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. 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.

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 inte-gral part of the Ca 2ϩ 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 ϳ0.1% 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 Ca 2ϩ -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, E 1 I, to an activated one, E 1 A (4 -6).
The current kinetic model for the PMCA proposes that the enzyme exists in two main conformations, E 1 and E 2 . E 1 has a high affinity for Ca 2ϩ and is readily phosphorylated by ATP, whereas E 2 has a low affinity for Ca 2ϩ and can be phosphorylated by P i . After binding of intracellular Ca 2ϩ to high affinity sites, E 1 can be phosphorylated by ATP with formation of the intermediate E 1 P. A subsequent conformational transition to E 2 P would allow Ca 2ϩ to be released to the extracellular medium from low affinity sites, followed by the hydrolysis of the phosphoenzyme to E 2 and a new conformational transition to E 1 (7). During some stages of the reaction cycle, Ca 2ϩ 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 E 1 Ca state, ATP also functions in a non-phosphorylating mode by enhancing the rates of the steps involved in phosphoenzyme turnover (E 1 PCa 3 E 2 P and E 2 P 3 E 2 ) as well as the E 2 3 E 1 Ca transition of the dephosphoenzyme (9 -20). In PMCA, the mechanisms underlying these modulatory effects of ATP remain largely unresolved. By using [ 125 I]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 (E 1 CaI, in the presence of Ca 2ϩ alone, hereafter denoted as E 1 Ca), a second one in which protein-lipid interactions are markedly decreased corresponding to an activated state (E 1 CaA, presence of Ca 2ϩ and calmodulin), and a third one in the absence of Ca 2ϩ (E 2 ) (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 Ca 2ϩ 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 Ca 2ϩ 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 E 2 P. Conformational movement of the ATP binding domain was determined using the fluorescent analog TNP-ATP. To assess the conformational behavior of the Ca 2ϩ domain, we also studied the occlusion of Ca 2ϩ 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 g/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 Ca 2ϩ -ATPase Activity-Ca 2ϩ -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 M DMPC, 120 M C 12 E 10 , 120 mM KCl, 30 mM MOPS-K (pH 7.4 at 37°C), 2 mM ATP, 3.75 mM MgCl 2 , 2 mM EGTA, and enough CaCl 2 to give 100 M final free [Ca 2ϩ ].
When necessary, sodium orthovanadate ((VO 4 ) 3Ϫ , 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 32 P incorporated into the enzyme (0.9 g/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 MgCl 2 , 120 M C 12 E 10 , 40 M DMPC, 2 mM EGTA, 30 M [␥-32 P]ATP, and enough CaCl 2 to obtain 100 M final free [Ca 2ϩ ]. The reaction was started by the addition of [␥-32 P]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 H 3 PO 4 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).
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/C 12 E 10 mixed micelles containing 10 g/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 ( ϭ 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 ␥-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 Ca 2ϩ 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 MgCl 2 , 2 mM ATP, and enough [ 45 Ca 2ϩ ]CaCl 2 to obtain the concentrations of free Ca 2ϩ indicated in the figures. For some experiments, either 50 M (VO 4 ) 3Ϫ or 50 M La III (in this case, to minimize the possibility of precipitation with La III , ATP was 25 M 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-m 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 45 Ca 2ϩ radioactivity in a scintillation counter. This was converted into nmol of Ca 2ϩ using the specific activity value of the 45 Ca 2ϩ in the reaction mixture. Ca 2ϩ occluded was considered equal to the 45 Ca 2ϩ radioactivity retained by the enzyme after subtracting the blank values. These were estimated from the amount of 45 Ca 2ϩ 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 ϫ 3 mm using an SLMspectrofluorometer 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 g/ml) was reconstituted in a medium containing 40 M DMPC, 120 M C 12 E 10 , 120 mM KCl, 30 mM MOPS-K (pH 7.4 at 25°C), 1 mM MgCl 2 and then incubated with one of the following components for 10 min at 25°C: (i) 2 mM EGTA, (ii) 100 M free Ca 2ϩ , (iii) 2 mM EGTA plus 50 M (VO 4 ) 3Ϫ , or (iv) 100 M free Ca 2ϩ plus 50 M (VO 4 ) 3Ϫ . 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.
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 [ 125 I]TID-PC/16 in the PMCA intermediates E 1 Ca and E 1 PCa. The crystal structures used for comparison were those of E 1 ⅐2Ca (Protein Data Bank code 1su4) (38) and Ca 2 E 1 P (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) 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.

Effect of Vanadate and Lanthanum on PMCA Ca 2ϩ -ATPase
Activity-Ca 2ϩ -ATPase activity of purified PMCA was measured in the presence of 100 M free Ca 2ϩ 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 (VO 4 ) 3Ϫ or La III ), V 0 is the Ca 2ϩ -ATPase activity in the absence of inhibitor, and K I is the concentration of inhibitor needed for halfmaximal effect. The value of K I for (VO 4 ) 3Ϫ was 1.5 Ϯ 0.1 M, and it was similar to that reported previously (43), and the value of K I found for La III was 8.2 Ϯ 0.5 M.
Although these inhibitors have similar effects on the PMCA activity, vanadate mimics the phosphoryl group in the transition state of E 2 P, preventing the enzyme phosphorylation ( Fig.  1A, inset) (43)(44)(45), whereas La III inhibits by blocking the transition E 1 P 3 E 2 P, acting noncompetitively with respect to Ca 2ϩ 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 (VO 4 ) 3Ϫ 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 [ 125 I]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 [ 125 I]TID-PC/16 labeling of the PMCA in its major known conformational states. The E 1 Ca conformer is obtained by incubating the enzyme in the presence of Ca 2ϩ alone, whereas in the presence of 2 mM EGTA, the conformational equilibrium is shifted toward the E 2 state (in the absence of Ca 2ϩ ), whose level of [ 125 I]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 Ca 2ϩ as E 1 Ca. In the E 1 Ca conformer, the incorporation of [ 125 I]TID-PC/16 increases by 50% as compared with that of E 2 . The E 1 PCa⅐La conformer was obtained by phosphorylation in the presence of La III , whereas E 2 P was mimicked by the complexes of PMCA with (VO 4 ) 3Ϫ , both with and without Ca 2ϩ . Fig. 2  In order to test these hypotheses, we measured the specific incorporation of [ 125 I]TID-PC/16 as a function of vanadate concentration (Fig. 3A) in media with (closed circles) and without (open circles) Ca 2ϩ . It can be seen that in the latter case,   Table 1 (Reaction iii). Specific incorporation of the probe in the presence of 2 mM EGTA was taken as 100%. specific incorporation is independent of [(VO 4 ) 3Ϫ ], indicating that the E 2 and the E 2 P analog conformations have similar areas accessible to lipids. The average value of the specific incorporation is around 100% (99.3 Ϯ 0.5%; Table 1). In the presence of Ca 2ϩ , 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 [(VO 4 ) 3Ϫ ], where PC is the specific incorporation of [ 125 I]TID-PC/16; [X] is the concentration of vanadate; PC 0 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 E 2 V i and E 1 Ca were two mutually exclusive species in equilibrium, the addition of enough vanadate should bring the entire enzyme to the E 2 V i state. We therefore conclude that there exists a ternary complex, E*V i Ca, which is confirmed by results of titration by Ca 2ϩ of E 2 V i (Fig. 3B) (Fig. 3B) and the addition of enough vanadate to the E 1 Ca complex (Fig.  3A, closed circles) lead to the same ternary complex, E*V i Ca. These results suggest that, despite the apparent lack of effect of vanadate in Fig. 3A (open circles), the addition of Ca 2ϩ reveals that E 2 and E 2 V i are different conformers of the pump. From the results in Fig. 3, we postulate the existence of two phosphoenzyme analogs with vanadate: E 2 V i in the absence of calcium and E*V i Ca 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 i Ca 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 Ca 2ϩ , 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 M ATP plus 50 M La III . As expected, experimental data for each condition can be described by a rectangular hyperbola as a function of [Ca 2ϩ ]. 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 Ca 2ϩ .  The apparent affinity for Ca 2ϩ 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 E 1 Ca but also in the E*V i Ca 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 i Ca to form a quaternary complex, E*V i CaATP.
Effects of ATP-In addition to its role as substrate in the phosphorylation of the E 1 state, ATP also operates in a non-phosphorylating mode (modulatory ATP), enhancing the rates of the steps involved in phosphoenzyme turnover (E 1 P 3 E 2 P and E 2 P 3 E 2 ) as well as the E 2 3 E 1 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. E 1 PCa State-In order to study the effect of ATP on the conformation of the transmembrane domain during formation of E 1 PCa, we measured the specific incorporation of [ 125 I]TID-PC/16 into PMCA as a function of [ATP] in the presence of La III and Ca 2ϩ (Fig. 5). In the initial condition, PMCA is in the E 1 Ca conformation (46,47), and as ATP concentration increases, the reaction is shifted toward the formation of the phosphorylated intermediate, E 1 PCa⅐La (see Fig. 1B, inset). Although ATP can also bind to this intermediate with low affinity (55), only marginal effects in specific incorporation of [ 125 I]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 M. However, these changes are difficult to ascertain, and it seems safer to describe the phenomenon by saying that the level of [ 125 I]TID-PC/16 incorporation remains approximately constant around the value obtained for E 1 Ca (155 Ϯ 1%).  Table 1. These results show that ATP can bind to PMCA, at micromolar concentrations, in the absence of Ca 2ϩ , causing a conformational change in the transmembrane domain. Fig. 6, B and C, shows the specific incorporation of [ 125 I]TID-PC/16 to PMCA as a function of [ATP] when the enzyme was preincubated in the presence of vanadate with and without Ca 2ϩ , 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 Ca 2ϩ 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 Ca 2ϩ .
When the pump was incubated in the absence of Ca 2ϩ and in the presence of saturating [ATP], the specific incorporation of [ 125 I]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 , and a precise value of K x could not be determined (see Table 1, Reaction ix).  Table 1 (Reactions iv, v, and viii, respectively). D, the specific incorporation of [ 125 I]TID-PC/16 to PMCA in media with 2 mM EGTA and 2 mM ATP did not vary with the concentration of (VO 4 ) 3Ϫ (continuous line), and thus K x could not be experimentally determined (see Table 1, Reaction vii).
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 Ca 2ϩ , PMCA is an equilibrium mixture of 10% E 1 and 90% E 2 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 E 2 and E 1 forms. A similar practice is used for the equilibrium between E 1 ATP and E 2 ATP, 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 i Ca is different from E 1 Ca and E 2 V i and is therefore denoted with an asterisk, as described above.
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 [ 125 I]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 Ca 2ϩ , 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 Ca 2ϩ to E 2 V i ATP, 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 Ca 2ϩ , (ii) the apparent affinity of PMCA for Ca 2ϩ decreases in the presence of vanadate, (iii) ATP does not modify the apparent affinity for Ca 2ϩ of the E 2 V i form, and (iv) the apparent affinity of E 2 V i and E*V i Ca forms for ATP were similar, meaning that Ca 2ϩ does not modify the apparent affinity for ATP of E 2 V i .

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 Ca 2ϩ , the catalytic role of ATP, because this nucleotide is rapidly hydrolyzed. Therefore, in order to obtain more information on the interaction of E 1 Ca or E 2 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.  Table 2. 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,

TABLE 2 Best fitting values of specific incorporation of [ 125 I]TID-PC/16 and dissociation constants for calcium, vanadate, and ATP predicted by the global model shown in
is the apparent dissociation constant for the probe. The best fitting values of K D and ⌬F max are shown in Table 3.
Results show that the apparent affinity of PMCA for TNP-ATP is 10-fold higher in the presence of Ca 2ϩ 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 Ca 2ϩ and was similar to that of E 1 Ca 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 Ca 2ϩ -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 E 1 Ca and E 2 , 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 ⌬F 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 cytoplas-mic domain, thus justifying the hypothesis of the existence of a different conformation, E*V i CaATP.

DISCUSSION
There are several crystal structures of the Ca 2ϩ pump of sarcoplasmic reticulum corresponding to different conformations reached during the reaction cycle (2, 3), and crystal structures of the H ϩ -ATPase (59) and of the Na ϩ /K ϩ -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 E 1 PCa form. This phenomenon is consistent with the fact that specific incorporation of [ 125 I]TID-PC/16 in the presence of Ca 2ϩ , La III , and ATP was 155.0 Ϯ 1.0%, a maximal value that is similar to that obtained in the absence of ATP. Like the phosphoenzyme level, occlusion of Ca 2ϩ 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 [ 125 I]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 Ca 2 E 1 P (our E 1 PCa) is known as 3ba6, whereas E 1 ⅐2Ca (our E 1 Ca) is represented by the structure known as 1su4. Results in Fig. 9 show  Table 3.  that most significant changes are found in helices M1-M3, with an increase of 338 Å 2 (32%) for M1, a decrease of 285 Å 2 (30%) for M2, and an increase of 131 Å 2 (12%) for M3. Thus, the overall change in ASA values when the enzyme goes from E 1 ⅐2Ca (7113.9 Å 2 ) to Ca 2 E 1 P (7349.6 Å 2 ) is 236 Å 2 , 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.
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 [ 125 I]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 E 2 in a reaction that requires Mg 2ϩ (43,44). In the absence of Ca 2ϩ , vanadate does not produce any detectable change in the specific incorporation of [ 125 I]TID-PC/16 (Fig. 3A, open circles). However, in the presence of Ca 2ϩ , increasing concentrations of vanadate produce a decrease in the specific incorporation of [ 125 I]TID-PC/16 (Fig. 3A, closed circles). A similar result was obtained when PMCA was incubated with vanadate, and increasing Ca 2ϩ concentrations were added (Fig. 3B). Inhibition of Ca 2ϩ -ATPase activity by vanadate is commonly explained as the result of its binding to the enzyme, leading to a conformation analogous to E 2 P. Early experiments on the kinetics of vanadate in SERCA showed that the inhibition is antagonized by Ca 2ϩ (44,53), a result consistent with the idea that Ca 2ϩ 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 "E 1 -like" rather than "E 2 -like." Our results confirm that in the presence of Ca 2ϩ and vanadate, PMCA forms a stable ternary complex, E*V i Ca, whose conformational state is different from that of E 2 and E 1 Ca 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 Ca 2ϩ , the ternary complex E*V i Ca could be interpreted as an intermediate state interposed between E 1 PCa and the Ca 2ϩ -free state E 2 P.
While forming the E*V i Ca complex from E 2 V i , we found that the apparent dissociation constant for Ca 2ϩ is higher than in the absence of vanadate (5.1 M versus 1 M; 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 E 2 conformation, support the conclusion that the dephosphorylation transition state of E 2 P represents a proton-occluded state. This idea arises from the fact that in contrast to E 2 , this state has a low affinity for Ca 2ϩ . Our results show that a similar state could exist for PMCA.
Ca 2ϩ Occlusion-We report here results on Ca 2ϩ 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 E 1 P and that the release of the occluded cation toward the opposite side takes place after the E 1 P to E 2 P 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 E 1 P to E 2 P, we found that Ca 2ϩ becomes occluded concomitantly with the formation of E 1 P with a stoichiometry of one Ca 2ϩ per phosphorylated enzyme unit (8). However, this experimental evidence does not exclude the possibility of occlusion of Ca 2ϩ in non-phosphorylated intermediates. In this sense, in the absence of ATP, occlusion of Ca 2ϩ in SERCA (67) and of Na ϩ 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 Ca 2ϩ 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 E 1 P to E 2 P 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 Ca 2ϩ alone. ATP can bind to the enzyme even when Ca 2ϩ is absent, but without Ca 2ϩ , the reaction cycle cannot progress. Binding of Ca 2ϩ will cause the movement of the transmembrane helices. Fig. 6 shows that ATP binds to PMCA in the absence of Ca 2ϩ , and this triggers a change in the transmembrane domain. The apparent dissociation constant for ATP measured with [ 125 I]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 E 2 V i (an E 2 P-like state) and to E*V i Ca (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 Ca 2ϩ . This is less than the 10-fold increase of the apparent affinity for ATP in SERCA. However, the structural and kinetic analyses of the E 2 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 Ca 2ϩ , 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 E 2 conformation. On the con-trary, our approach measures the actual substrate affinity through the binding of trace amounts of [ 125 I]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 E 1 Ca cannot be measured directly due to the fast setting of a steady state of transport of Ca 2ϩ 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 Ca 2ϩ , although with a higher apparent dissociation constant than that observed in the presence of Ca 2ϩ (E 1 Ca). 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 E 2 Vi and E*V i Ca states were similar to that obtained for the E 1 Ca state but 7-fold lower than for E 2 .
The maximal fluorescence intensity of the high affinity component for the binding of TNP-ATP was similar for E 2 V i and E*V i Ca states but significantly lower than that obtained for E 1 Ca. This indicates that the N-domain environment is more hydrophobic in the presence of Ca 2ϩ (E 1 Ca).
A Functional Cycle for PMCA-Scarborough (73) has observed the inadequacy of the E 1 /E 2 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 ([ 125 I]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 E 1 ⅐2Ca and Ca 2 E 1 P 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 E 1 -E 2 model is that E 2 V i , E 2 V i ATP, and E*V i CaATP, all of them intermediates that in principle would be thought of as E 2 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 E 1 Ca 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 E 2 state to circumvent the E 1 Ca conformation and to transition directly into the E 1 ATPCa 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 E 1 P(Ca)ATP and E 2 PATP⅐Ca, whose conformation is different from that of E 1 and E 2 , 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, Ca 2ϩ can be occluded in or simply bound to the enzyme. In its resting condition, PMCA would mainly exist as two intermediates in equilibrium, E 1 ATP and E 2 ATP, until a signal of Ca 2ϩ or calmodulin-Ca 2ϩ initiates the transport of the cation driven by the ATP hydrolysis.
In this study, we focus on the role of ATP in the reaction cycle, leaving aside the effect of cofactors like Mg 2ϩ and activators such as calmodulin, which are worthy of future study using the findings of this work as a basis.