Mg²⁺, K⁺, and protonation of transport sites.

Figure 4: Hydrophobic property at nucleotide/catalytic site in E2·BeF₃⁻ revealed by TNP-AMP superfluorescence.

E2·BeF₃⁻ or the BeF₃⁻-free Ca²⁺-ATPase (E2) in SR vesicles (0.06 mg protein/ml) were incubated at 25 °C for 3 min in 0.5 mM EGTA, 30 mM MES/Tris (pH 6.0) or MOPS/Tris (pH 7.4), 0.1 M KCl or LiCl, and 0–60 mM MgCl₂ with or without 0.02 mg/ml C₁₂E₈ and/or 2.5 μM A23187, as indicated in the figure. Subsequently, TNP-AMP at saturating 4 μM was added. The fluorescence intensity was obtained by subtracting the protein background level without TNP-AMP and the level of 4 μM TNP-AMP without SR vesicles, and plotted versus Mg²⁺ concentration.

E2·BeF₃⁻ in C₁₂E₈ and Mg^2 +is resistant to luminal Ca²⁺-induced reverse conversion to E1Ca₂·BeF₃⁻

The E2P ground state possesses luminally partially open low affinity transport sites and luminal Ca²⁺ at sub-mM to ~mM concentration is able to bind and cause reverse isomerization E2P + 2Ca²⁺ → E2PCa₂ → E1PCa₂, which contributes to the proper setting of luminal Ca²⁺ concentration through “back-door inhibition”. This reverse process as well as the forward EP isomerization is mimicked and characterized with the structural analogs E2·BeF₃⁻ (E2P), E2·BeF₃⁻·Ca₂ (E2PCa₂, the transient intermediate state before the Ca²⁺-release), and E1Ca₂·BeF₃⁻ (E1PCa₂)^4,17,18,19. In Figs 5 and 6, the effect of luminal Ca²⁺ on E2·BeF₃⁻ was examined at pH 7.4 in C₁₂E₈ or A23187, various concentrations of Mg²⁺, and 0.1 M K⁺ or Li⁺. Here it should be noted that the E1Ca₂·BeF₃⁻ complex is not stable and rapidly decomposes to E1Ca₂ in the presence of a high concentration of Ca²⁺ (due to Ca²⁺-substitution at the unoccluded catalytic Mg²⁺ site in E1Ca₂·BeF₃⁻¹⁷), on the other hand, it is very rapidly isomerized to E2·BeF₃⁻ releasing Ca²⁺ upon the removal or reduction of luminal free Ca²⁺ concentration (to below ~100 μM) as the process mimics the isomeric transition E1PCa₂ → E2P + 2Ca²⁺¹⁷. Also, the E1Ca₂·BeF₃⁻ complex decomposes to E1Ca₂ upon ADP binding, mimicking the ADP-induced reverse dephosphorylation of E1PCa₂, and upon TNP-AMP binding probably analogous to the ADP-induced process, in contrast to a stable E2·BeF₃⁻ with bound ADP or TNP-AMP¹⁷.

Figure 5: Luminal Ca²⁺-induced reverse conversion and decomposition of E2·BeF₃⁻ determined by loss of TNP-AMP superfluorescence.

E2·BeF₃⁻ in SR vesicles was incubated at 25 °C for 3 min in 30 mM MOPS/Tris (pH 7.4), 0.1 M KCl or LiCl, 0.5 mM EGTA, 0–60 mM MgCl₂ with or without 0.15 mg/ml C₁₂E₈ or 15 μM A23187, as indicated. Subsequently Ca²⁺ was added to give 0.5 mM free concentration and incubated for various times, then diluted 10-fold with the above solution containing 5 mM EGTA without Ca²⁺. At 30 s after dilution, 4 μM TNP-AMP was added to determine the superfluorescence intensity. The representative time courses of loss of superfluorescence in C₁₂E₈ in 0.1 M K⁺ are shown in inset. The rate of Ca²⁺-induced E2·BeF₃⁻ decomposition was determined by least-squares fit of a single exponential to the time course and plotted versus the Mg²⁺ concentration.

Figure 6: Luminal Ca²⁺-effect on E2·BeF₃⁻ in C₁₂E₈ (a) and formation and stabilization of E2·BeF₃⁻ in forward conversion from E1Ca₂·BeF₃⁻ in C₁₂E₈ (b,c).

(a) E2·BeF₃⁻ in SR vesicles was incubated without or with 0.15 mg/ml C₁₂E₈ or with 15 μM A23187 at 25 °C for 3 min in 30 mM MOPS/Tris (pH 7.4), 0.1 M LiCl, 0.5 mM EGTA, and 0 (upper panel) or 30 mM MgCl₂ (lower panel), then Ca²⁺ was added to give 0.5 mM free concentration. After 10 s, prtK was added at 0.5 mg/ml and incubated for indicated times. As a control, the BeF₃⁻-free Ca²⁺-ATPase in SR vesicles (“E1Ca₂”) was subjected to the proteolysis in 0.5 mM free Ca²⁺. (b) The prtK proteolysis was performed under the conditions that produce and perfectly stabilize E1Ca₂·BeF₃^{− 17}, i.e. 30 mM MOPS/Tris (pH 7.0), 0.1 M KCl, 15 mM MgCl₂, and 0.7 mM CaCl₂ in the presence of 100 μM BeCl₂ and 2 mM KF without and with 15 μM A23187, and the effect of C₁₂E₈ was examined by including C₁₂E₈ without A23187, otherwise as in (a). The BeF₃⁻-free Ca²⁺-ATPase (“E1Ca₂”) in A23187 and in C₁₂E₈ was subjected to proteolysis otherwise as above. Note that the slow decomposition of E2·BeF₃⁻ in Ca²⁺ in the absence of A23187 and C₁₂E₈ (a) is probably due to slow Ca²⁺ permeation into the SR vesicles lumen¹⁷. (c) E1Ca₂·BeF₃⁻ was produced by incubating SR vesicles for 30 min with 100 μM BeCl₂ and 2 mM KF in the absence of A23187 and C₁₂E₈ otherwise as in (b), then C₁₂E₈ or A23187 was added to give 0.02 mg/ml and 2.5 μM, respectively. At 10 s after this addition, TNP-AMP was added to give a saturating 4 μM, and the fluorescence monitored; trace b, without C₁₂E₈ and A23187; traces c and d, in A23178 and in C₁₂E₈, respectively. Trace e, the fluorescence monitored with E2·BeF₃⁻ in the presence of 2 mM EGTA without adding Ca²⁺. Trace a, the non-superfluorescent E1Ca₂ level (BeF₃⁻-free Ca²⁺-ATPase) in 4 μM TNP-AMP.

In Fig. 5, taking these known properties into account, we first determined the overall time course of the Ca²⁺-induced E2·BeF₃⁻ reverse conversion and decomposition to E1Ca₂ (E2·BeF₃⁻ + 2Ca²⁺ → E2·BeF₃⁻·Ca₂ → E1Ca₂·BeF₃⁻ → E1Ca₂) by adding an excess EGTA after various times of incubation with 0.5 mM Ca²⁺ thereby converting the remaining E1Ca₂·BeF₃⁻ to the stable E2·BeF₃⁻ species, and in addition adding TNP-AMP to determine superfluorescence development to estimate the total amount of E2·BeF₃⁻ and E1Ca₂·BeF₃⁻ species remaining at the time of EGTA addition. In Fig. 6, prtK proteolysis was performed for a short period during the 0.5 mM Ca²⁺ incubation and without the EGTA addition to identify the structural states of EP species under representative conditions in Fig. 5 (although the Ca²⁺-induced process proceeds).

First in Fig. 5 where TNP-AMP superfluorescence is examined, we found both with K⁺ and without K⁺ (with LiCl) that the Ca²⁺-induced reverse conversion/decomposition of E2·BeF₃⁻ is considerably slower in C₁₂E₈ than in A23187, and increasing Mg²⁺ to ~20 mM in C₁₂E₈ causes a marked retardation or almost complete inhibition. The retardation by Mg²⁺ in C₁₂E₈ is much stronger and occurs at much lower Mg²⁺ concentration than in A23187. In the absence of both C₁₂E₈ and A23187, i.e. with an impermeable SR membrane, no conversion nor decomposition of E2·BeF₃⁻ occurs with Ca²⁺, therefore the Ca²⁺-induced decomposition is due to the Ca²⁺ access from the luminal side as found previously^4,17. Regarding the K⁺ effect, the luminal Ca²⁺-induced conversion/decomposition of E2·BeF₃⁻ is considerably faster in K⁺ than in its absence, therefore specific K⁺ binding^20,21 accelerates the process.

Then in Fig. 6a, prtK-proteolysis was performed to identify the structural state stabilized in C₁₂E₈ with, most typically, 30 mM Mg²⁺ in the absence of K⁺ during luminal Ca²⁺-induced E2·BeF₃⁻ reverse conversion and decomposition. Here, the sample was incubated first with 0.5 mM Ca²⁺ for 10 s, and then with a high concentration of prtK for various times without removal of Ca²⁺. The proteolytic pattern was compared with those of BeF₃⁻-free E1Ca₂ and of E1Ca₂·BeF₃⁻ that is formed and stabilized perfectly under the previously identified most appropriate conditions, i.e. at pH 7.0 with 0.7 mM Ca²⁺ and 15 mM Mg²⁺ in 0.1 M K⁺ in the absence or presence of A23187 ¹⁷; in these states, p81/p83 fragments are produced due to cleavage at Thr²⁴² (p83) and Ala⁷⁴⁶ (p81) without production of the p95-fragment (Fig. 6b). In C₁₂E₈ and Ca²⁺ (Fig. 6a), E2·BeF₃⁻ both without and with 30 mM Mg²⁺ is degraded slowly as compared with E1Ca₂, producing the stable p95 fragment as seen with E2·BeF₃⁻ in C₁₂E₈ without Ca²⁺ (cf. Fig. 3) and a small amount of p81/p83 fragments, which degrade rapidly as the BeF₃⁻-free E1Ca₂ state. Note also that the 110-kDa ATPase chain degradation is much slower and formation of the rapidly degrading p81/p83 fragments is much less in 30 mM Mg²⁺ than without Mg²⁺. The results show that E2·BeF₃⁻ in C₁₂E₈ and Ca²⁺ is resistant to the luminal Ca²⁺-induced reverse conversion to E1Ca₂·BeF₃⁻, which can be interpreted as very slow Ca²⁺ binding to luminal transport sites and what slow conversion occurs is markedly retarded by 30 mM Mg²⁺. These results accord with those using superfluorescence as the indicator in Fig. 5.

In the presence of A23187, as seen in Fig. 6a, formation of the p81/p83 fragments from E2·BeF₃⁻ in Ca²⁺ occurs without any p95 fragment, as with E1Ca₂ and E1Ca₂·BeF₃⁻ in A23187 (cf. Fig. 6b) indicating a fast conversion of E2·BeF₃⁻ to E1Ca₂·BeF₃⁻ without the detergent and with the ionophore. These results together with the retardation by Mg²⁺ of loss of TNP-AMP superfluorescence (Fig. 5) indicate that E1Ca₂·BeF₃⁻ is formed from E2·BeF₃⁻ without detergent on luminal Ca²⁺ binding and further decomposed to E1Ca₂, and that Mg²⁺ at a high concentration retards the decomposition of E1Ca₂·BeF₃⁻ to E1Ca₂ probably by inhibiting the Ca²⁺-replacement of Mg²⁺ at the unoccluded catalytic subsite¹⁷.

Forward conversion of E1Ca₂·BeF₃⁻ to E2·BeF₃⁻ is favored in C₁₂E₈

Also in Fig. 6b, it can be seen that under conditions where E1Ca₂·BeF₃⁻ is perfectly stable in A23187¹⁷, the addition of C₁₂E₈ in place of A23187 produces the same proteolytic pattern as developed with E2·BeF₃⁻ in C₁₂E₈ and Ca²⁺. The results reveal that the E2·BeF₃⁻ state is produced and stabilized in C₁₂E₈ even under conditions that perfectly stabilize E1Ca₂·BeF₃⁻ in the absence of C₁₂E₈. This was further verified by superfluorescence development and loss upon TNP-AMP addition in Fig. 6c, which was performed on the basis of previous findings¹⁷ that E1Ca₂·BeF₃⁻ rapidly decomposes to the non-fluorescent E1Ca₂ state upon TNP-AMP binding whereas E2·BeF₃⁻ with bound TNP-AMP is stable, and also that the superfluorescence intensity is greater in E2·BeF₃⁻ than in E1Ca₂·BeF₃⁻ (by approximately 25%). In Fig. 6c, E1Ca₂·BeF₃⁻ was first formed under the conditions in Fig. 6b without A23187 and C₁₂E₈, and then A23187 or C₁₂E₈ added. After 10 s, superfluorescence upon TNP-AMP addition was recorded. In A23187 or in its absence, superfluorescence development is followed by its rapid loss, which is due to E1Ca₂·BeF₃⁻ decomposition to E1Ca₂ on TNP-AMP binding¹⁷. In C₁₂E₈, greater superfluorescence develops and its loss is considerably slower than in A23187. The results show again that in C₁₂E₈, E2·BeF₃⁻ is formed even under conditions that perfectly stabilize E1Ca₂·BeF₃⁻ (although E2·BeF₃⁻ is slowly decomposed to the non-fluorescent E1Ca₂ state via E1Ca₂·BeF₃⁻ in high Ca²⁺ and decomposition by TNP-AMP).

E2P hydrolysis

In Fig. 7, the effects of C₁₂E₈, K⁺, and Mg²⁺ on the forward E2P hydrolysis rate were examined at pH 7.4 and 6.0. Here E2P was first formed in the reverse reaction of hydrolysis from the Ca²⁺-deprived E2 state and ³²P_i in 7 mM Mg²⁺ without or with C₁₂E₈ (or with A23187) in 20% (v/v) Me₂SO, conditions that favor E2P formation. Then hydrolysis was initiated by a 20-fold dilution in non-radioactive P_i, various concentrations of Mg²⁺, and 0.1 M K⁺ (Fig. 7a) or Li⁺ (Fig. 7b) at the desired pH. In K⁺ at pH 7.4, C₁₂E₈ markedly retards hydrolysis as found previously at pH 7.5²⁷, and increasing Mg²⁺ concentration in C₁₂E₈ hardly affects the rate (perhaps a slight increase), but the cation decreases the rate in the absence of C₁₂E₈. Because this decrease is observed both without and with A23187 (an ionophore for Ca²⁺ and Mg²⁺) and because Me₂SO (used for the P_i-induced E2P formation) does not permeabilize the SR membrane, the hydrolysis reaction rate itself is likely affected by Mg²⁺ at the cytoplasmic side. At pH 6.0 in K⁺, hydrolysis is much slower than at pH 7.4, as is well known²⁸, and C₁₂E₈ and Mg²⁺ have almost no effect on the slowed rate.

Figure 7: Effects of C₁₂E₈ and various factors on E2P hydrolysis.

SR vesicles were phosphorylated with 0.1 mM ³²P_i at 25 °C for 10 min in 5 μl of a mixture containing 0.3 mg protein/ml with or without 3 μM A23187 as indicated, 1 mM EGTA, 7 mM MgCl₂, 30 mM MOPS/Tris (pH 7.4) or MES/Tris (pH 6.0), and 20% (v/v) Me₂SO. The mixture was then cooled, and a small volume of C₁₂E₈ was added to give 0.1 mg/ml (1/3 (w/w) of the protein) to the indicated samples. Subsequently, the samples were diluted at 0 °C by the addition of 95 μl of a mixture containing 0.1 mM non-radioactive P_i, 105 mM KCl (a) or LiCl (b), 1 mM EGTA, 1–30 mM MgCl₂, and 50 mM MOPS/Tris (pH 7.4) or MES/Tris (pH 6.0), as indicated with different symbols. The E2P hydrolysis rate was determined as described under “METHODS” and plotted versus Mg²⁺concentration. Note the difference in the scale of the ordinate in (a) and (b).

In the absence of K⁺ (Fig. 7b), E2P hydrolysis at both pH 7.4 and 6.0 is much slower than in 0.1 M K⁺ (by ~10-fold at the respective pH), in agreement with the well-known acceleration of hydrolysis by specific K⁺ binding on the P domain^20,21. In the absence of K⁺, hydrolysis in C₁₂E₈ is only slightly slower than that without C₁₂E₈. Mg²⁺ at ~10 mM somewhat increases the rate although the rate is still much slower than that in the presence of K⁺. In summary, induction of the detergent-stabilized state strongly inhibits hydrolysis at pH 7.4, but not following protonation of the transport sites at pH 6.0, and only in the presence of K⁺.

Discussion

Ca²⁺ transport by Ca²⁺-ATPase includes phosphorylated intermediates where Ca²⁺ is occluded at the transport sites and then released to the lumen, i.e. E1P[Ca₂] → E2P + Ca²⁺. During this process the A domain swings around and engages with the P domain and neck region of the protein at the cytoplasmic part of M2 (Fig. 1). The A-domain rotation inclines the P-domain by pulling an A/M1′-link, pushing M4 down towards the lumen to release the Ca^{2+ 8,18,19}. There is evidence that the gathering and interaction of A and P domains at the cytoplasmic part of M2 occurs progressively. Namely, changes, which are linked to deocclusion and opening of the luminal access channel with an affinity reduction, are followed by constrictions to limit access, protonation, and finally closure, and all these changes are synchronized with catalytic site preparations for hydrolysis^{4,13,15,17,18,19}. Part of the development is seen with the Leu¹¹⁹ prtK cleavage site, being exposed in E2PCa₂, hidden in E2P, E2~P^‡ and E2·P_i, and exposed again in E2^4,18,19. We found here that non-solubilizing concentrations of C₁₂E₈ uncovers the Leu¹¹⁹ prtK site of E2P, as depicted in its analog E2·BeF₃⁻. This indicates that membrane perturbation drives the intermediate towards one more like that with bound Ca²⁺, and points to an earlier catalytic intermediate with a looser arrangement in the head region, as expected for early engagement of the rotated A domain. The responsiveness of E2P to membrane perturbation and the detergent-induced state to ligand binding (Ca²⁺, Mg²⁺, K⁺, H⁺, and TG) through changes in exposure of the Leu¹¹⁹ prtK site at the cytoplasmic part of M2 points to flexible and rather unstable forms. These properties are due most probably not only to its unoccupied transport sites and associated circle of negative charges, but also to a loose meeting of domains and neck region with largely unsecured interactions at the cytoplasmic part of M2. The downward thrust of M4 (by a full turn of an α-helix⁸), together with M3, is probably partly stabilized by surrounding phospholipids and insertion of non-ionic detergent between them could be disruptive. In the head region the interactions at Leu¹¹⁹ involve the formation of Y122-HC, a hydrophobic interaction network of Tyr¹²²/Leu¹¹⁹ with the A and P domains and A/M3-linker involving seven residues (Fig. 2). As mentioned above, the interactions are likely progressive, loose at first as the A domain engages followed by incremental tightening in E2P to the fully stabilized state in E2~P^‡ and E2·P_i. Indeed, in the E2·BeF₃⁻ crystal structures (formed in the presence of C₁₂E₈) with the bound Mg²⁺ or with protonation without the Mg²⁺, Leu¹¹⁹/Tyr¹²² on M2 are close but not yet associated with the five other gathered residues involved in Y122-HC formation. The knitting of Leu¹¹⁹ and Tyr¹²² with the other residues is seen in the crystal structures of analogs of the next intermediates, E2~P^‡ and E2·P_i. Accumulating interactions fit perfectly with the staggered changes at the luminal transport sites, from closed to open to closed again.

Stabilization of the early detergent-induced state is seen in the forward direction of catalysis coming from E1PCa₂ (E1Ca₂·BeF₃⁻) and in the backward direction with Ca²⁺ binding to the luminal sites of E2P (E2·BeF₃⁻), using both TNP-AMP superfluorescence and the prtK sites as probes. Our results suggest that the E2·BeF₃⁻ structural state favored in C₁₂E₈ and stabilized by Mg²⁺ represents one between E1PCa₂ and Ca²⁺-released E2P, i.e. the transient E2P state immediately following Ca²⁺ release denoted as E2P^∗ with luminally open and vacant low affinity transport sites (E2P^∗Ca₂ → E2P^∗ in Fig. 8). C₁₂E₈ stabilizes the E2P^∗ state and thereby retards both the luminal Ca²⁺-induced reverse conversion and the forward hydrolysis of E2P at pH 7.4. Mg²⁺ binding probably prevents luminal Ca²⁺-access and consequent reverse conversion (Figs 3,5 and 6). This Mg²⁺ is likely at or near the luminally open Ca²⁺ transport sites (in addition to Mg²⁺ occluded at the catalytic subsite in E2·BeF₃⁻ and E2P) as actually seen in the E2·BeF₃⁻ crystal produced in a high concentration of Mg²⁺¹¹. The Mg²⁺ probably manifests itself in the competitive inhibition by Mg²⁺ of luminal Ca²⁺-induced reverse isomerization E2P+ 2Ca²⁺ → E1PCa₂²⁹. Notably also, the dephosphorylated E1 state is able to accommodate one Mg²⁺ at the transport sites and forms E1·Mg, which favors high affinity Ca²⁺-binding resulting in a rapid E2 → E1·Mg → E1Ca₂ transition^30,31 (Fig. 1). Thus it seems that Mg²⁺ binds at the empty transport sites both in the unphosphorylated and phosphorylated states and modifies transport function.

Figure 8: EP processing and gating.

The effects of C₁₂E₈, luminal Ca²⁺, and Mg²⁺ found in this study are summarized. E2P[Ca₂] (E2P with occluded Ca²⁺) and ^∗E2PCa₂ (E2P with luminally open gate and with bound Ca²⁺ yet at a high affinity) were previously identified by the elongation of the A/M1′-linker^18,19 and by substitutional mutation of Leu¹¹⁹ and Tyr¹²²¹⁵, but they are transient intermediates and have never been trapped or identified in wild type^15,18,19; therefore they are shown in brackets. The E2P structural states found in this study at pH 7.4, the Leu¹¹⁹-cleavable state and the prtK-resistant state are denoted as E2P^∗ and E2P, respectively. The prtK-resistant state found in C₁₂E₈ at pH 6.0 (i.e. with protonation of the transport sites) is denoted as E2P(^∗). Note that Y122-HC formation on gathering of Leu¹¹⁹/Tyr¹²² on M2 with engaged A and P domains occurs progressively during E2P processing and couples with luminal gating (see more in “Discussion”).

The E2·BeF₃⁻ structures revealed in C₁₂E₈ and in A23187 at pH 7.4 reflect E2P^∗ and E2P respectively in Fig. 8 on the basis of prtK-resistance. Analysis of the Mg²⁺ inhibition of luminal Ca²⁺-induced reverse conversion of E2·BeF₃⁻ in Figs 5 and 6 indicates that Mg²⁺ accesses E2P^∗ with a much higher affinity than E2P. Thus the transport sites appear more open and accessible to Mg²⁺ on the luminal side in the Leu¹¹⁹-site cleavable E2P^∗ state than in the prtK-resistant E2P ground state. In fact, in the E2·BeF₃⁻ crystal with bound Mg²⁺ at the transport sites, the sites are actually more open to the lumen than in the structure without Mg²⁺ (Fig. 2). Note also that in E2·AlF₄⁻ and E2·MgF₄²⁻ (E2~P^‡ and E2·P_i) and in E2·BeF₃⁻ with bound TG, Ca²⁺ cannot bind as the luminal gate is tightly closed^4,7,8, and the Leu¹¹⁹-site is completely resistant to prtK regardless of the presence of C₁₂E₈ (Fig. 3). These findings suggest that the structural change reflected by prtK resistance at Leu¹¹⁹ is associated with luminal gating, supporting the above conclusion that substantial luminal gate closure occurs in E2P^∗ → E2P, which probably involves gathering of Leu¹¹⁹/Tyr¹²² with the engaged A and P domains to accomplish the Y122-HC network. Then the passage is completely sealed in E2~P^‡ and E2·P_i (E2·AlF₄⁻ and E2·MgF₄²⁻)⁴.

Previous kinetic analysis of the luminal Ca²⁺-induced reverse isomerization E2P + 2Ca²⁺ → E1PCa₂ indicated¹⁴ that the luminal Ca²⁺ access to the transport sites in E2P is rate-limiting. This is described in Fig. 8 with the equilibrium E2P^∗ ↔ E2P, where the former state is more open and the latter relatively closed. This view agrees with our finding on the Ca²⁺ release kinetics E1PCa₂ → E2PCa₂ → E2P+ 2Ca^{2+ 15} that the E2P structure proceeds from a luminally open state for Ca²⁺ release (corresponding to E2P^∗ in Fig. 8) to a closed state (E2P) with the structural contribution of Leu¹¹⁹/Tyr¹²². The observation that Mg²⁺ hardly alters the forward E2P hydrolysis rate in C₁₂E₈ (Fig. 7a) can be accounted for by a rapid Mg²⁺ binding/release relative to the hydrolysis reaction process, and implies that Mg²⁺ binding favors the forward reaction.

At pH 6.0 in which the transport sites are protonated, the Leu¹¹⁹-site is completely resistant to prtK regardless of the presence of C₁₂E₈, and the E2P hydrolysis rate is not affected by C₁₂E₈. In Fig. 8, the protonated structural state with the prtK-resistance revealed in C₁₂E₈ is denoted as E2P(^∗) to be discriminated from the prtK-cleavable E2P^∗ state without protonation. Protonation neutralizes charges at the Ca²⁺-binding sites and stabilizes the arrangement of transmembrane helices via a hydrogen bonding network⁸, which lowers Ca²⁺-accessibility (without completely closing the gate as seen in the E2·BeF₃⁻ crystal formed at pH 5.7⁸). The protonated state proceeds promptly to subsequent hydrolysis with tight gate closure E2P+ H₂O → E2~P^‡ (E2·BeF₃⁻ → E2·AlF₄⁻), as indicated previously by kinetic analysis of E2P hydrolysis⁵.

K⁺ in the presence of C₁₂E₈ accelerates both forward E2P hydrolysis and luminal Ca²⁺-induced reverse conversion of E2·BeF₃⁻ (Figs 5 and 7). These findings are in complete agreement with the known role of specific K⁺ binding on the P domain in accelerating both forward hydrolysis^20,21 and luminal Ca²⁺-induced reverse conversion of E2P¹⁴. K⁺ binding likely destabilizes both E2P and E2P^∗ in Fig. 8, thus promoting rapid transport.

Finally, induction of the detergent-stabilized state, an early intermediate to ground state E2P, shows how phospholipids are intimately involved in the latter’s stabilization. Membrane perturbation effects during the transport cycle may be under-appreciated as fundamental to the mechanism.

Methods

Preparation of SR vesicles and treatment with BeF_x, AlF_x, and MgF_x

SR vesicles were prepared from rabbit skeletal muscle as described^32,33, in which all the methods were carried out in accordance with institutional laws and regulations of the Asahikawa Medical University and the experimental protocols were approved by the Animal Experimentation Ethics Committee of the Asahikawa Medical University (license number 16006). The content of the phosphorylation site in the vesicles and the Ca²⁺-dependent ATPase activity were determined as described^32,33. E2·BeF₃⁻, E2·AlF₄⁻, and E2·MgF₄²⁻ were produced by incubating the SR vesicles with the respective metal fluoride and by washing the unbound ligands as described previously⁴.

Formation and hydrolysis of E2P

The SR vesicles were phosphorylated with 0.1 mM ³²P_i at 25 °C for 10 min in 20% (v/v) Me₂SO in the absence of Ca²⁺, after which the samples were cooled and diluted 20-fold by a solution containing 2.1 mM non-radioactive P_i to initiate the hydrolysis of ³²P_i-labeled E2P, otherwise as described in detail in the legend to Fig. 7. The reaction was quenched with ice-cold trichloroacetic acid containing P_i. The precipitated proteins were separated by 5% SDS-PAGE at pH 6.0 according to Weber and Osborn³⁴. The radioactivity associated with the separated Ca²⁺-ATPase was quantified by digital autoradiography as described³⁵. Rapid kinetics measurement of hydrolysis was performed with a handmade rapid mixing apparatus and the rate of hydrolysis was determined with the least-squares fit to a single exponential, as described³⁵.

Proteolytic analysis

SR vesicles (0.45 mg/ml protein) were subjected to proteolysis at 25 °C by addition of trypsin (at 0.3 mg/ml, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) or proteinase K (prtK, at 0.1 mg/ml, Sigma) as described previously^6,16, otherwise as indicated in the figure legends. The proteolysis was terminated by trichloroacetic acid, and the samples were subjected to Laemmli SDS-polyacrylamide gel electrophoresis³⁶, and densitometric analyses of the gels stained with Coomassie Brilliant Blue R-250, as described^6,16. The degradation rate of 110-kDa ATPase chain with prtK was determined by least-squares fit of a single exponential to the time course (0–150 min) as described previously¹⁶.

Fluorescence measurements

The TNP-AMP fluorescence of the Ca²⁺-ATPase (0.06 mg/ml protein, TNP-AMP from Molecular Probes® Life Technologies) was measured on a RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) with excitation and emission wavelengths 408 and 540 nm (with band widths 5 and 10 nm), as described previously⁴.

Miscellaneous

Protein concentrations were determined by the method of Lowry et al.³⁷ with bovine serum albumin as a standard. Three-dimensional models of the enzyme were reproduced by the program VMD³⁸. The values presented are the mean ± s.d. (n = 3–4).