Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

Background: The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes.

Results: We identified different conformations after the association of Ca²⁺, ATP, and vanadate to PMCA.

Conclusion: PMCA forms a stable complex with Ca²⁺ and vanadate; ATP can bind to all pump conformations.

Significance: This study found a new intermediate in the PMCA reaction cycle; all of the intermediates interact with ATP.

Abstract

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca²⁺ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca²⁺ binding domain, we also studied the occlusion of Ca²⁺, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca²⁺ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E₁-E₂ model devised using kinetics methodology only.

Introduction

P-type ATPases are a group of enzymes responsible for active transport of cations across the cell membrane. They use the hydrolysis of ATP as a source of energy and share in common the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The plasma membrane calcium pump (PMCA)⁴ is a P-type ATPase that participates as an integral part of the Ca²⁺ signaling mechanism from eukaryotic cells (1) and is thus a crucial component of cell function. Detailed structural information about PMCA is currently lacking. Its abundance is ∼0.1% of the total protein in the red cell membrane, although it is much more abundant in some specialized cells. Unfortunately, these latter cells are not available in quantity, hampering efforts to produce suitable crystals for x-ray structure analysis. Although PMCA could not yet be crystallized, insight into the structural organization of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), a member of the same family, has come from the elucidation of several crystal structures at atomic resolution, representing the pump in various intermediate states (see Refs. 2 and 3). The membrane-buried region of SERCA is made up of 10 membrane-spanning helices and is connected to a large cytoplasmic headpiece, which is further separated into three distinct domains, denoted A (“actuator”), P (“phosphorylation”), and N (“nucleotide binding”). However, unlike SERCA, PMCA is highly regulated by calmodulin, which activates this protein by binding to an autoinhibitory region (4) and changes the conformation of the pump from an inhibited state, E₁I, to an activated one, E₁A (4–6).

The current kinetic model for the PMCA proposes that the enzyme exists in two main conformations, E₁ and E₂. E₁ has a high affinity for Ca²⁺ and is readily phosphorylated by ATP, whereas E₂ has a low affinity for Ca²⁺ and can be phosphorylated by P_i. After binding of intracellular Ca²⁺ to high affinity sites, E₁ can be phosphorylated by ATP with formation of the intermediate E₁P. A subsequent conformational transition to E₂P would allow Ca²⁺ to be released to the extracellular medium from low affinity sites, followed by the hydrolysis of the phosphoenzyme to E₂ and a new conformational transition to E₁ (7). During some stages of the reaction cycle, Ca²⁺ becomes occluded (i.e. trapped in the enzyme machinery) while it is transported from one side to the other side of the membrane (8). It has been described that in addition to being the substrate in the phosphorylation of the E₁Ca state, ATP also functions in a non-phosphorylating mode by enhancing the rates of the steps involved in phosphoenzyme turnover (E₁PCa → E₂P and E₂P → E₂) as well as the E₂ → E₁Ca transition of the dephosphoenzyme (9–20). In PMCA, the mechanisms underlying these modulatory effects of ATP remain largely unresolved.

By using [¹²⁵I]TID-PC/16, we were previously able to assess different transmembrane conformations in PMCA: a first one in which the protein displays maximum lipid exposure corresponding to an autoinhibited state of the enzyme (E₁CaI, in the presence of Ca²⁺ alone, hereafter denoted as E₁Ca), a second one in which protein-lipid interactions are markedly decreased corresponding to an activated state (E₁CaA, presence of Ca²⁺ and calmodulin), and a third one in the absence of Ca²⁺ (E₂) (21). Using the same experimental approach, we were also able to measure equilibrium constants for different ligands through the change of transmembrane conformations in PMCA (22). In order to obtain a complete understanding of the physical processes that involve ATP hydrolysis and Ca²⁺ transport, it is necessary to know more about the structure of the enzyme. The above results highlight the convenience of directly exploring the effects of different ligands on the PMCA transmembrane region. Applying this technique to PMCA, we were able to measure equilibrium constants for the dissociation of ligands from PMCA complexes and to draw structural conclusions about the regulation of the transport of Ca²⁺ in the presence of different ligands, such as ATP and vanadate, a well known inhibitor of P-ATPases, which forms a complex analogous to the phosphorylated intermediate E₂P. Conformational movement of the ATP binding domain was determined using the fluorescent analog TNP-ATP. To assess the conformational behavior of the Ca²⁺ domain, we also studied the occlusion of Ca²⁺ in experimental conditions that lead the PMCA to different intermediates of the reaction cycle.

EXPERIMENTAL PROCEDURES

Reagents

All chemicals used in this work were of analytical grade and purchased mostly from Sigma. Recently drawn human blood for the isolation of PMCA was obtained from the Hematology Section of Fundacion Fundosol (Argentina). Blood donation in Argentina is voluntary, and therefore the donor provides informed consent for the donation of blood and for the subsequent legitimate use of the blood by the transfusion service. None of the experiments described in this paper included calmodulin in the incubation media, and therefore, all results refer to the autoinhibited form of the PMCA.

Purification of PMCA from Human Erythrocytes

PMCA was isolated from calmodulin-depleted erythrocyte membranes by the calmodulin affinity chromatography procedure (23). Protein concentration after purification was about 10 μg/ml. No phospholipids were added at any step along the purification procedure. The purification procedure described preserves transport activity and the kinetic properties and regulatory characteristics of the enzyme in its native milieu (23, 24).

Measurement of Ca²⁺-ATPase Activity

Ca²⁺-ATPase activity was measured at 37 °C as the initial velocity of release of P_i from ATP, as described previously (7). The incubation medium contained 40 μm DMPC, 120 μm C₁₂E₁₀, 120 mm KCl, 30 mm MOPS-K (pH 7.4 at 37 °C), 2 mm ATP, 3.75 mm MgCl₂, 2 mm EGTA, and enough CaCl₂ to give 100 μm final free [Ca²⁺]. When necessary, sodium orthovanadate ((VO₄)³⁻, also named vanadate hereafter) or lanthanum chloride (La^III) was added at the concentrations indicated in the figures. Release of P_i was estimated according to the procedure of Fiske and SubbaRow (25). Measurements were performed in a Jasco V-630 Bio spectrophotometer.

Determination of Phosphorylated Intermediates

The phosphorylated intermediates (EP) were measured as the amount of acid-stable ³²P incorporated into the enzyme (0.9 μg/ml), according to the method described by Echarte et al. (26). The phosphorylation was measured at 4 °C in a medium containing 30 mm MOPS-K (pH 7.4 at 4 °C), 120 mm KCl, 3.75 mm MgCl₂, 120 μm C₁₂E₁₀, 40 μm DMPC, 2 mm EGTA, 30 μm [γ-³²P]ATP, and enough CaCl₂ to obtain 100 μm final free [Ca²⁺]. The reaction was started by the addition of [γ-³²P]ATP under vigorous stirring, and after 1 min, it was stopped with an ice-cold solution of TCA (10% (w/v) final concentration). The tubes were centrifuged at 7000 rpm for 3.5 min at 4 °C. The samples were then washed once with 7% TCA, 150 mm H₃PO₄ and once with double-distilled water and processed for SDS-PAGE. For this purpose, the pellets were dissolved in a medium containing 150 mm Tris-HCl (pH 6.5 at 14 °C), 5% SDS, 5% DTT, 10% glycerol, and bromphenol blue (sample buffer). Electrophoresis was performed at pH 6.3 (14 °C) in a 7.5% polyacrylamide gel. The reservoir buffer was 0.1 m sodium phosphate, pH 6.3, with 0.1% SDS. Migration of the sample components took place at 14 °C, with a current of 60 mA until the tracking dye reached a distance of about 10 cm from the top of the gel. Gels were stained, dried, and exposed to a Storage Phospho Screen (Amersham Biosciences). Unsaturated autoradiograms and stained gels were scanned with an HP Scanjet G2410 scanner. Analysis of the images was performed with GelPro Analyzer. EP quantification was achieved as described by Echarte et al. (26).

Preparation of [¹²⁵I]TID-PC/16

TTD-PC/16 (tin precursor) was a kind gift of Dr. J. Brunner (ETH Zentrum, Zurich, Switzerland). [¹²⁵I]TID-PC/16 was prepared by radioiodination of its tin precursor according to Weber and Brunner (27).

Labeling

A photolabeling assay was carried out as described by Mangialavori et al. (21, 22). A dried film of the photoactivatable reagent was suspended in PC/C₁₂E₁₀ mixed micelles containing 10 μg/ml of the membrane protein. The samples were incubated in the presence of necessary components for 10 min at 25 °C before being irradiated for 10 min with light from a filtered UV source (λ = 360 nm).

Radioactivity and Protein Determination

Electrophoresis was performed according to the Tris-Tricine SDS-PAGE method (28). Polypeptides were stained with Coomassie Blue R; the isolated bands were excised from the gel, and the incorporation of radioactivity was directly measured on a γ-counter. The amount of protein was quantified by eluting each stained band, as described previously (29), including bovine serum albumin in each gel as a standard for protein quantification. Specific incorporation was calculated as the ratio between the measured radioactivity and the amount of protein determined for each band. In all cases, a sample in the presence of 2 mm EGTA was included as a control and was taken as 100% of the specific incorporation. This condition was included in the figures only when it was necessary for the result analysis.

Measurements of Occluded Calcium

The procedure for measuring the occlusion of Ca²⁺ in microsomes containing PMCA involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of calcium occluded (8, 30). In a typical experiment, one volume of a microsomal preparation suspended in a solution with 30 mm MOPS-K (pH 7.4 at 25 °C), 120 mm KCl, and 400 nm thapsigargin was mixed with the same volume of a solution containing the same concentrations of MOPS-K and KCl plus 3.75 mm MgCl₂, 2 mm ATP, and enough [⁴⁵Ca²⁺]CaCl₂ to obtain the concentrations of free Ca²⁺ indicated in the figures. For some experiments, either 50 μm (VO₄)³⁻ or 50 μm La^III (in this case, to minimize the possibility of precipitation with La^III, ATP was 25 μm instead of 2 mm) was also included in the latter solution. Measurements were carried out in equilibrium conditions at 25 °C. To ensure the achievement of equilibrium, a reaction time of 3 s was selected after measuring the time courses of occluded calcium in different incubation media. Reactions were quenched by injecting the reaction mixture into the filtration chamber (quenching and washing chamber) at a flow rate of 1–5 ml/s. During the injection process, the fluid was mixed with an ice-cold washing solution flowing at a rate of 30–40 ml/s and then filtered through a Millipore filter (AA, 0.8-μm pore size) placed in the quenching and washing chamber in order to retain the microsomal suspension that included the enzyme. To ensure that the initial temperature in the quenching and washing chamber was 1–2 °C and that the flow was constant, about 50 ml of washing solution was allowed to run through the filter prior to the injection of the reaction mixture, and 240 ml of washing solution was applied to the filter thereafter. The composition of the washing solution was 10 mm Tris (pH 7.4 at 2 °C) and 10 mm EDTA. After the washing solution was drained, the filter was removed, dried under a lamp, and counted for ⁴⁵Ca²⁺ radioactivity in a scintillation counter. This was converted into nmol of Ca²⁺ using the specific activity value of the ⁴⁵Ca²⁺ in the reaction mixture. Ca²⁺ occluded was considered equal to the ⁴⁵Ca²⁺ radioactivity retained by the enzyme after subtracting the blank values. These were estimated from the amount of ⁴⁵Ca²⁺ retained by the filters in the presence of enzyme that was heat-inactivated for 2 h at 50 °C.

Spectroscopic Measurements

The fluorescence measurements were made in a quartz cell of 3 × 3 mm using an SLM- spectrofluorometer AMINCO BOWMAN Series 2 (Spectronic Instrument Inc., Rochester, NY). The excitation and emission bandwidths were set at 4 nm.

Determination of PMCA Apparent Affinity for TNP-ATP

Purified PMCA (10 μg/ml) was reconstituted in a medium containing 40 μm DMPC, 120 μm C₁₂E₁₀, 120 mm KCl, 30 mm MOPS-K (pH 7.4 at 25 °C), 1 mm MgCl₂ and then incubated with one of the following components for 10 min at 25 °C: (i) 2 mm EGTA, (ii) 100 μm free Ca²⁺, (iii) 2 mm EGTA plus 50 μm (VO₄)³⁻, or (iv) 100 μm free Ca²⁺ plus 50 μm (VO₄)³⁻. After incubation, increasing concentrations of TNP-ATP were added to each condition as described previously by Qu et al. (31). The fluorescence emission was measured at 539 nm after excitation at 495 nm and corrected for light scattering by background subtraction. An aliquot of the same solution without protein was titrated as a control in all experiments. In this case, the fluorescence emission behavior of TNP-ATP was linear and less than 15% of the fluorescence emission in the presence of protein.

As described previously by Pérez-Gordones et al. (32), in the presence of calcium, no hydrolysis was observed of TNP-ATP. As was reported for other ATPases (33–37), PMCA Ca²⁺-ATPase activity is completely inhibited by this analog.

Analysis of SERCA Structure and Accessible Surface Area (ASA)

The ASAs of two crystal structures of SERCA were compared as an approach to analyze the changes in incorporation of [¹²⁵I]TID-PC/16 in the PMCA intermediates E₁Ca and E₁PCa. The crystal structures used for comparison were those of E₁·2Ca (Protein Data Bank code 1su4) (38) and Ca₂E₁P (Protein Data Bank code 3ba6) (39). The transmembrane regions were taken as explicitly defined in Uniprot for sarcoplasmic/endoplasmic reticulum calcium ATPase 2, accession number P20647. The ASA of the transmembrane helices was calculated with MolMol (40, 41) with a solvent radius of 1.4 Å.

Data Analysis

Theoretical equations were fitted to the results by nonlinear regression based on the Gauss-Newton algorithm using commercial programs (Excel and Sigma-Plot for Windows, the latter providing not only the best fitting values of the parameters but also their S.E.). The goodness of fit of a given equation to the experimental results was evaluated by the corrected AIC criterion (42) defined as AIC_C = N ln(SS/N) + 2P N/(N − P − 1), where N is the number of data, P is the number of parameters plus one, and SS is the sum of weighted square residual errors. Unitary weights were considered in all cases, and the best equation was chosen as that giving the lower value of AIC_C. The AIC criterion is based on information theory and selects an equation among several possible equations on the basis of its capacity to explain the results using a minimal number of parameters.

RESULTS

Effect of Vanadate and Lanthanum on PMCA Ca²⁺-ATPase Activity

Ca²⁺-ATPase activity of purified PMCA was measured in the presence of 100 μm free Ca²⁺ and increasing concentrations of vanadate (Fig. 1A) or La^III (Fig. 1B). Equation 1 was fitted to the experimental data (continuous lines),

where [I] is the concentration of inhibitor (either (VO₄)³⁻ or La^III), V₀ is the Ca²⁺-ATPase activity in the absence of inhibitor, and K_I is the concentration of inhibitor needed for half-maximal effect. The value of K_I for (VO₄)³⁻ was 1.5 ± 0.1 μm, and it was similar to that reported previously (43), and the value of K_I found for La^III was 8.2 ± 0.5 μm.

FIGURE 1.

Effect of vanadate (A) and lanthanum (B) on PMCA Ca²⁺-ATPase activity. PMCA Ca²⁺-ATPase activity was determined as described under “Experimental Procedures” and normalized according to the activity obtained in the absence of (VO₄)³⁻ or La^III. The continuous line represents the fit of Equation 1 to the experimental values. The apparent K_i for the complexes of PMCA with (VO₄)³⁻ and La^III were 1.5 ± 0.1 and 8.2 ± 0.5 μm, respectively. Insets, gel autoradiographs of the phosphorylated intermediates of PMCA obtained in the absence of vanadate (control (C)) or in the presence of 50 μm (VO₄)³⁻ (A) or 100 μm La^III (B).

Although these inhibitors have similar effects on the PMCA activity, vanadate mimics the phosphoryl group in the transition state of E₂P, preventing the enzyme phosphorylation (Fig. 1A, inset) (43–45), whereas La^III inhibits by blocking the transition E₁P → E₂P, acting noncompetitively with respect to Ca²⁺ and ATP and bringing the enzyme phosphorylation level to a maximum (Fig. 1B, inset) (46, 47).

Effects of Vanadate and Lanthanum on the PMCA Transmembrane Domain Conformation

To investigate the structure-function relationship of intermediates of the reaction cycle of PMCA, we studied the effects of (VO₄)³⁻ and La^III on the conformation of the transmembrane domain of the pump under conditions similar to those used in the inhibition experiments of Fig. 1, except for the fact that all experiments were performed in equilibrium. To this end, we used a hydrophobic photolabeling strategy with [¹²⁵I]TID-PC/16, a photoactivatable reagent that has been previously shown to behave as phosphatidylcholine with regard to protein-lipid interactions (48, 49). It is thus possible to assess lipid exposure of transmembrane protein regions by quantifying the amount of reagent that becomes covalently attached to the protein upon photolysis (6, 21, 22, 48–52). In order to ensure equilibrium conditions, ATP was omitted from the media except when La^III was present.

First, we determined the extent of [¹²⁵I]TID-PC/16 labeling of the PMCA in its major known conformational states. The E₁Ca conformer is obtained by incubating the enzyme in the presence of Ca²⁺ alone, whereas in the presence of 2 mm EGTA, the conformational equilibrium is shifted toward the E₂ state (in the absence of Ca²⁺), whose level of [¹²⁵I]TID-PC/16 incorporation was taken as 100% (21). Because in this work we do not analyze the effect of calmodulin, we simply refer to the state of PMCA plus Ca²⁺ as E₁Ca. In the E₁Ca conformer, the incorporation of [¹²⁵I]TID-PC/16 increases by 50% as compared with that of E₂. The E₁PCa·La conformer was obtained by phosphorylation in the presence of La^III, whereas E₂P was mimicked by the complexes of PMCA with (VO₄)³⁻, both with and without Ca²⁺. Fig. 2 shows the specific incorporation of [¹²⁵I]TID-PC/16 to PMCA in the presence of EGTA (E₂); Ca²⁺ (E₁Ca); (VO₄)³⁻ plus EGTA (E₂V_i); (VO₄)³⁻ plus Ca²⁺ (E*V_iCa); or La^III, Ca²⁺, and ATP (E₁PCa·La). In agreement with reported data (21) specific incorporation of [¹²⁵I]TID-PC/16 to PMCA was about 55% higher in the E₁Ca conformation than in E₂. In the presence of EGTA, vanadate has no significant effect on the specific incorporation of [¹²⁵I]TID-PC/16 (98 ± 2.3%). However, whenever Ca²⁺ was present together with vanadate, the area accessible to lipids was different from that obtained in the presence of vanadate or Ca²⁺ alone (115 ± 3.4%). On the other hand, the specific incorporation of [¹²⁵I]TID-PC/16 in the presence of Ca²⁺, La^III, and ATP was 155.0 ± 1.0%, a value similar to that obtained in the presence of Ca²⁺ alone.

FIGURE 2.

Effect of different ligands on the conformation of PMCA. Specific incorporation of [¹²⁵I]TID-PC/16 into PMCA was determined as described under “Experimental Procedures” in the presence of 2 mm EGTA (E₂); 100 μm free Ca²⁺ (E₁Ca); 50 μm (VO₄)³⁻ plus 2 mm EGTA (E₂V_i); 50 μm (VO₄)³⁻ plus 100 μm free Ca²⁺ (E*CaV_i); or 50 μm La^III, 100 μm free Ca²⁺ plus 25 μm ATP (E₁PCa·La). Specific incorporation of the probe in the presence of 2 mm EGTA (E₂) was taken as 100%. Error bars, S.E.

Earlier experiments on the kinetics of vanadate inhibition (53) showed antagonism by Ca²⁺, consistent with Ca²⁺ and (VO₄)³⁻ binding to alternate conformations. Results obtained with vanadate plus calcium might be explained by two possible hypotheses: (i) the state denoted as E*V_iCa is actually an equilibrium mixture of mutually exclusive states of PMCA, E₂V_i, and E₁Ca, or (ii) both Ca²⁺ and (VO₄)³⁻ can bind together to PMCA in a state, E*V_iCa, where the asterisk indicates a distinct conformation with characteristics of both E₁ and E₂.

In order to test these hypotheses, we measured the specific incorporation of [¹²⁵I]TID-PC/16 as a function of vanadate concentration (Fig. 3A) in media with (closed circles) and without (open circles) Ca²⁺. It can be seen that in the latter case, specific incorporation is independent of [(VO₄)³⁻], indicating that the E₂ and the E₂P analog conformations have similar areas accessible to lipids. The average value of the specific incorporation is around 100% (99.3 ± 0.5%; Table 1). In the presence of Ca²⁺, however, there is a decrease of specific incorporation that tends to a level significantly higher than 100%. Results in Fig. 3A (closed circles) were analyzed by nonlinear fitting of the following decreasing function of [(VO₄)³⁻],

where PC is the specific incorporation of [¹²⁵I]TID-PC/16; [X] is the concentration of vanadate; PC₀ and PC_∞ are specific incorporations in the absence of vanadate and at nonlimiting concentration of the inhibitor, respectively; and K_X is the concentration of vanadate for half-maximal effect. This analysis shows (Table 1) that the value of PC_∞ is significantly higher than 100%, making hypothesis (i) above untenable because if E₂V_i and E₁Ca were two mutually exclusive species in equilibrium, the addition of enough vanadate should bring the entire enzyme to the E₂V_i state. We therefore conclude that there exists a ternary complex, E*V_iCa, which is confirmed by results of titration by Ca²⁺ of E₂V_i (Fig. 3B). To analyze these results, Equation 2 above was rearranged to the following increasing hyperbolic function of [Ca²⁺],

where [X] is now the concentration of Ca²⁺, PC₀ and PC_∞ are specific incorporations of [¹²⁵I]TID-PC/16 in the absence of Ca²⁺ and at [Ca²⁺] tending to infinity, respectively, and K_X is the concentration of Ca²⁺ for half-maximal effect. In this case, the value of PC_∞ is 114.1 ± 2.2%, which is not significantly different from the value of 112 ± 0.8% obtained from the titration of E₁Ca with vanadate. These values indicate that the addition of enough Ca²⁺ to the enzyme-vanadate complex (Fig. 3B) and the addition of enough vanadate to the E₁Ca complex (Fig. 3A, closed circles) lead to the same ternary complex, E*V_iCa.

FIGURE 3.

Effect of vanadate on the conformation of the transmembrane domain of PMCA. A, specific incorporation of [¹²⁵I]TID-PC/16 to PMCA in the presence of 2 mm EGTA (open circles) or 100 μm free Ca²⁺ (closed circles) at increasing (VO₄)³⁻ concentrations. The continuous line in the presence of calcium is the graph of Equation 2 using the best fitting parameters in Table 1 (Reaction ii). Specific incorporation of [¹²⁵I]TID-PC/16 to PMCA in the presence of 2 mm EGTA as a function of [(VO₄)³⁻] was constant (continuous line; Reaction vi), and thus K_x could not be experimentally determined. B, specific incorporation of [¹²⁵I]TID-PC/16 to PMCA in the presence of 50 μm (VO₄)³⁻ and increasing free Ca²⁺ concentrations. The continuous line is the graph of Equation 3 using the best fitting parameters shown in Table 1 (Reaction iii). Specific incorporation of the probe in the presence of 2 mm EGTA was taken as 100%.

TABLE 1.

Parameters for the interaction of PMCA with calcium, vanadate, and ATP determined from the specific incorporation of [¹²⁵I]TID-PC/16 under equilibrium conditions (Equations 2 and 3)

E represents the enzyme in the absence of calcium and is supposed to be an equilibrium mixture of the conformational states E₁ and E₂ ([E] = [E₁] + [E₂]), which is shifted toward E₂. E* represents a state formed in the presence of vanadate and calcium whose conformation is different from that of E₁ and E₂. The parameters PC₀ and PC_∞ are the specific incorporation of [¹²⁵I]TID-PC/16 in the absence of reactants or when their concentration tends to infinity, respectively. K_x is the apparent dissociation constant of the complex of PMCA formed through the corresponding equilibrium reaction on the left. Data for reaction (i), which were included for comparison, were taken from Ref. 22.

These results suggest that, despite the apparent lack of effect of vanadate in Fig. 3A (open circles), the addition of Ca²⁺ reveals that E₂ and E₂V_i are different conformers of the pump. From the results in Fig. 3, we postulate the existence of two phosphoenzyme analogs with vanadate: E₂V_i in the absence of calcium and E*V_iCa in its presence.

Effect of Vanadate on Occluded Intermediates of PMCA

We have previously identified and characterized the calcium occluded intermediate(s) of PMCA using a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and the quantification of occluded calcium (8).

To confirm the existence of the E*V_iCa intermediate in equilibrium and steady-state conditions, with or without ATP, we measured the amount of occluded calcium in the absence and in the presence of vanadate for different concentrations of Ca²⁺, in media either without (Fig. 4A) or with 2 mm ATP (Fig. 4B). We also included a control experiment performed in the absence of vanadate and in the presence of 25 μm ATP plus 50 μm La^III. As expected, experimental data for each condition can be described by a rectangular hyperbola as a function of [Ca²⁺]. In the absence of ATP, vanadate slightly reduces (around 20%) the amount of intermediate with calcium occluded (Fig. 4A), whereas the inhibitor has no significant effect in the presence of ATP (Fig. 4B). In accordance with the increase of phosphorylated intermediate shown in Fig. 1B, the maximal level of occluded intermediate obtained in the presence of La^III and ATP is about 30% higher than in the absence of the inhibitor. The addition of lanthanum also increased more than 2.5-fold the apparent affinity for Ca²⁺.

FIGURE 4.

Effect of vanadate on the level of calcium occluded by the PMCA. Occluded calcium was determined for increasing Ca²⁺ concentrations, with (triangles) or without (circles and squares) 50 μm (VO₄)³⁻ (see “Experimental Procedures” for details). Determinations where performed in the absence (A) and in the presence (B) of 2 mm ATP, except for a control experiment (closed squares), where media contained 25 μm ATP and 50 μm La^III. The continuous lines represent the fit of a Michaelis-Menten-like function of [Ca²⁺] to the experimental data. Best fitting values of the parameters were as follows. A, closed circles, Ca_occ,max = 0.058 ± 0.002 nmol of ⁴⁵Ca (mg of protein)⁻¹, K_0.5 = 19.7 ± 1.9 μm; closed triangles, Ca_occ,max = 0.046 ± 0.001 nmol of ⁴⁵Ca (mg of protein)⁻¹, K_0.5 = 17.4 ± 1.4 μm. B, open circles, Ca_occ,max = 0.064 ± 0.001 nmol of ⁴⁵Ca (mg of protein)⁻¹, K_0.5 = 17.2 ± 0.7 μm; open triangles, Ca_occ,max = 0.063 ± 0.005 nmol of ⁴⁵Ca (mg of protein)⁻¹, K_0.5 = 19.1 ± 4 μm; closed squares, Ca_occ,max = 0.084 ± 0.003 nmol of ⁴⁵Ca (mg of protein)⁻¹, K_0.5 = 6.8 ± 0.9 μm.

The apparent affinity for Ca²⁺ obtained from these experiments was lower than that observed for the purified enzyme, and the effect of vanadate on this was only marginal. Despite the difficulty of comparing data coming from very different preparations (see “Experimental Procedures”), these results confirm that calcium is occluded not only in E₁Ca but also in the E*V_iCa ternary complex. The higher level of calcium occlusion obtained in the presence of ATP (Fig. 4B) would indicate that the nucleotide is able to bind to E*V_iCa to form a quaternary complex, E*V_iCaATP.

Effects of ATP

In addition to its role as substrate in the phosphorylation of the E₁ state, ATP also operates in a non-phosphorylating mode (modulatory ATP), enhancing the rates of the steps involved in phosphoenzyme turnover (E₁P → E₂P and E₂P → E₂) as well as the E₂ → E₁ transition of the dephosphoenzyme (7, 54, 55) The mechanisms underlying these modulatory effects of ATP have been extensively studied in SERCA (9–20). However, for PMCA, this question remains unsolved. Therefore, we employed a strategy similar to that described above to study the transmembrane conformational changes associated with the binding of ATP to different states of PMCA.

E₁PCa State

In order to study the effect of ATP on the conformation of the transmembrane domain during formation of E₁PCa, we measured the specific incorporation of [¹²⁵I]TID-PC/16 into PMCA as a function of [ATP] in the presence of La^III and Ca²⁺ (Fig. 5). In the initial condition, PMCA is in the E₁Ca conformation (46, 47), and as ATP concentration increases, the reaction is shifted toward the formation of the phosphorylated intermediate, E₁PCa·La (see Fig. 1B, inset). Although ATP can also bind to this intermediate with low affinity (55), only marginal effects in specific incorporation of [¹²⁵I]TID-PC/16 are detected. There appears to be a slight increase at low concentrations of ATP followed by a low affinity decrease at concentrations higher than 50 μm. However, these changes are difficult to ascertain, and it seems safer to describe the phenomenon by saying that the level of [¹²⁵I]TID-PC/16 incorporation remains approximately constant around the value obtained for E₁Ca (155 ± 1%).

FIGURE 5.

Effect of ATP on the conformation of the transmembrane domain of the E₁PCa analog. Specific incorporation of [¹²⁵I]TID-PC/16 to PMCA was determined as a function of increasing ATP concentrations in the presence of 100 μm free Ca²⁺ plus 50 μm La^III. Specific incorporation of the probe into PMCA in the presence of 2 mm EGTA was taken as 100%. Results were approximately constant (continuous line, but see “Discussion”), and a precise value of K_x could not be determined (see Table 1, Reaction ix).

E₂ and E₂P Analog States

On the contrary, titration of the E₂ state with ATP in the absence of Ca²⁺ (Fig. 6A) leads to a decrease in specific incorporation of [¹²⁵I]TID-PC/16. Results were analyzed by nonlinear fitting of Equation 2, where the variable X stands for ATP. The best fitting values of the parameters are shown in Table 1. These results show that ATP can bind to PMCA, at micromolar concentrations, in the absence of Ca²⁺, causing a conformational change in the transmembrane domain.

FIGURE 6.

Effect of ATP on the conformation of the PMCA transmembrane domain. Specific incorporation of [¹²⁵I]TID-PC/16 to PMCA was determined as a function of increasing ATP concentrations in the presence of 2 mm EGTA (A), 2 mm EGTA plus 50 μm (VO₄)³⁻ (B), or 100 μm free Ca²⁺ plus 50 μm (VO₄)³⁻ (C). The continuous lines are the graphs of Equation 2 for the best fitting parameter values shown in Table 1 (Reactions iv, v, and viii, respectively). D, the specific incorporation of [¹²⁵I]TID-PC/16 to PMCA in media with 2 mm EGTA and 2 mm ATP did not vary with the concentration of (VO₄)³⁻ (continuous line), and thus K_x could not be experimentally determined (see Table 1, Reaction vii).

Fig. 6, B and C, shows the specific incorporation of [¹²⁵I]TID-PC/16 to PMCA as a function of [ATP] when the enzyme was preincubated in the presence of vanadate with and without Ca²⁺, respectively. Nonlinear fitting of Equation 2 (Table 1) yields values of K_X for ATP that are at least 50% lower than that obtained in the absence of vanadate. In addition, the simultaneous presence of vanadate and Ca²⁺ causes the levels of specific incorporation at both zero and non-limiting [ATP] to be higher than the corresponding levels obtained in the absence of Ca²⁺.

When the pump was incubated in the absence of Ca²⁺ and in the presence of saturating [ATP], the specific incorporation of [¹²⁵I]TID-PC/16 to PMCA was around 85% (Fig. 6, A and D). The addition of increasing vanadate concentrations has no effect on the incorporation of the probe (Fig. 6D). This result shows that in the absence of calcium, vanadate does not produce changes in the area accessible to the lipid environment of PMCA transmembrane domain, regardless of the conformation reached by the pump in the presence of ATP. As will be shown below, this lack of effect does not imply the absence of binding of vanadate to PMCA.

A Model for the Interaction of PMCA with Calcium, Vanadate, and ATP

Our findings can be summarized by the scheme shown in Fig. 7. In the absence of Ca²⁺, PMCA is an equilibrium mixture of 10% E₁ and 90% E₂ forms (56). However, because our experiments do not include information on this equilibrium, we refer to the enzyme in the presence of EGTA as “E”, which is an equilibrium mixture of the E₂ and E₁ forms. A similar practice is used for the equilibrium between E₁ATP and E₂ATP, which will depend on the apparent affinities of these forms for the nucleotide, and thus in the absence of calcium, these will simply be denoted as “EATP.” On the other hand, our results show that, regarding the conformation of the transmembrane domain, the ternary complex E*V_iCa is different from E₁Ca and E₂V_i and is therefore denoted with an asterisk, as described above.

FIGURE 7.

Scheme of the model proposed for the interaction of PMCA with calcium, vanadate, and ATP. The model was used to perform a global fitting analysis of the results shown in Figs. 3, 5, and 6, and the best fitting values of the parameters are shown in Table 2.

Each step in the model represents the equilibrium binding of a single ligand to PMCA. A global fitting of the parameters of the equilibrium equations arising from the model was performed on the results shown in Figs. 3, 5, and 6. The best fitting values for the specific incorporation of [¹²⁵I]TID-PC/16 (percentage) in the presence of the different reactants and their apparent dissociation constants are shown in Table 2. The parameter values obtained from this global fitting correlate very well with those obtained from each experiment (Table 1), which were taken as initial values for the fitting. The correlation is indicative that this global model is appropriate to describe the interactions of PMCA with Ca²⁺, vanadate, and ATP. The model allows the determination of the apparent dissociation constants for steps 6, 7, and 8 (i.e. for the binding of Ca²⁺ to E₂V_iATP, for the binding of vanadate to E, and for the binding of vanadate to EATP, where the latter two could not be experimentally measured). Results summarized in Table 2 show that (i) the apparent affinity of PMCA for vanadate decreases in the presence of Ca²⁺, (ii) the apparent affinity of PMCA for Ca²⁺ decreases in the presence of vanadate, (iii) ATP does not modify the apparent affinity for Ca²⁺ of the E₂V_i form, and (iv) the apparent affinity of E₂V_i and E*V_iCa forms for ATP were similar, meaning that Ca²⁺ does not modify the apparent affinity for ATP of E₂V_i.

TABLE 2.

Best fitting values of specific incorporation of [¹²⁵I]TID-PC/16 and dissociation constants for calcium, vanadate, and ATP predicted by the global model shown in Fig. 7

Constants K_V, K_t^ATP and K_Ca^VATP were calculated as a combination of the rest of the equilibrium constants forming part of the same closed reaction cycle according to the equations, K_>V = K_V^CaK_Ca/K_Ca^V, K_V^ATP = K_ATP^VK_V/K_ATP, and K_Ca^VATP = K_ATP^CaV K_Ca^V/K_ATP^V.

Interaction of TNP-ATP with Different PMCA Intermediates

The previous strategy does not allow for obtaining information about the relationship between ATP and PMCA in the presence of Ca²⁺, the catalytic role of ATP, because this nucleotide is rapidly hydrolyzed. Therefore, in order to obtain more information on the interaction of E₁Ca or E₂ with ATP, we studied the binding of its non-hydrolyzable, fluorescent analog, TNP-ATP, to PMCA in different conditions. This approach was used for studying the interaction with ATP in several proteins (reviewed in Ref. 57). As was previously reported for PMCA (32) and other proteins (57), TNP-ATP is not a substrate for the pump and inhibits its ATPase activity. Fig. 8 shows the fluorescence at 539 nm as a function of [TNP-ATP] in media where PMCA was incubated with and without 100 μm Ca²⁺ and without (A) or with (B) 50 μm vanadate. Experimental data can be described by the equation,

which represents the interaction with a single type of binding sites, where ΔF represents the change in fluorescence intensity following the addition of TNP-ATP at a concentration [S], ΔF_max is the maximum change in fluorescence intensity, and K_D is the apparent dissociation constant for the probe. The best fitting values of K_D and ΔF_max are shown in Table 3.

FIGURE 8.

Binding of TNP-ATP to PMCA. Fluorescence intensity of bound TNP-ATP to PMCA as a function of the concentration of TNP-ATP was determined as described under “Experimental Procedures” in the presence of 100 μm free Ca²⁺ (closed circles) or 2 mm EGTA (open circles) (A) and in the presence of 50 μm (VO₄)³⁻ and either 100 μm free Ca²⁺ (closed triangles) or 2 mm EGTA (open triangles) (B). The continuous line is the graph of Equation 4 using the best fitting parameters shown in Table 3.

TABLE 3.

PMCA apparent affinity for TNP-ATP under equilibrium conditions

ΔF_max corresponds to the fluorescence intensity when the concentration of TNP-ATP tends to infinity, and K_D represents the apparent dissociation constant for the complex formed between PMCA and TNP-ATP. For the meaning of E and E*, see the legend to Table 1.

Results show that the apparent affinity of PMCA for TNP-ATP is 10-fold higher in the presence of Ca²⁺ than that in its absence. However, in the presence of vanadate, the apparent affinity for this ATP analog did not change with the addition of Ca²⁺ and was similar to that of E₁Ca in Fig. 8A. These results can be compared with those in Fig. 6, A–C, where the affinity for ATP is clearly higher in the presence of vanadate (B and C) than in its absence (A), showing a parallel behavior for the affinities of TNP-ATP. It could be surprising that the affinity for ATP observed in Fig. 6 is considerably lower than that for TNP-ATP in Fig. 8. However, Moutin et al. (58) reported that the apparent affinity of sarcoplasmic reticulum Ca²⁺-ATPase for TNP-ATP is significantly higher than that for the unmodified nucleotide.

On the other hand, TNP-ATP fluorescence intensity at saturating concentrations of the probe was similar for E₁Ca and E₂, whereas this value was 40% lower in the presence of vanadate. These results suggest that there is a change in the hydrophobic environment of the TNP-ATP and therefore a different environment for the ATP binding domain as a consequence of the different conformations.

The change in ΔF_max upon the addition of vanadate proves that the lack of response to the inhibitor observed in Fig. 3A is not due to its inability to bind to PMCA. In other words, vanadate apparently does not modify the transmembrane domain, but it does produce a conformational change on the cytoplasmic domain, thus justifying the hypothesis of the existence of a different conformation, E*V_iCaATP.

DISCUSSION

There are several crystal structures of the Ca²⁺ pump of sarcoplasmic reticulum corresponding to different conformations reached during the reaction cycle (2, 3), and crystal structures of the H⁺-ATPase (59) and of the Na⁺/K⁺-ATPase were reported as well (60, 61). However, there are as yet no high resolution structures for the majority of the P-type ATPases, including the family of PMCAs. This underlines the continued need to develop new tools to probe the structure-function relationship in these proteins by alternative and complementary methods. Of particular interest for a better understanding of the pump mechanism are methods that yield information on the membrane-embedded part of the pump, which is the most difficult region to study by traditional methods of protein expression and analysis.

Effects of Lanthanum and Vanadate on the Transmembrane Domain

For PMCA, the amount of EP obtained in the presence of lanthanum is usually considered as a valid calculation of the total active enzyme concentration (55). It has been proposed that La^III would act as a dead end inhibitor capable of bringing the entire amount of functionally active enzyme into the E₁PCa form. This phenomenon is consistent with the fact that specific incorporation of [¹²⁵I]TID-PC/16 in the presence of Ca²⁺, La^III, and ATP was 155.0 ± 1.0%, a maximal value that is similar to that obtained in the absence of ATP. Like the phosphoenzyme level, occlusion of Ca²⁺ is also maximal in the presence of La^III and ATP (8). Fig. 5 shows that increasing ATP concentrations produced only marginal changes in the incorporation of [¹²⁵I]TID-PC/16. This can mean that either (i) significant changes occur in the transmembrane segments, but these are internally counterbalanced, or (ii) La^III hinders the transmembrane conformational changes elicited by ATP. As an approach to answer this question, we evaluated the possible extent of this change by calculating the ASA of the transmembrane region using crystallographic data from SERCA. Note that in Protein Data Bank code, the structure of Ca₂E₁P (our E₁PCa) is known as 3ba6, whereas E₁·2Ca (our E₁Ca) is represented by the structure known as 1su4. Results in Fig. 9 show that most significant changes are found in helices M1–M3, with an increase of 338 Ų (32%) for M1, a decrease of 285 Ų (30%) for M2, and an increase of 131 Ų (12%) for M3. Thus, the overall change in ASA values when the enzyme goes from E₁·2Ca (7113.9 Ų) to Ca₂E₁P (7349.6 Ų) is 236 Ų, just about 3% of the total ASA. These results for SERCA support the idea that our method could be not precise enough to resolve such a small overall difference in areas exposed to lipids in PMCA, despite the important changes that can occur in some of the transmembrane segments.

FIGURE 9.

ASAs of the transmembrane domains (helices M1–M10) calculated from the crystal structures 1su4 (E₁Ca₂) and 3ba6 (E₁P(Ca₂)) of SERCA. For details, see “Experimental Procedures.”

An important conclusion from the above analysis is that, although a difference in the area accessible to lipids can only occur as a consequence of the existence of different conformations of the pump, the opposite is not necessarily true (i.e. a similar value of specific incorporation of [¹²⁵I]TID-PC/16 does not necessarily imply similar conformations), because different arrangements of the transmembrane helices could present a similar surface accessible to lipids.

Vanadate is considered to mimic a pentacoordinated transition state of the phosphoryl group, binding to E₂ in a reaction that requires Mg²⁺ (43, 44). In the absence of Ca²⁺, vanadate does not produce any detectable change in the specific incorporation of [¹²⁵I]TID-PC/16 (Fig. 3A, open circles). However, in the presence of Ca²⁺, increasing concentrations of vanadate produce a decrease in the specific incorporation of [¹²⁵I]TID-PC/16 (Fig. 3A, closed circles). A similar result was obtained when PMCA was incubated with vanadate, and increasing Ca²⁺ concentrations were added (Fig. 3B). Inhibition of Ca²⁺-ATPase activity by vanadate is commonly explained as the result of its binding to the enzyme, leading to a conformation analogous to E₂P. Early experiments on the kinetics of vanadate in SERCA showed that the inhibition is antagonized by Ca²⁺ (44, 53), a result consistent with the idea that Ca²⁺ and vanadate bind to different, mutually exclusive conformations. Experiments by Krebs et al. (62) performed in calmodulin-activated PMCA show that vanadate prevents the fluorescence changes that accompany phosphorylation by ATP. However, fluorescence experiments in SERCA (63) suggest the existence of a calcium-enzyme-vanadate complex whose fluorescence properties are “E₁-like” rather than “E₂-like.” Our results confirm that in the presence of Ca²⁺ and vanadate, PMCA forms a stable ternary complex, E*V_iCa, whose conformational state is different from that of E₂ and E₁Ca and that we tentatively designated “E*”. Importantly, this ternary complex could be formed in the absence and in the presence of ATP. In agreement with results reported by Daiho et al. (64, 65), who obtained a stable transition phosphorylation state of SERCA in the presence of Ca²⁺, the ternary complex E*V_iCa could be interpreted as an intermediate state interposed between E₁PCa and the Ca²⁺-free state E₂P.

While forming the E*V_iCa complex from E₂V_i, we found that the apparent dissociation constant for Ca²⁺ is higher than in the absence of vanadate (5.1 μm versus 1 μm; see Ref. 22). In SERCA (66), biochemical data obtained in the absence of thapsigargin, a potent inhibitor that binds to the transmembrane domain and leads the pump to the E₂ conformation, support the conclusion that the dephosphorylation transition state of E₂P represents a proton-occluded state. This idea arises from the fact that in contrast to E₂, this state has a low affinity for Ca²⁺. Our results show that a similar state could exist for PMCA.

Ca²⁺ Occlusion

We report here results on Ca²⁺ occlusion obtained in equilibrium conditions, in the absence of ATP or in the presence of ATP plus vanadate. It is a generally accepted idea that in P-type ATPases, occlusion of the cation transported from the cytoplasmic side occurs concomitantly with the formation of E₁P and that the release of the occluded cation toward the opposite side takes place after the E₁P to E₂P conformational transition. This has been proposed both for the occlusion of two calcium ions in the SERCA and of three sodium ions in the Na,K-ATPase. Using membrane-bound PMCA preparations in media with ATP and lanthanum, which blocks the conformational transition from E₁P to E₂P, we found that Ca²⁺ becomes occluded concomitantly with the formation of E₁P with a stoichiometry of one Ca²⁺ per phosphorylated enzyme unit (8). However, this experimental evidence does not exclude the possibility of occlusion of Ca²⁺ in non-phosphorylated intermediates. In this sense, in the absence of ATP, occlusion of Ca²⁺ in SERCA (67) and of Na⁺ in oligomycin-inhibited Na,K-ATPase (68) has been reported. This is not surprising if one admits the idea that cation occlusion precedes phosphorylation (or dephosphorylation, depending on the transported cation), thus triggering the reaction (e.g. see Refs. 60 and 69).

The increase in affinity for Ca²⁺ observed in the presence of La^III can be explained on the basis of the irreversibility of the phosphorylation reaction and the blocking effect of this inhibitor on the E₁P to E₂P conformational transition, which prevents the return of the enzyme to calcium-free states.

Binding of ATP

Toyoshima et al. (70) described that in SERCA, the reaction cycle is regulated essentially by Ca²⁺ alone. ATP can bind to the enzyme even when Ca²⁺ is absent, but without Ca²⁺, the reaction cycle cannot progress. Binding of Ca²⁺ will cause the movement of the transmembrane helices. Fig. 6 shows that ATP binds to PMCA in the absence of Ca²⁺, and this triggers a change in the transmembrane domain. The apparent dissociation constant for ATP measured with [¹²⁵I]TID-PC/16 is in agreement with previous reports for SERCA (20). Our results (Fig. 6, B and C) show that ATP also binds to E₂V_i (an E₂P-like state) and to E*V_iCa (an E*P(Ca)-like state), producing conformational changes in the PMCA transmembrane domain. Vanadate increases 3-fold the apparent affinity for ATP, both in the presence and in the absence of Ca²⁺. This is less than the 10-fold increase of the apparent affinity for ATP in SERCA. However, the structural and kinetic analyses of the E₂ conformation of SERCA were performed in the presence of thapsigargin. It was recently suggested (71) that thapsigargin produces stiffness at the transmembrane domain of SERCA, making it unresponsive to conformational changes occurring within the cytosolic domain, and it is unclear how closely these inhibitor-bound structures resemble other, perhaps more physiological, states of the enzyme. In addition, it has been described (72) that in the absence of Ca²⁺, affinity of SERCA for ATP is lower when thapsigargin is present.

Therefore, those results should not be compared directly with our studies, which were performed in the absence of an inhibitor that leads PMCA to the E₂ conformation. On the contrary, our approach measures the actual substrate affinity through the binding of trace amounts of [¹²⁵I]TID-PC/16, which is directly proportional to the transmembrane surface of PMCA exposed to surrounding lipids.

A most important point of interest is whether binding of nucleotide produces an induced fit prior to the phosphoryl transfer reaction. However, binding of ATP to E₁Ca cannot be measured directly due to the fast setting of a steady state of transport of Ca²⁺ and ATP hydrolysis. We therefore extended our studies of specific conformational effects produced by ATP using its analog TNP-ATP. This fluorescent probe has been used for studying the interaction of several proteins with ATP (53) and is not hydrolyzed by PMCA.

Results shown in Fig. 8 confirm that TNP-ATP binds to PMCA in the absence of Ca²⁺, although with a higher apparent dissociation constant than that observed in the presence of Ca²⁺ (E₁Ca). At saturating TNP-ATP concentrations, the fluorescence intensity was similar in both conditions, indicating that the hydrophobic environment of the N-domain is also similar, in agreement with results reported in SERCA (2). In PMCA, the apparent K_D values for TNP-ATP of the E₂Vi and E*V_iCa states were similar to that obtained for the E₁Ca state but 7-fold lower than for E₂.

The maximal fluorescence intensity of the high affinity component for the binding of TNP-ATP was similar for E₂V_i and E*V_iCa states but significantly lower than that obtained for E₁Ca. This indicates that the N-domain environment is more hydrophobic in the presence of Ca²⁺ (E₁Ca).

A Functional Cycle for PMCA

Scarborough (73) has observed the inadequacy of the E₁/E₂ nomenclature to describe the intermediates involved in the reaction cycle of P-type ATPases. In this work, we have demonstrated the existence of species of PMCA that can be seen as different conformers, depending on whether the measurements were sensing changes in the cytoplasmic (experiments with TNP-ATP) or in the transmembrane ([¹²⁵I]TID-PC/16 photolabeling) domains of the enzyme. For instance, the apparent lack of effect of vanadate on the transmembrane domain of EATP contrasts with the effect of the inhibitor on the maximal fluorescence change upon binding of TNP-ATP. Although this could mean that significant changes in the transmembrane domain cancel each other regarding their exposure to lipids, as shown above in the case of the structures of E₁·2Ca and Ca₂E₁P of SERCA, a possible uncoupling between conformational changes in the cytoplasmic and membrane domains cannot be ruled out and is worth considering. Evidence adding important features to the E₁-E₂ model is that E₂V_i, E₂V_iATP, and E*V_iCaATP, all of them intermediates that in principle would be thought of as E₂ conformers, actually exhibit different surface areas accessible to phospholipids, which reveals that they should at least be considered as different subconformations.

We have also studied in this work for the first time the effect of ATP on the structure of these subconformations, showing that they are all able to bind ATP at physiological concentrations (see Figs. 6 (A–C) and 8). This notion questions the physiological relevance of the E₁Ca conformation as an intermediate in the transport scheme, because under physiological conditions, with a concentration of ATP high enough and a large ATP/ADP ratio, the modulatory site will be saturated by ATP, allowing the E₂ state to circumvent the E₁Ca conformation and to transition directly into the E₁ATPCa state.

Taking this evidence together, Fig. 10 shows a functional cycle of ATP binding and hydrolysis by PMCA. Note that (i) we have included a new intermediate, E*P(Ca)ATP, between E₁P(Ca)ATP and E₂PATP·Ca, whose conformation is different from that of E₁ and E₂, and (ii) with the exception of a transient ADP-bound state, all conformers are bound to a molecule of ATP and can be simultaneously phosphorylated. Conversely, Ca²⁺ can be occluded in or simply bound to the enzyme. In its resting condition, PMCA would mainly exist as two intermediates in equilibrium, E₁ATP and E₂ATP, until a signal of Ca²⁺ or calmodulin-Ca²⁺ initiates the transport of the cation driven by the ATP hydrolysis.

FIGURE 10.

Scheme of the model proposed for the PMCA functional reaction cycle. Parentheses denote cation occlusion.

In this study, we focus on the role of ATP in the reaction cycle, leaving aside the effect of cofactors like Mg²⁺ and activators such as calmodulin, which are worthy of future study using the findings of this work as a basis.