Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca²⁺-ATPase Is Critical for Ca²⁺ Binding on Both Sides of the Membrane

Abstract

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.

Introduction

The Ca²⁺-ATPase² of sarco(endo)plasmic reticulum (1–3) is an ion-translocating ATPase of P-type that mediates active transport of Ca²⁺ from the cytoplasm to the endoplasmic reticulum lumen, thereby allowing rapid oscillation of Ca²⁺ during cellular activation events. Ca²⁺ pumping is achieved by means of a reaction cycle (Scheme 1) involving the formation and decomposition of an aspartyl-phosphorylated intermediate coupled to protein conformational changes that facilitate the binding of two Ca²⁺ ions at cytoplasmically facing high affinity binding sites and subsequent dissociation from luminally facing low affinity sites. Because of extensive efforts that in recent years have involved x-ray crystallography (4–7), an increasingly detailed picture of the structural changes relating to Ca²⁺ transport by the Ca²⁺ pump is steadily emerging. The Ca²⁺-ATPase consists of 10 membrane-spanning helices (M1 through M10) connecting three major cytoplasmic domains named A (actuator), P (phosphorylation), and N (nucleotide binding) and some smaller luminal loops. Transmembrane helices M4-M6 and M8 contain the residues that coordinate the two Ca²⁺ ions bound side-by-side in a binding pocket in the Ca₂E1 and Ca₂E1P states. A key element in the transport cycle is the action of the A domain, which is directly linked to M1-M3 via flexible linkers (see Fig. 1). Thus, occlusion of the Ca²⁺ ions at the high affinity sites of Ca₂E1 is achieved through an ATP-induced ∼30° tilting of the A domain and accompanying partial unfolding and bending of the N-terminal part of M1 (3). After transfer of the γ-phosphate of ATP to Asp³⁵¹, a further ∼90° rotation of the A domain during the Ca₂E1P → Ca₂E2P transition propagates to the transmembrane domain, disrupting the high affinity Ca²⁺ sites and exposing the Ca²⁺ ions to the lumen. The subsequent release of the Ca²⁺ ions to the lumen is succeeded by dephosphorylation of the aspartyl phosphate in E2P, catalyzed by the ¹⁸¹TGES phosphatase motif of the A domain (8) that has been brought into position in the catalytic site in E2P (5, 6). The cycle is completed by reversal of the A domain rotation and restoration of the cytoplasmically facing high affinity Ca²⁺ sites (E2 → E1 in Scheme 1).

SCHEME 1.

Ca²⁺-ATPase reaction cycle. Major conformational changes and substrate binding and dissociation steps are shown.

FIGURE 1.

Structural arrangement of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ in Ca²⁺-ATPase crystallized in the Ca₂E1 state (left panel) and in the E2·BeF₃⁻E2P analog state (right panel). The respective Protein Data Bank accession codes corresponding to the structures shown are 1SU4 (4) and 3B9B (7). Amino acid side chains are shown for residues discussed in the text, and relevant distances are listed in supplemental Table S1. Carbon atoms are shown in gray, nitrogen in blue, oxygen in red, and Mg²⁺ and Ca²⁺ in green. The two arrows indicate the rotation of the actuator domain and the rearrangement of M1/M2 relative to M3/M4 during the conformational transition. The phosphorylation, nucleotide binding, and actuator domains are indicated by P, N, and A, respectively.

The molecular nature and properties of the Ca²⁺ binding pocket in the membrane-spanning region of the Ca₂E1 and Ca₂E1P states are known in great detail. Little, however, is known about the exact entry and exit pathways to and from the Ca²⁺ binding pocket and the structural elements involved in extrusion of the Ca²⁺ ions to the endoplasmic reticulum lumen. The crystal structure of Ca²⁺-ATPase in the E2·BeF₃⁻ state (presumed E2P ground state analog) (7) provides some important clues. Thus, in the E2·BeF₃⁻ structure, part of the transmembrane domain near the lumen is in a considerably more open conformation compared with other crystal structures of the Ca²⁺-ATPase. The M4-residue Glu³⁰⁹, an essential residue for Ca²⁺ coordination in the occluded binding pocket of the Ca₂E1 and Ca₂E1P states (4, 7, 9), is exposed to the lumen and associated with a Mg²⁺ ion in the E2·BeF₃⁻ structure (Fig. 1). The E2·BeF₃⁻ crystals were formed in the presence of a high concentration of Mg²⁺ (50 mm) and absence of Ca²⁺, and it may be speculated that the Mg²⁺ ion is bound in place of Ca²⁺ at a luminally facing low affinity Ca²⁺ site. Ser⁷² and Glu⁹⁰ at the luminal ends of transmembrane helices M1 and M2, respectively, are positioned very close to Glu³⁰⁹ in E2·BeF₃⁻, and the Glu⁹⁰ side-chain carboxylate seems to contribute to coordination of the luminal Mg²⁺ ion. In the Ca₂E1 and Ca₂E1P states, on the other hand, Ser⁷² and Glu⁹⁰ are 10–20 Å apart from Glu³⁰⁹ and the Ca²⁺ sites due to translational movements of M1/M2 and M3/M4 relative to each other, and Ser⁷² and Glu⁹⁰ instead interact with Lys²⁹⁷ at the luminal end of M4 (Fig. 1 and supplemental Table S1.

In the present study we investigated the significance of the above-described luminal interaction networks seen in the various crystal structures of the Ca²⁺-ATPase. Thus, mutants with alterations to Ser⁷², Glu⁹⁰, and Lys²⁹⁷ were compared with wild type Ca²⁺-ATPase by analyzing the partial reactions in transient and steady-state kinetic measurements. We found that the mutations to Glu⁹⁰ affect the Ca²⁺ binding properties profoundly both on the luminal side of the membrane, where Glu⁹⁰ is situated, as well as on the cytoplasmic side. Furthermore, single-substitution of either Ser⁷² or Glu⁹⁰ with arginine gives rise to Ca²⁺ pumps with a remarkably stable E2P state, compatible with the hypothesis that the positive charge of the arginine side chain interacts directly with the luminal Ca²⁺ outlet, possibly mimicking Ca²⁺ and thereby blocking further processing of the phosphoenzyme.

EXPERIMENTAL PROCEDURES

Site-directed mutagenesis of cDNA encoding the rabbit fast twitch muscle Ca²⁺-ATPase (SERCA1a isoform) inserted into the pMT2 vector (10) was carried out using the QuikChange site-directed mutagenesis kit (Stratagene), and the mutant cDNA was sequenced throughout. To express wild type or mutant cDNA, COS-1 cells were transfected using the calcium phosphate precipitation method (11). Microsomal vesicles containing either expressed wild type or mutant Ca²⁺-ATPase were isolated by differential centrifugation (12). The concentration of expressed Ca²⁺-ATPase was determined by an enzyme-linked immunosorbent assay (13) and by determination of the maximum capacity for phosphorylation with ATP or P_i (“active site concentration”; see Ref. 14). Transport of ⁴⁵Ca²⁺ into the microsomal vesicles and ⁴⁵Ca²⁺ binding at 25 °C were measured by filtration, and the ATPase activity was determined by following the liberation of P_i (15) in the presence of 4 μm calcium ionophore A23187 to prevent inhibition caused by rebinding of Ca²⁺ to the luminally facing Ca²⁺ sites (14). Measurements of phosphorylation and dephosphorylation were generally carried out by manual mixing at 0 °C (14, 16). Transient state kinetics at 25 °C was analyzed using the Bio-Logic quench-flow module QFM-5 (Bio-Logic Science Instruments, Claix, France) with mixing protocols as previously described (17, 18). The determination of the phosphorylation level by acid quenching followed by acid SDS-polyacrylamide gel electrophoresis and quantification of the radioactivity associated with the Ca²⁺-ATPase band was carried out using the previously established procedures (14, 16).

The experiments were conducted at least twice, and average values are shown in Figs. 2–9. Generally, the complete data set (i.e. including all experimental points before averaging) was analyzed by nonlinear regression using the SigmaPlot program (SPSS, Inc.) with the equations described in the figure legends, giving the lines in the figures and the S.E. shown in the tables. To analyze the phosphorylation time courses in Fig. 2, the kinetic simulation software SimZyme was applied, allowing computation of the phosphorylation overshoot as detailed below and in previous publications (17, 18). The best fit was in this case determined manually by trial and error, comparing the computed time courses with the experimental data points for various choices of rate constants. For any choice of reaction cycle and rate constants, SimZyme solves the relevant differential equations using the fourth order Runge-Kutta numerical method and provides a graphical representation of the time dependence of the concentration of the reaction intermediates (17).

FIGURE 2.

Transient kinetics of phosphorylation of Ca₂E₁ with [γ- ³²P]ATP. Using a QFM-5 quench-flow module at 25 °C, phosphorylation was carried out by mixing the enzyme contained in microsomal vesicles suspended in 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 5 mm MgCl₂, and 100 μm CaCl₂ with an equal amount of the same medium containing [γ-³²P]ATP to produce a final concentration of 5 μm followed by acid quenching at the indicated time intervals. The lines show fits to the data obtained by computation based on the reaction scheme shown in the bottom right corner using the SimZyme program (17), giving the rate constants for Ca₂E₁ → Ca₂E₁P (k_A), Ca₂E₁P → E₂ (k_B), and E₂ → Ca₂E₁ (k_C) indicated in each panel as (k_A, k_B, k_C). The maximum phosphorylation level obtained in each case was taken as 100%.

FIGURE 3.

Rate of the Ca₂E1P → Ca₂E2P conformational transition. Mixing protocols are illustrated by the diagrams above the panels. A, shown is processing of phosphoenzyme accumulated by phosphorylation from ATP. The enzyme contained in microsomal vesicles was phosphorylated by incubation for 15 s at 0 °C in 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 5 mm MgCl₂, 1 mm EGTA, 0.955 mm CaCl₂ (giving a free Ca²⁺ concentration of 10 μm during phosphorylation), 2 μm calcium ionophore A23187, and 5 μm [γ-³²P]ATP. Dephosphorylation was then studied at 0 °C by the addition of excess EGTA (to remove Ca²⁺ and, thus, terminate phosphorylation) followed by acid quenching at the indicated times (open symbols). The lines show the best fits of a monoexponential decay function EP = EP_max·e^−kt (extracted rate constants are listed in Table 1 for the wild type, S72A, and K297A). The closed symbols represent experiments in which 1 mm ADP was added together with the EGTA chase medium. The symbol code is the same as for the corresponding open symbols. B, shown is the rate of loss of ADP sensitivity (i.e. Ca₂E1P → Ca₂E2P) for S72R and E90R. The enzyme was phosphorylated for the indicated time intervals at 0 °C in 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 5 mm MgCl₂, 50 μm CaCl₂, 2 μm calcium ionophore A23187, and 5 μm [γ-³²P]ATP followed by the addition of an equal volume of 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 5 mm MgCl₂, 10 mm EGTA, and 2 mm ADP and acid quenching 4 s later. The lines show the best fits of a monoexponential rise to maximum function EP = EP_max·(1 − e^−kt), giving the rate constants listed in Table 1 for S72R and E90R. In each case the 100% value corresponds to the steady-state level of phosphoenzyme present 10 s after the initiation of phosphorylation without the addition of EGTA and ADP.

FIGURE 4.

Rate of E2P dephosphorylation. Dephosphorylation of E2P accumulated by phosphorylation with P_i is shown. The enzyme contained in microsomal vesicles was phosphorylated with 0.5 mm ³²P_i for 10 min at 25 °C in 100 mm MES/Tris (pH 6.0), 10 mm MgCl₂, 2 mm EGTA, and 30% (v/v) dimethyl sulfoxide (the organic solvent ensuring maximal amount of phosphoenzyme). The phosphorylated enzyme was then chilled on ice, and dephosphorylation was studied at 0 °C by a 19-fold dilution into ice-cold medium containing 40 mm MOPS/Tris (pH 7.0), 10 mm KCl, 2 mm MgCl₂, 2 mm EGTA, and 0.5 mm nonradioactive P_i followed by acid quenching at the indicated time intervals. The lines show the best fits of a monoexponential decay function EP = EP_max·e^−kt, giving the rate constants listed in Table 1.

FIGURE 5.

Occlusion of ⁴⁵Ca²⁺. ADP·AlF₃⁻-induced ⁴⁵Ca²⁺ occlusion (open symbols) was accomplished by incubating the enzyme, contained in microsomal vesicles, for 30 min at 25 °C in 30 mm MOPS/Tris (pH 7.0), 100 mm KCl, 0.2 mm MgCl₂, 10 μm ⁴⁵CaCl₂, 100 μm ADP, 50 μm AlCl₃, 2 mm NaF, and 2 μm calcium ionophore A23187. For measurement of ⁴⁵Ca²⁺ occlusion in the phosphoenzyme (closed symbols), the same amount of microsomal vesicles, pre-equilibrated in 30 mm MOPS/Tris (pH 7.0), 100 mm KCl, 5 mm MgCl₂, 10 μm ⁴⁵CaCl₂, and 2 μm calcium ionophore A23187, was phosphorylated for 5 s at 25 °C with 5 μm ATP. In either case the reaction was terminated by the addition of EGTA to a final concentration of 2 mm. At the indicated times after EGTA addition, the samples were filtered through a 0.45-μm nitrocellulose membrane filter and washed rapidly four times with 2 ml of a buffer containing 50 mm MOPS/Tris (pH 7.0), 100 mm KCl, 7 mm MgCl₂, 2 mm EGTA, and 5 μm calcium ionophore A23187. The filters were then dried, and radioactivity on each filter was quantified by autoradiography. The amount of ⁴⁵Ca²⁺ specifically bound to the Ca²⁺-ATPase (indicated on the ordinate) was determined by subtracting the amount of nonspecific ⁴⁵Ca²⁺ binding measured in parallel experiments in which ADP·AlF₃⁻ and ATP were excluded from the respective reaction mixtures. The amount of Ca²⁺-ATPase-specific ADP·AlF₃⁻-induced ⁴⁵Ca²⁺ occlusion obtained at zero time was taken as 100%. S.E. is indicated by error bars. The phosphorylation state of the samples is documented in supplemental Fig. S2.

FIGURE 6.

Function of the luminal Ca²⁺ sites of the E2P state. Microsomal vesicles containing wild type or mutant Ca²⁺-ATPase were loaded with Ca²⁺ in the lumen by incubation overnight (∼18 h) on ice in 150 mm MOPS/Tris (pH 7.0), 125 mm sucrose, 75 mm KCl, and the indicated CaCl₂ concentration ([Ca²⁺]_lum). The phosphorylation by P_i was determined at 25 °C by a 50-fold dilution of the Ca²⁺-loaded vesicles into a phosphorylation medium containing 150 mm MOPS/Tris (pH 7.0), 125 mm sucrose, 77 mm KCl, 10 mm EGTA (removing Ca²⁺ from the medium outside the vesicles), and 0.5 mm ³²P_i followed by acid quenching 1 min later. The ordinate shows the results as percentage of the maximal phosphorylation level (max EP) obtained by phosphorylation with P_i under optimal conditions for E2P formation in the presence of dimethyl sulfoxide as described in the legend to Fig. 4. The lines show the best fits of the equation, EP = EP_min + (EP_∞ − EP_min)·[Ca²⁺]ⁿ/(K_0.5ⁿ + [Ca²⁺]ⁿ), to the data with EP_min accounting for the basic phosphorylation level in the absence of a Ca²⁺ gradient, and EP_∞ representing the extrapolated phosphorylation level at infinite intravesicular Ca²⁺. The K_0.5 (Ca_lum²⁺) values obtained are listed in Table 2. The bottom panel shows ³²P_i-phosphorylated Ca²⁺-ATPase bands on SDS-polyacrylamide separation gels in experiments assessing the ADP sensitivity of the phosphoenzyme accumulated with wild type and mutants S72R, E90R, and 4Gi-46/47 either under conditions of maximal E2P formation in the presence of dimethyl sulfoxide (max EP) or after phosphorylation of the Ca²⁺-loaded vesicles in the medium without dimethyl sulfoxide described above. The ADP sensitivity was studied by a 2-s incubation with 1 mm ADP at 25 °C before the acid quenching (ADP +). For comparison, the phosphorylation of samples that have not been incubated with ADP is also shown (ADP −). The samples indicated by bg represent background phosphorylation obtained in the presence of excess Ca²⁺ (absence of EGTA) in the phosphorylation medium.

FIGURE 7.

E2·BeF₃⁻ and E2·vanadate complexes of wild type and mutants S72R, E90R, and 4Gi-46/47. A and B, affinity of E2 for BeF₃⁻ and vanadate, respectively, were determined by inhibition of phosphorylation from [γ-³²P]ATP. Wild type and mutants were incubated for 30 min (A) or 60 min (B) at 25 °C followed by 10 min at 0 °C in media containing 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 2 mm EGTA, and 200 μm MgCl₂, 2 mm NaF, and varying concentrations of BeSO₄ (represented as BeF to indicate that most Be²⁺ reacts with fluoride) (A) or 5 mm MgCl₂ and varying concentrations of orthovanadate (vanadate) (B). The degree of inhibition was then determined by phosphorylation for 10 s at 0 °C with 5 μm [γ-³²P]ATP after the addition of CaCl₂ to a final concentration of 2.1 mm (A) or 2.5 mm (B) (giving a free Ca²⁺ concentration of 100 and 500 μm, respectively) and MgCl₂ to a final concentration of 5 mm. The level of phosphoenzyme obtained in the absence of inhibitor was taken as 100%. The lines show the best fits of the Hill equation for inhibition, EP = EP_max·(1 − [L]ⁿ/(K_0.5ⁿ + [L]ⁿ)), in which L is either BeF or vanadate, giving the following K_0.5 values. For BeF₃⁻: wild type, 0.58 ± 0.03 μm; S72R, 0.55 ± 0.03 μm; E90R, 7.8 ± 1.1 μm; 4Gi-46/47, 0.74 ± 0.10 μm. For vanadate: wild type, 0.13 ± 0.006 μm; S72R, 0.074 ± 0.003 μm; E90R, 0.80 ± 0.05 μm; 4Gi-46/47, 0.12 ± 0.021 μm. C and D, rates of Ca²⁺-induced dissociation of E2·BeF₃⁻ and E2·vanadate, respectively. The inhibited enzyme complexes were formed by incubation for 30 min at 25 °C followed by 10 min at 0 °C in media containing 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 2 mm EGTA, 5 μm calcium ionophore A23187, and 200 μm MgCl₂, 2 mm NaF, and 100 μm BeSO₄ (C) or 5 mm MgCl₂ and 50 μm orthovanadate (D). Enzyme-inhibitor dissociation at 0 °C was then initiated by the addition of 2.5 mm CaCl₂ (giving a free Ca²⁺ concentration of 500 μm), and after the indicated time intervals, the level of reactivated enzyme was assessed by phosphorylation for 10 s at 0 °C in the presence of 5 μm [γ-³²P]ATP, and MgCl₂ at a final concentration of 5 mm. The level of phosphoenzyme obtained in the absence of inhibitor was taken as 100%. The lines show the best fits of a monoexponential rise to maximum function EP = EP_max·(1 − e^−kt), giving rate constants for dissociation of the complexes as follows. For E2·BeF₃⁻: wild type, 0.16 ± 0.014 min⁻¹; for S72R, E90R, and 4Gi-46/47, no Ca²⁺-induced dissociation of E2·BeF₃⁻ was detected within the time frame of the experiment. E2·vanadate: wild type, 0.22 ± 0.026 min⁻¹; S72R, 0.04 ± 0.004 min⁻¹; E90R, 0.008 ± 0.007 min⁻¹; 4Gi-46/47, 0.52 ± 0.057 min⁻¹. The symbol code for all panels is indicated in panel A.

FIGURE 8.

Ca²⁺ dependence of phosphorylation from [γ-³²P]ATP. The enzyme was pre-equilibrated at 0 °C in 40 mm MOPS/Tris (pH 7.0), 80 mm KCl, 5 mm MgCl₂, 1 mm EGTA and various concentrations of CaCl₂ to obtain the free Ca²⁺ concentrations indicated followed by phosphorylation for 15 s at 0 °C with 5 μm [γ-³²P]ATP and subsequent acid quenching. The lines show the best fits of the Hill equation, EP = EP_max·[Ca²⁺]ⁿ/(K_0.5ⁿ + [Ca²⁺]ⁿ), to the data, giving the K_0.5 (Ca_cyt²⁺) values listed in Table 2.

FIGURE 9.

Kinetics of binding and dissociation of Ca²⁺ at cytoplasmically facing sites. Quench-flow experiments were carried out with mixing protocols as illustrated by the diagrams above the panels using a QFM-5 module at 25 °C. Left panels, the Ca²⁺ binding transition taking place in the presence of 40 mm MES/Tris (pH 6.0), 80 mm KCl, and 100 μm free Ca²⁺ is shown. The enzyme, preincubated in 40 mm MES/Tris (pH 6.0), 80 mm KCl, and 2 mm EGTA (to accumulate E2), was mixed with an equal volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, and 2.2 mm CaCl₂. At the indicated time intervals, the amount of phosphorylatable Ca₂E1 was determined by adding the double volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, 10 mm MgCl₂, 1 mm EGTA, 10 μm [γ-³²P]ATP, and 1.1 mm CaCl₂ followed by acid quenching 34 ms later. To obtain the point corresponding to zero time, the enzyme was preincubated in 40 mm MES/Tris (pH 6.0), 80 mm KCl, and 2 mm EGTA and mixed with an equal volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, 10 mm MgCl₂, 10 μm [γ-³²P]ATP, and 2.2 mm CaCl₂ followed by acid quenching 34 ms later. The lines represent the best fits of a monoexponential rise to maximum function with an initial offset, EP = EP_min + (EP_max − EP_min)·(1 − e^−kt), giving the rate constants of E2 → Ca₂E1 listed in Table 2. In each case the EP_max extracted from the fit was taken as 100%. Right panels, Ca²⁺ dissociation from the high affinity Ca²⁺ sites of Ca₂E1 back toward the cytoplasm is shown. The enzyme, preincubated in 40 mm MES/Tris (pH 6.0), 80 mm KCl, 5 mm MgCl₂, and 100 μm CaCl₂ (to accumulate Ca₂E1), was mixed with an equal volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, 5 mm MgCl₂, and 4 mm EGTA to initiate Ca²⁺ dissociation. At the indicated time intervals, the amount of phosphorylatable Ca₂E1 remaining was determined by adding the double volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, 5 mm MgCl₂, 2 mm EGTA, and 10 μm [γ-³²P]ATP followed by acid quenching 34 ms later. To obtain the point corresponding to zero time, 4 mm EGTA was replaced by 100 μm CaCl₂. The lines show the best fits of a monoexponential decay function EP = EP_max·e^−kt, giving the rate constants of Ca²⁺ dissociation from Ca₂E1 listed in Table 2. In each case, the phosphorylation level corresponding to zero time was taken as 100%.

RESULTS

Expression and Assays of Overall Function

To study the potential roles of Glu⁹⁰ and Ser⁷² for Ca²⁺ interaction at the luminal ion gate in the E2P state as well as to address the significance of the Glu⁹⁰-Lys²⁹⁷-Ser⁷² interaction network seen in the Ca₂E1 structures, we produced six point mutants with alterations to Ser⁷², Glu⁹⁰, and Lys²⁹⁷ of Ca²⁺-ATPase. Glu⁹⁰ was substituted with alanine, leucine, and arginine, Ser⁷² was substituted with alanine and arginine, and Lys²⁹⁷ was substituted with alanine. The five mutants with alterations to Ser⁷² or Glu⁹⁰ could be expressed in COS-1 cells to levels comparable with that obtained for the wild type, whereas the expression level in COS-1 cells of mutant K297A was generally only ∼30% that obtained with wild type (supplemental Table S2). A reduced expression level of mutants with alterations to Lys²⁹⁷ has previously been reported by Chen et al. (19). Thus, K297G was not expressed (despite wild type-like levels of mRNA transcript in the cells), and K297F yielded low expression levels, whereas K297M, K297R, and K297E were expressed at wild type-like levels (19). Fortunately, the expression level of K297A was sufficiently high for us to carry out reliable functional measurements.

To assess the overall function of the mutants, the rate of ATP hydrolysis was measured at 37 °C in the presence of 5 mm MgATP and 3 μm free Ca²⁺. The catalytic turnover rates (ATP hydrolysis activity per enzyme molecule (14)) of S72A and K297A were wild type-like (80 and 87%, respectively, that obtained with wild type), whereas the remaining mutants displayed little or no ATPase activity (19% of the wild type rate for E90A and <10% for S72R, E90L, and E90R, supplemental Table S2). The results of measuring the rate of ⁴⁵Ca²⁺ transport into the microsomal vesicles at 37 °C with 5 mm MgATP present were similar to those obtained in the ATPase activity assay, i.e. wild type-like Ca²⁺ transport rates for S72A and K297A and much less for the remaining mutants (supplemental Table S2).

Transient State Kinetics of Phosphorylation of Ca₂E1 by ATP

To determine the rate of phosphorylation of the Ca²⁺-bound enzyme from ATP and to obtain an initial overview of the effects of the mutations on the succeeding partial reactions of the pump cycle, we used rapid kinetic instrumentation at 25 °C to examine the transient state kinetics of phosphorylation at 5 μm [γ-³²P]MgATP at pH 7 of enzyme pre-equilibrated with Ca²⁺ (Fig. 2). Under these conditions, the wild type and some of the mutants displayed overshoots of phosphorylation that could be reproduced by computation based on the simplified three-intermediate reaction cycle shown in the bottom right corner of Fig. 2 (for a detailed description of this approach as used previously in case of wild type and other mutants, see Refs. 17 and 18). We find that the best fit to the wild type data is obtained for the rate constants k_A ∼ 50 s⁻¹ for phosphorylation of Ca₂E1, k_B = 6 s⁻¹ for phosphoenzyme processing (i.e. Ca₂E1P → Ca₂E2P → E2P → E2), and k_C = 20 s⁻¹ for the Ca²⁺ binding transition of the dephosphoenzyme (i.e. E2 → Ca₂E1). As seen in Fig. 2, for all six mutants a good fit to the data could be obtained with a phosphorylation rate constant (k_A) similar to that of wild type. Mutants E90A and E90L displayed large overshoots of phosphorylation reflecting accumulation of a considerable amount of dephosphoenzyme at steady state. In such cases where the phosphorylation overshoot is large, fairly accurate values of the rate constants k_B and k_C can be extracted from the computational analysis. Thus, the increased levels of dephosphoenzyme accumulating at steady state with E90A and E90L were found to result from 2-fold increased rates of phosphoenzyme processing (k_B) combined with 10- and 20-fold reduced rates of the Ca²⁺ binding transition of the dephosphoenzyme (k_C), respectively (Fig. 2). Direct measurements of the rates of the partial reactions involved in phosphoenzyme and dephosphoenzyme processing are presented below.

The Ca₂E1P → Ca₂E2P Conformational Transition

After the phosphorylation of Ca₂E1 by ATP, the subsequent turnover of the phosphoenzyme occurs in at least three distinct steps comprising the Ca₂E1P → Ca₂E2P conformational transition, Ca²⁺ dissociation from the Ca²⁺ sites (now opening toward the luminal side and exhibiting low affinity), i.e. Ca₂E2P → E2P, and the dephosphorylation of the Ca²⁺-free E2P intermediate (E2P → E2).

The time course of forward processing of the phosphoenzyme formed by phosphorylation of Ca²⁺-saturated enzyme with [γ-³²P]ATP was determined at 0 °C by chasing the accumulated phosphoenzyme with an excess of the Ca²⁺ chelator EGTA (terminating phosphorylation by removal of Ca²⁺ from Ca₂E1) followed by acid quenching at serial times (Fig. 3A, open symbols). Mutants S72A and K297A displayed phosphoenzyme turnover rates differing less than 2-fold from that of the wild type, whereas the phosphoenzyme turnover in mutants S72R and E90R was extremely slow (no dephosphorylation detected within the time frame of the experiment). Similar direct measurements of phosphoenzyme turnover could not be performed for E90A and E90L because of their low steady-state phosphoenzyme levels (cf. Fig. 2), but as noted above, reliable rates of phosphoenzyme processing could be extracted by computational analysis of the time course of phosphorylation from ATP measured by rapid quench instrumentation at 25 °C (Fig. 2), showing that the phosphoenzyme processing was ∼2-fold accelerated for E90A and E90L relative to wild type, in sharp contrast to the block of phosphoenzyme turnover observed for S72R and E90R.

The phosphoenzyme intermediates E1P and E2P can be distinguished experimentally by their differential sensitivities to ADP, E1P being able to transfer the phosphoryl group back to ADP forming ATP and E2P being insensitive to added ADP and dephosphorylating only in the forward direction by hydrolysis of the phosphoryl bond. The ADP sensitivity of the phosphoenzyme accumulated at steady state was determined by a 5-s chase with excess EGTA and 1 mm ADP (Fig. 3A, closed symbols). For the wild type, the ADP chase resulted in a complete disappearance of the phosphoenzyme, indicating that all the accumulated phosphoenzyme belonged to the ADP-sensitive E1P type, as expected because the Ca₂E1P → Ca₂E2P transition is known to be the rate-limiting step in the phosphoenzyme turnover of the wild type. The phosphoenzyme accumulated for mutant S72A was also exclusively E1P, and for mutant K297A ∼90% of the phosphoenzyme was E1P (∼10% phosphoenzyme left after ADP chase, Fig. 3A, closed diamond). Thus, for the wild type, S72A, and K297A, the rate constant extracted from fits of a monoexponential function to the data shown in Fig. 3A, open symbols, reflects the rate of the Ca₂E1P → Ca₂E2P transition. In contrast, the phosphoenzyme accumulated for S72R and E90R was almost exclusively ADP-insensitive (Fig. 3A, closed triangles), showing that the block of phosphoenzyme turnover in these two mutants results from a block of a step in the reaction sequence subsequent to the Ca₂E1P → Ca₂E2P conformational transition, i.e. a step in the sequence Ca₂E2P → E2P → E2. For these two mutants it was feasible to determine the rate of Ca₂E1P → Ca₂E2P by following the formation of ADP-insensitive phosphoenzyme during incubation with [γ-³²P]ATP (Fig. 3B), assuming that Ca₂E1 → Ca₂E1P is as rapid as in the wild type (documented in Fig. 2, k_A) and, therefore, not rate-limiting for the reaction sequence Ca₂E1 → Ca₂E1P → Ca₂E2P. Hence, the data in Fig. 3B were obtained by incubating Ca²⁺-saturated enzyme with [γ-³²P]ATP for varying time intervals under conditions similar to those used in Fig. 3A followed by determination of the amount of ADP-insensitive phosphoenzyme accumulated (i.e. the sum of Ca₂E2P and E2P) by adding 1 mm ADP for 4 s to remove any Ca₂E1P present before acid quenching. The rate of the Ca₂E1P → Ca₂E2P transition obtained for S72R and E90R in this way was close to 10-fold faster than the rate of Ca₂E1P → Ca₂E2P obtained with the wild type as illustrated in Fig. 3A under the same buffer and temperature conditions. A summary of the relative Ca₂E1P → Ca₂E2P rate constants derived from the data of Figs. 2 and 3 is presented in Table 1.

TABLE 1.

Summary of relative rate constants of the phosphoenzyme processing steps

The values extracted by regression analysis or computation are shown relative to that of wild type. The S.E. is indicated, and the value n refers to the number of experimental data points on which the analysis is based.

	Ca2E1P → Ca2E2P	E2P → E2a
Wild type	1.0 ± 0.05 (n = 40b, 52c)	1.0 ± 0.04 (n = 89)
S72A	0.74 ± 0.04 (n = 16)b	0.71 ± 0.06 (n = 18)
S72R	11 ± 0.6 (n = 17)d	∼0 (n = 25)
E90A	2.2 (n = 54)c	2.8 ± 0.16 (n = 18)
E90L	1.7 (n = 54)c	1.8 ± 0.09 (n = 18)
E90R	7.8 ± 0.6 (n = 17)d	∼0 (n = 26)
K297A	1.6 ± 0.03 (n = 16)b	0.29 ± 0.01 (n = 18)
E309Q	NDe	0.065 ± 0.001 (n = 17)

^a Rate constant of dephosphorylation of phosphoenzyme formed by phosphorylation with P_i (experimental details are in the legend to Fig. 4) relative to that obtained with wild type (k = 0.11 s⁻¹).

^b Rate constant of dephosphorylation of Ca₂E1P formed by phosphorylation of Ca₂E1 with ATP (at 0 °C, experimental details are in the legend to Fig. 3A) relative to that obtained with the wild type (k = 0.10 s⁻¹).

^c For mutants E90A and E90L, the rate constant of Ca₂E1P → Ca₂E2P was extracted by analysis of the time course of phosphorylation by ATP (at 25 °C, experimental details in the legend to Fig. 2) using the SimZyme simulation software (see “Experimental Procedures” and Ref. 17). Because the dephosphorylation of E2P in mutants E90A and E90L is at least as fast as that of wild type (cf. Fig. 4 and the right column of this table), it can be assumed that Ca₂E1P → Ca₂E2P is rate limiting for the phosphoenzyme turnover. The rate constants listed are relative to that obtained with the wild type (k_B = 6 s⁻¹). Because SimZyme does not provide a regression analysis, no S.E. is shown in this case.

^d For mutants S72R and E90R the rate constant of Ca₂E1P → Ca₂E2P was determined by measuring the rate of loss of ADP sensitivity of the phosphoenzyme formed by phosphorylation with ATP (at 0 °C, experimental details are in the legend to Fig. 3B). The rate constants listed in the table are relative to that obtained with wild type in the experiments corresponding to Fig. 3A (directly comparable to those of Fig. 3B because the conditions during phosphoenzyme processing are identical in the two experimental setups).

^e ND, not determined, because E309Q does not undergo phosphorylation under the present conditions for reaction with ATP (21).

The Stability of E2P and Ca₂E2P

All six mutants were able to form E2P phosphoenzyme by phosphorylation of Ca²⁺-deprived enzyme from P_i in the backward direction of normal turnover and displayed apparent affinities for P_i at least as high as that of the wild type (supplemental Fig. S1). To measure the rate of E2P dephosphorylation (i.e. the forward reaction E2P → E2), E2P was formed in the presence of a saturating concentration of ³²P_i and subsequently chased by dilution into a buffer containing excess nonradioactive P_i followed by acid quenching at serial times (Fig. 4). Mutants S72A and K297A displayed dephosphorylation rates 1.4- and 3.4-fold lower than that of the wild type, respectively, whereas the dephosphorylation rates of mutants E90A and E90L were enhanced (2.8- and 1.8-fold, respectively) relative to wild type. Importantly, the dephosphorylation of E2P was completely blocked in mutants S72R and E90R, with no phosphoenzyme decay detected within the time frame of the experiment (Fig. 4 and Table 1).

Such a block of dephosphorylation of E2P has previously been noted for mutations of some of the residues involved in Ca²⁺ binding from the cytoplasmic side in the E1 conformation, which in E2P are candidates for luminal interaction sites for Ca²⁺ or protons to be countertransported such as Glu³⁰⁹ (9, 20, 21). For direct comparison with S72R and E90R, we also conducted a dephosphorylation experiment with mutant E309Q under the presently applied conditions (Fig. 4). In accordance with the previous result (20, 21), the dephosphorylation of E2P was also markedly slowed in E309Q, although not quite as much as seen for S72R and E90R (Fig. 4 and Table 1).

For S72R and E90R, the results summarized in Table 1 demonstrate that the first and the last reaction step in the sequence Ca₂E1P → Ca₂E2P → E2P → E2 is enhanced and inhibited, respectively. It is, however, unclear from these data how the intermediate reaction step Ca₂E2P → E2P, i.e. the dissociation of Ca²⁺ from the luminally exposed Ca²⁺ sites, is affected by the mutations. To answer the question of whether the inserted arginine residue inhibits the luminal Ca²⁺ dissociation such that Ca₂E2P → E2P contributes to rate limitation of phosphoenzyme turnover, we examined whether Ca²⁺ remained tightly bound (“occluded”) in the accumulated phosphoenzyme (Fig. 5). A mutant previously studied by Daiho et al. (22), with four glycines inserted in the A/M1 linker between Gly⁴⁶ and Lys⁴⁷ (4Gi-46/47), was included as a positive control in which the luminal Ca²⁺ dissociation step Ca₂E2P → E2P is blocked. For reference we first demonstrated the ability of the mutants to occlude Ca²⁺ in the stable Ca₂E1P-like Ca₂E1·AlF₃⁻·ADP complex. The enzyme was incubated with ⁴⁵Ca²⁺ in the presence of AlF₃⁻ and ADP followed by a chase with excess EGTA, removing free Ca²⁺ from the medium (on both sides of the membrane due to the presence of calcium ionophore). At varying time intervals after the chase, the samples were rapidly filtered and washed to determine the amount of ⁴⁵Ca²⁺ associated with the Ca²⁺-ATPase. As seen in Fig. 5, open symbols, S72R, E90R, and 4Gi-46/47 were all able to form a stable Ca²⁺-occluded Ca₂E1·AlF₃⁻·ADP complex similar to the wild type (23, 24). Then in another set of experiments without AlF₃⁻ and ADP, the enzyme was phosphorylated by ATP in the presence of ⁴⁵Ca²⁺ followed by the same EGTA chase and filtration procedure as described above (closed symbols in Fig. 5). In parallel experiments using [γ-³²P]ATP, the phosphoenzyme decay was followed under comparable conditions at 25 °C (supplemental Fig. S2). In the wild type, the phosphoenzyme decayed too rapidly for any Ca²⁺ occlusion in the phosphoenzyme to be detected by the manual filtration technique used here. However, in mutants 4Gi-46/47 and E90R, the phosphoenzyme was very stable even at 25 °C (supplemental Fig. S2), and reliable measurements of Ca²⁺ occlusion in the phosphoenzyme could easily be performed. For 4Gi-46/47, a high level of ⁴⁵Ca²⁺ occlusion similar to that obtained in the presence of ADP·AlF₃⁻ was seen after the phosphorylation by ATP, in accordance with the previously described block of luminal Ca²⁺ dissociation from the Ca₂E2P state in this mutant (22). Notably, the E2P phosphoenzyme of E90R was not in the ⁴⁵Ca²⁺-occluded state, unlike that of 4Gi-46/47 (Fig. 5), thus indicating that in E90R the luminal Ca²⁺ dissociation does not contribute to the rate limitation in the Ca₂E2P → E2P → E2 reaction sequence. Likewise, no ⁴⁵Ca²⁺ occlusion could be detected in S72R during the phosphorylation experiment, but in the latter case the conclusion is uncertain, because much of the phosphoenzyme may have disappeared during the measurement due to the higher dephosphorylation rate of S72R (compare Fig. 5 with supplemental Fig. S2).

Function of the Luminally Exposed Low Affinity Ca²⁺ Sites

To examine the accessibility and affinity of the luminally exposed Ca²⁺ sites in E2P, we applied a recently devised assay (25) that takes advantage of the well known Ca²⁺ gradient-dependent formation of E2P from P_i (26, 27). Microsomal vesicles containing wild type or mutant Ca²⁺-ATPase were loaded passively with various Ca²⁺ concentrations by overnight incubation and then diluted into a medium containing ³²P_i and EGTA. The latter chelator was present to remove Ca²⁺ in the medium outside the vesicles, thereby preventing Ca²⁺ binding from the outside (corresponding to the cytoplasmic side), which would inhibit phosphorylation by P_i by shifting the E2-E1 equilibrium away from E2 (cf. Scheme 1). On the contrary, a high luminal Ca²⁺ concentration facilitates the reaction of the wild type Ca²⁺-ATPase with P_i, even under conditions (neutral pH, presence of K⁺) that in the absence of a Ca²⁺ gradient favor dephosphorylation (27). A K_0.5 value of 7.5 mm for Ca²⁺ activation from the luminal side was obtained for the wild type from the dependence of the phosphorylation by P_i on the luminal Ca²⁺ concentration (Fig. 6). For mutants S72A and K297A the apparent Ca²⁺ affinity on the luminal side determined in this way was wild type-like, whereas E90A and E90L displayed markedly reduced apparent Ca²⁺ affinities (K_0.5 increased ∼3- and ≥5-fold, respectively). Mutant 4Gi-46/47 displayed a 9-fold increased apparent Ca²⁺ affinity (K_0.5 reduced) relative to wild type (Fig. 6 and Table 2), which likely is associated with the high stability of the Ca₂E2P state in this mutant (22). Remarkably, S72R and E90R were maximally phosphorylated even at 0.01 mm luminal Ca²⁺, where almost no phosphoenzyme is obtained with wild type, and remained maximally phosphorylated throughout the range of luminal Ca²⁺ concentrations applied in the experiment (Fig. 6). As further seen in Fig. 6, E309Q likewise showed a rather constant phosphorylation level independent of the luminal Ca²⁺ concentration. The phosphorylation level of E309Q was lower than that of S72R and E90R, consistent with the slightly higher rate of dephosphorylation of E2P in E309Q as compared with S72R and E90R (Fig. 4).

TABLE 2.

Summary of apparent affinities and rate constants relating to Ca²⁺ interaction

The values extracted by regression analysis are shown relative to that of wild type. The S.E. is indicated, and the value n refers to the number of experimental data points on which the analysis is based.

	K0.5 (Calum2+)a	K0.5 (Cacyt2+)b	Rate constant of Ca2+ binding transition E2 → Ca2E1c	Rate constant of Ca2+ dissociation from Ca2E1d
Wild type	1.0 ± 0.04 (n = 62)	1.0 ± 0.03 (n = 45)	1.0 ± 0.05 (n = 62)	1.0 ± 0.06 (n = 70)
S72A	0.9 ± 0.11 (n = 16)	0.67 ± 0.02 (n = 18)	1.2 ± 0.08 (n = 32)	0.46 ± 0.02 (n = 32)
S72R	(n = 14)e	0.83 ± 0.03 (n = 27)	0.48 ± 0.04 (n = 32)	0.64 ± 0.03 (n = 34)
E90A	2.6 ± 0.46 (n = 24)	5.9 ± 0.43 (n = 18)	0.005 ± 0.011 (n = 32)	0.042 ± 0.002 (n = 36)
E90L	≥5 (n = 16)e	7.6 ± 0.61 (n = 18)	0.006 ± 0.007 (n = 29)	0.18 ± 0.014 (n = 34)
E90R	(n = 16)e	3.9 ± 0.28 (n = 18)	0.56 ± 0.05 (n = 30)	2.4 ± 0.17 (n = 35)
K297A	1.0 ± 0.10 (n = 31)	1.8 ± 0.07 (n = 18)	2.2 ± 0.05 (n = 14)	0.35 ± 0.014 (n = 18)
4Gi-46/47	0.11 ± 0.01 (n = 32)	NDf	NDf	NDf
E309Q	(n = 16)e	NDg	NDg	NDg

^a K_0.5 values for Ca²⁺ activation from the luminal side of the membrane of phosphorylation from P_i, relative to that obtained with wild type (7.5 mm). For experimental details, see the legend to Fig. 6.

^b K_0.5 values for Ca²⁺ activation from the cytoplasmic side of the membrane of phosphorylation from ATP, relative to that obtained with wild type (0.9 μm). For experimental details, see the legend to Fig. 8.

^c Rate constants relative to that obtained with wild type (0.9 s⁻¹). For experimental details, see the legend to Fig. 9, left panels.

^d Rate constants relative to that obtained with wild type (2.7 s⁻¹). For experimental details, see the legend to Fig. 9, right panels.

^e Extraction of an apparent affinity for luminal Ca²⁺ was not feasible for mutants S72R, E90R, and E309Q, because the phosphoenzyme levels of these three mutants were rather independent of the luminal Ca²⁺ concentration, and for E90L the affinity was too low for an accurate determination (cf. Fig. 6).

^f ND, not determined in the present study. See Ref. 22 for a detailed functional study of mutant 4Gi-46/47.

^g ND, not determined, because E309Q does not undergo phosphorylation under the present conditions for reaction with ATP (21).

The phosphoenzyme that accumulates for the wild type upon phosphorylation of Ca²⁺-loaded vesicles with P_i is ADP-sensitive Ca₂E1P, due to the equilibrium E2P + 2Ca_lum²⁺ ↔ Ca₂E2P ↔ Ca₂E1P (25). In contrast, as demonstrated by the experiment shown in the bottom panel of Fig. 6, the phosphoenzyme accumulated for mutants S72R, E90R, and 4Gi-46/47 remained ADP-insensitive even when the vesicles had been loaded with high amounts of Ca²⁺, implying either a block of Ca²⁺ binding at the luminal Ca²⁺ sites and/or of the Ca₂E2P → Ca₂E1P transition.

The Stability and Ca²⁺ Sensitivity of E2P-like Analog States

We furthermore studied the properties of E2 in complex with the two inhibitory phosphoryl analogs BeF₃⁻ and vanadate. E2·BeF₃⁻ is believed to mimic the E2P ground state (7, 28), whereas vanadate is generally thought to capture the ATPase in a state similar to the transition state of E2P dephosphorylation (29). Both inhibitors bind to the Ca²⁺-deprived E2 state in a slow reaction that requires Mg²⁺. Panels A and B of Fig. 7 show the BeF₃⁻ and vanadate dependences, respectively, of the inhibition of the wild type and mutants S72R, E90R, and 4Gi-46/47 by reaction with the phosphoryl analog. S72R and 4Gi-46/47 deviated only marginally from the wild type with respect to the apparent affinities for BeF₃⁻ and vanadate. In contrast, E90R displayed markedly reduced apparent affinities for both BeF₃⁻ and vanadate (13- and 6-fold, respectively) relative to the wild type.

The E2·BeF₃⁻ complex is destabilized by luminal Ca²⁺ binding at the transport sites in the luminally exposed configuration (28), unlike the complexes of the E2 state of Ca²⁺-ATPase with AlF₃⁻ and MgF₄²⁻ in which the transport sites are occluded inside the transmembrane domain and inaccessible to Ca²⁺ present on either side of the membrane (5, 6). E2·vanadate is also sensitive to Ca²⁺, although it seems that the dissociation of vanadate results from Ca²⁺ binding at the cytoplasmically exposed Ca²⁺ sites (30). Panels C and D of Fig. 7 show the rates of dissociation of E2·BeF₃⁻ (Fig. 7C) and E2·vanadate (Fig. 7D) for the wild type and mutants S72R, E90R, and 4Gi-46/47 after supplementation with 500 μm excess Ca²⁺ in the presence of calcium ionophore A23187 (to allow Ca²⁺ access to the luminally exposed Ca²⁺ sites). The E2·BeF₃⁻ complexes of S72R, E90R, and 4Gi-46/47 were, in contrast to that of the wild type, completely insensitive to Ca²⁺. The dissociation of E2·vanadate was also markedly slowed for S72R and E90R, whereas for mutant 4Gi-46/47 the dissociation of E2·vanadate was 2.4-fold accelerated relative to wild type.

Function of the Cytoplasmically Facing High Affinity Ca²⁺ Sites

Phosphorylation of the Ca²⁺-ATPase by ATP depends on the binding of Ca²⁺ at the high affinity cytoplasmically exposed sites of the E1 form. Fig. 8 shows the Ca²⁺ concentration dependence of the steady-state level of phosphorylation from ATP. Mutants S72A, S72R, and K297A deviated less than 2-fold from the wild type with respect to the K_0.5 of Ca²⁺ activation. The apparent Ca²⁺ affinities of E90A, E90L, and E90R were more markedly affected, being 6-, 8-, and 4-fold reduced, respectively, relative to wild type (K_0.5 increased, see Table 2).

The left panels of Fig. 9 show the kinetics of the Ca²⁺ binding transition measured by use of rapid kinetic instrumentation at 25 °C. This transition comprises Ca²⁺ binding to the dephosphoenzyme with accompanying enzyme conformational changes, i.e. reactions E2 → E1 → CaE1 → Ca₂E1 in Scheme 1. The assay takes advantage of the fact that only the Ca₂E1 state is able to be phosphorylated by ATP (31). Ca²⁺-deprived enzyme is incubated with Ca²⁺ for varying time intervals (t in the mixing protocol at the top left of Fig. 9), and the amount of phosphorylatable Ca₂E1 is determined for each time interval by a 34-ms incubation with [γ-³²P]ATP followed by acid quenching (32). The mutations S72R and E90R reduced the rate of the Ca²⁺ binding transition 2-fold, whereas mutation K297A led to a 2-fold enhanced rate. Mutant S72A displayed a wild type-like rate of Ca²⁺ binding. Importantly, the rate of the Ca²⁺ binding transition was extremely slow in mutants E90A and E90L, being reduced by more than 2 orders of magnitude relative to wild type. This is in keeping with the large phosphorylation overshoots seen for E90A and E90L in Fig. 2 as a result of the accumulation of dephosphoenzyme at steady state and with the resulting small values for the derived rate constants k_B. It should be emphasized that the rate constant of the Ca²⁺ binding transition extracted from the data in Fig. 9 is independent of the rate of phosphorylation of Ca₂E1 (32). The data in Fig. 2 indicate that the phosphorylation of Ca₂E1, unlike the Ca²⁺ binding transition, proceeds with roughly the same rate constant (k_A ∼50 s⁻¹) in mutants and wild type. To examine whether this is the case for E90A and E90L also under the conditions (pH 6.0) applied for Fig. 9, we repeated for these mutants the phosphorylation of Ca₂E1 corresponding to Fig. 2 but this time at pH 6.0. The result was again that k_A ∼ 50 s⁻¹ for mutants as well as wild type (supplemental Fig. S3).

The dissociation of Ca²⁺ from the high affinity sites of the Ca₂E1 state back toward the cytosol was also examined by the rapid-quench technique (Fig. 9, right panels). Again, the assay used takes advantage of the dependence of the reaction with ATP on Ca²⁺ occupancy of the Ca²⁺ sites, as illustrated by the mixing protocol in the top right of Fig. 9. Thus, when an excess of EGTA is added to Ca²⁺-saturated enzyme, the ability to become phosphorylated by ATP will disappear at a rate corresponding to the rate of Ca²⁺ dissociation from the enzyme. After incubation with EGTA for varying time intervals, the amount of enzyme still in the phosphorylatable Ca₂E1 state is determined by a 34-ms incubation with [γ-³²P]ATP before acid quenching. Moderate deviations from wild type of the rate of Ca²⁺ dissociation from the high affinity Ca²⁺ sites of the Ca₂E1 state were seen with mutants S72R, S72A, and E90R (1.5- and 2-fold reduced rates and 2.4-fold enhanced rate, respectively, Table 2). K297A displayed an ∼3-fold slowing of Ca²⁺ dissociation. Again the mutations E90A and E90L proved most detrimental, reducing the rates of Ca²⁺ dissociation from Ca₂E1 24- and 6-fold, respectively, relative to wild type. Thus, mutations E90A and E90L markedly inhibit the Ca²⁺ binding transition as well as the dissociation of Ca²⁺ from the high affinity cytoplasmically exposed Ca²⁺ sites. Ca²⁺ dissociation is also slowed by mutation K297A, but unlike E90A and E90L, K297A enhances the Ca²⁺ binding transition slightly. These data on Ca²⁺ interaction are summarized in Table 2. Caution is generally required in the interpretation of the K_0.5 values determined at steady state (Fig. 8) because they depend not only on the rate constants of Ca²⁺ binding and Ca²⁺ dissociation but also on the rate constants of all the other partial reaction steps in the Ca²⁺ transport cycle, as previously discussed in detail (33). The shifts toward higher K_0.5 values for Ca²⁺ activation observed for E90A and E90L are, however, in good accordance with the much larger inhibitory effects seen for the Ca²⁺ binding transition as compared with Ca²⁺ dissociation from Ca₂E1 in these mutants. For E90R, the 2-fold slowing of Ca²⁺ binding and 2-fold enhancement of Ca²⁺ dissociation may both contribute to the increase of the K_0.5 for Ca²⁺ activation of steady-state phosphorylation.

DISCUSSION

Here we have investigated the interaction network involving Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminally protruding ends of the transmembrane helices M1, M2, and M4 of the Ca²⁺-ATPase. Our functional analysis of mutants with alterations to Glu⁹⁰ implicates this residue as a critical element at the luminal ion gate for extrusion of the Ca²⁺ ions from the E2P state to the endoplasmic reticulum lumen. It is furthermore of note that some of the mutations to Glu⁹⁰ also had a pronounced impact on the function of the cytoplasmically exposed high affinity Ca²⁺ sites of the E1 state, i.e. events that take place at the opposite side of the membrane relative to where Glu⁹⁰ is located.

Glu⁹⁰ and Glu³⁰⁹ Provide Ligands for Luminal Ca²⁺ Binding

The propagation of the movements of the A domain to the transmembrane region during the Ca₂E1 → Ca₂E1P → Ca₂E2P → E2P transitions leads to a considerable displacement of the transmembrane hairpins M1/M2 and M3/M4 relative to each other (cf. the arrows in Fig. 1). Consequently, in the presumed structural analog of the Ca²⁺-free E2P ground state, the E2·BeF₃⁻ crystal structure (7), Ser⁷² and Glu⁹⁰ are positioned near Glu³⁰⁹, which is known as an essential Ca²⁺ binding residue in Ca₂E1 and Ca₂E1P and probably plays a central role as well in the countertransport of protons (9, 20, 21). An issue of great importance for the understanding of the transport mechanism is where in the enzyme Ca²⁺ is bound before it leaves from E2P in exchange for the protons to be countertransported. In E2·BeF₃⁻ the luminally protruding end of the transmembrane domain is in an open configuration compared with other crystal structures of the Ca²⁺-ATPase, with Glu³⁰⁹ exposed to the lumen and associated with a Mg²⁺ ion (7). The hypothesis that this Mg²⁺ is bound in place of Ca²⁺ at a luminally facing low affinity Ca²⁺ site is consistent with the observation that under certain conditions Mg²⁺ in the lumen inhibits dephosphorylation of E2P, similar to the effect of Ca²⁺ transported into the lumen (34, 35). The close proximity of the side chains of Glu⁹⁰ and Ser⁷² to the bound Mg²⁺ ion (2.5 and 5.9 Å, respectively, see Fig. 1 and supplemental Table S1), therefore, led us to examine the functional roles of these two residues. The results in Fig. 6 suggest that the side chain of Glu⁹⁰ indeed does interact with Ca²⁺ in the lumen, thus identifying an essential locus (a leaving site) on the hitherto unknown luminal Ca²⁺ exit pathway. Hence, substitution of Glu⁹⁰ with alanine or leucine led to a marked reduction of the apparent affinity for luminal Ca²⁺, as indicated by the shift toward higher luminal Ca²⁺ concentrations of the Ca²⁺ activation curves for the reaction of E2 with P_i (Fig. 6 and Table 2). The substitution of Ser⁷² with alanine had no effect on the K_0.5 for Ca_lum²⁺, suggesting a more peripheral position of Ser⁷² relative to the luminal Ca²⁺ site in accordance with the 5.9 Å distance to the Mg²⁺ ion in the E2·BeF₃⁻ crystal structure. When, however, Ser⁷² or Glu⁹⁰ was replaced by an arginine, possessing a longer and positively charged side chain, high amounts of phosphoenzyme were obtained even at very low luminal Ca²⁺ concentrations, i.e. conditions where little or no phosphoenzyme was formed in the wild type or any of the other mutants examined here, except E309Q (Fig. 6). Like S72R and E90R, E309Q also displayed considerable activation of phosphorylation by P_i at low luminal Ca²⁺ concentrations and little further activation at higher luminal Ca²⁺ concentrations within the range studied (Fig. 6). Thus, in S72R and E90R as well as E309Q, the E2P formation from P_i appears to be constitutively activated without the need for Ca²⁺ binding from the lumen. For S72R and E90R, this may be explained by occupation of a luminally facing Ca²⁺ site by the guanidinium group of the arginine side chain, thus allowing the guanidinium group to mimic bound Ca²⁺ to some extent due to its positive charge. For E309Q, the neutralization of the negative charge of the glutamate likewise provides a possible explanation in terms of mimicking Ca²⁺. To examine whether such ideas are realistic, we made structural models of the S72R and E90R mutants based on the E2·BeF₃⁻ crystal structure (Fig. 10). In E90R, the position of the guanidinium group of the arginine substituent overlaps with the position of the Mg²⁺ ion for those rotamers of the arginine side chain with lowest energy, and after applying energy minimization to the arginine side chain as well as the side chain of Glu³⁰⁹, the resulting distance is consistent with bond formation between the guanidinium group and the side-chain carboxylate of Glu³⁰⁹ (Fig. 10B), in accordance with the hypothesis that Glu⁹⁰ and Glu³⁰⁹ provide oxygen ligands for a common luminally facing Ca²⁺ site, which is occupied by the guanidinium group of the arginine side chain in E90R. It is also clear that such a site is disturbed by the guanidinium group in S72R (Fig. 10C). This model also explains the block of the E2P → E2 phosphoenzyme hydrolysis in S72R, E90R, and E309Q (Fig. 4), because dephosphorylation of E2P during the normal Ca²⁺ transport cycle does not take place until the translocated Ca²⁺ has dissociated from the luminally exposed site(s).

FIGURE 10.

Structural models of mutants E90R and S72R in the E2·BeF₃⁻ state. A, shown is a structural arrangement of Glu⁹⁰, Ser⁷², and Glu³⁰⁹ relative to the luminal Mg² ion in Ca²⁺-ATPase crystallized in the E2·BeF₃⁻ state (same structure and view as in Fig. 1, right panel). B and C, procedures are the same as in panel A but with the luminal Mg²⁺ ion removed and Glu⁹⁰ (B) or Ser⁷² (C) substituted with arginine. The amino acid substitutions were carried out using the DeepView/Swiss-PdbViewer program (Swiss Institute of Bioinformatics). For E90R, the program provided a rotamer library of 28 possible conformations of the arginine side chain. The rotamer shown is one of three rotamers with the most favorable energy score, which all displayed arrangements where the arginine guanidinium group is held in place by the side chain carboxyl of Glu³⁰⁹, overlapping the region where the luminal Mg²⁺ ion is positioned in the wild type structure. For S72R, the program provided 26 possible rotamers, of which the rotamer with the most favorable energy score is shown. In both mutant structures there was no steric hindrance to the arginine side chain. After the arginine substitution, energy minimization was also carried out for the Glu³⁰⁹ side chain using the DeepView/Swiss-PdbViewer program and selecting the rotamer of the glutamate side chain with the most favorable energy score. Color codes as for Fig. 1.

It should be noted that E90A and E90L differ from E90R by not displaying a reduced rate of dephosphorylation of E2P (Fig. 4). The apparent affinity for luminal Ca²⁺ was nevertheless found markedly reduced in these mutants relative to wild type (Fig. 6), thus indicating that the reduced luminal Ca²⁺ affinity is not an indirect consequence of E2P stabilization. This supports the hypothesis that Glu⁹⁰ is directly involved in luminal Ca²⁺ binding. Furthermore, because E2P is stabilized in E309Q, as in E90R, it is likely that neutralization of the negative charge of Glu³⁰⁹, similar to what is obtained by luminal Ca²⁺ binding, is the reason for the E2P stabilization in E90R. This fits well with the hypothesis that in the normal Ca₂E2P state Glu⁹⁰ and Glu³⁰⁹ bind a Ca²⁺ ion that through this interaction prevents dephosphorylation, probably through substantial rearrangements of the transmembrane domain, resulting in long-range effects of the altered positions of the transmembrane helices on the insertion of the ¹⁸¹TGES phosphatase motif of the A domain into the catalytic site (5–7).

When phosphorylation was carried out with ATP in the forward-running mode of the Ca²⁺-ATPase cycle, the E2P phosphoenzyme accumulated for E90R (Fig. 3A) was Ca²⁺ free (Fig. 5), which excludes the possibility that the stabilization of E2P seen under these conditions is due to hindrance of luminal Ca²⁺ dissociation by the arginine side chain. This contrasts with the 4Gi-46/47 mutant that displayed a very stable E2P state with occluded Ca²⁺ due to impaired Ca²⁺ dissociation (Fig. 5 and Ref. 22). Like 4Gi-46/47, the S72R and E90R mutants were unable to form ADP-sensitive Ca₂E1P backward from the P_i-phosphorylated enzyme (bottom panel of Fig. 6). In S72R and E90R the reason may be the arginine guanidinium group occupying a Ca²⁺ site of E2P without being able to fulfill a similar role in E1P, because of the large distance between Ser⁷²/Glu⁹⁰ and Glu³⁰⁹ in this conformation (cf. Fig. 1). The hypothesis that E2P is stabilized by insertion of the arginine guanidinium group into a luminally exposed Ca²⁺ site is also consistent with the highly accelerated loss of ADP sensitivity of the phosphoenzyme of E90R and S72R (Fig. 3B and Table 1), because the measured rate of Ca₂E1P → Ca₂E2P is a net rate that depends both on the forward rate as well as the rate of the reverse reaction, which according to our hypothesis could be greatly reduced as a consequence of the guanidinium group interfering with luminal Ca²⁺ binding in E2P, thereby enforcing Ca²⁺ dissociation and depriving Ca₂E2P. E90A and E90L likewise showed some acceleration of Ca₂E1P → Ca₂E2P, although not to the extent of S72R and E90R (Table 1), which may again be explained by a reduced rate of the reverse reaction due to enhanced Ca²⁺ dissociation, as predicted from the reduced affinity of these mutants for luminal Ca²⁺ (Fig. 6 and Table 2).

The interaction of the arginine side chain with the luminal Ca²⁺ site in the E2P state of S72R and E90R is further reflected by the results shown in Fig. 7 in which the Ca²⁺ sensitivity of the E2P analog states E2·BeF₃⁻ and E2·vanadate were examined. Ca²⁺-induced dissociation of E2·BeF₃⁻ has been shown to result from Ca²⁺ binding at a luminally exposed Ca²⁺ site (28), in accordance with the idea that E2·BeF₃⁻ represents an E2P ground state-like conformation. The dissociation is most likely a three-step process in which Ca²⁺ first binds at the luminally exposed Ca²⁺ sites, forming Ca₂E2·BeF₃⁻ (Ca₂E2P analog) followed by a slow conformational transition to Ca₂E1·BeF₃⁻ (Ca₂E1P analog) and subsequent BeF₃⁻ release from Ca₂E1·BeF₃⁻ (36). E2·vanadate is generally believed to represent an E2P transition state-like conformation (29), and its dissociation is triggered by Ca²⁺ binding at the high affinity Ca²⁺ sites on the cytoplasmic side of the membrane allowed by the equilibrium of E2 with E1 (30). Accordingly, the Ca²⁺-induced dissociation of E2·BeF₃⁻ was very slow in mutant 4Gi-46/47 (Fig. 7C), reflecting the high stability of the occluded Ca₂E2P state in this mutant (22), whereas the Ca²⁺-induced dissociation of the E2·vanadate complex was rapid (Fig. 7D), reflecting the more wild type-like characteristics of the E2P transition state of 4Gi-46/47, allowing formation of E2 from E2·vanadate at a normal rate and subsequent transition to E1 (22). In contrast, the dissociation of E2·BeF₃⁻ as well as that of E2·vanadate were very slow in both S72R and E90R (Fig. 7, C and D), thus reflecting the stabilization of a Ca²⁺-free E2P-like state with the arginine side chain located in the luminal Ca²⁺ site.

Importance of Glu⁹⁰ for Cytoplasmic Ca²⁺ Binding

The results of Fig. 9 and Table 2 show that Glu⁹⁰ is an important player not only at the luminal ion gate but is required for normal function of the cytoplasmic Ca²⁺ sites as well.

The rate of Ca²⁺ dissociation back toward the cytosol from Ca₂E1 was markedly slowed by mutations E90A and E90L. Because Glu⁹⁰ is located at the luminal side of the membrane and, in the Ca₂E1 state, some 15–20 Å below the Ca²⁺ ions (Fig. 1 and supplemental Table S1), any direct interaction with the cytoplasmic Ca²⁺ sites seems to be excluded. In all the Ca₂E1 crystal structures (with or without bound nucleotide or phosphoryl analogs), the side-chain carboxylate of Glu⁹⁰ is closely associated with the side-chain amino group of Lys²⁹⁷ (Fig. 1 and supplemental Table S1). This led us to speculate that a Glu⁹⁰-Lys²⁹⁷ ion bond might be involved in control of cytoplasmic Ca²⁺ binding, and we, therefore, included mutation K297A in the study. The reduced rate of Ca²⁺ dissociation seen for K297A supports the hypothesis that the presence of an ion bond between Glu⁹⁰ and Lys²⁹⁷ influences the dissociation of Ca²⁺ from Ca₂E1. The finding that E90R displayed a 2-fold enhanced rate of Ca²⁺ dissociation indicates that a surplus of positive charge in this area will destabilize the Ca₂E1 state, thus pointing to a possible role for Glu⁹⁰ in neutralizing the positive charge of Lys²⁹⁷ in Ca₂E1. This influence on the cytoplasmically exposed Ca²⁺ sites of E1 must be exerted by long-range effects involving repositioning of the transmembrane helices.

The Ca²⁺ binding properties of E90A and E90L were further profoundly affected by a more than 2 orders of magnitude-slowing of the Ca²⁺ binding transition E2 → Ca₂E1. Because K297A showed a 2-fold-enhanced rate of E2 → Ca₂E1 (Table 2), the slow rates of E2 → Ca₂E1 in E90A and E90L seem to be unrelated to the disruption of the ion bond between Glu⁹⁰ and Lys²⁹⁷ in Ca₂E1. Moreover, E90R displayed only a 2-fold reduction of E2 → Ca₂E1 relative to the wild type, implying that the very slow rate of this transition in E90A and E90L is related to the hydrophobic nature of the side chain in these mutants. The various structures of the Ca²⁺-ATPase crystallized in E2 state (37–41) provide a possible explanation. Thus, common to these structures is that the Glu⁹⁰ side chain is located inside a hydrophobic pocket consisting of Val³⁰⁰ of M4 and Ile⁷⁸⁸, Pro⁷⁸⁹, and Val⁷⁹⁰ of M6 (Fig. 11). It is, therefore, quite conceivable that substitution of Glu⁹⁰ with a hydrophobic residue such as alanine or leucine would strengthen the interaction between M2 and M4/M6, hindering the displacement of M1/M2 relative to M3/M4 during the E2 → Ca₂E1 transition, thus effectively locking the enzyme in the E2 conformation.

FIGURE 11.

Structural arrangement around Glu⁹⁰ in Ca²⁺-ATPase crystallized in the E2 state. The Protein Data Bank accession code corresponding to the structure shown is 2C8L (39). Color codes are as for Fig. 1.

Importance of Lys²⁹⁷

In a previous mutational study predating the high resolution crystal structures of the Ca²⁺-ATPase, Chen et al. (19) carried out functional analysis of Ca²⁺ pumps with other mutations to Lys²⁹⁷ than the K297A mutation studied here and found that mutants K297M and K297F displayed slow E2P dephosphorylation, leading to the suggestion that Lys²⁹⁷ seals the luminal gate of the Ca²⁺ transport pathway (19). In the present study mutant K297A displayed a 3.4-fold reduction of the rate of E2P dephosphorylation (Fig. 4 and Table 1), resembling the effects observed by Chen et al. (19) with K297M and K297F. However, the apparent affinity of the E2P state of K297A for Ca²⁺ binding at the luminal sites was indistinguishable from that of the wild type (Fig. 6 and Table 2), making it unlikely that Lys²⁹⁷ is associated with the luminal ion gate of E2P. Rather, the effect of mutations to Lys²⁹⁷ on E2P stability could be due to critical ion bonding between Lys²⁹⁷ and the residue(s) in the loop connecting M1 and M2, one likely interaction partner candidate being Glu⁷⁹ (Fig. 1).

Conclusion and Perspective

In conjunction, all the data presented here seem to point to Glu⁹⁰ as a residue that participates together with Glu³⁰⁹ in Ca²⁺ binding at a luminally exposed site in the E2P state. This finding, together with previous functional (9) and structural (3, 7) evidence for an alternating exposure of Glu³⁰⁹ at the two sides of the membrane during the pump cycle is consistent with a mechanism in which Glu³⁰⁹ carries one of the two Ca²⁺ ions along from the cytoplasmic side to a leaving site, where Ca²⁺ is received by Glu⁹⁰ before the final exit to the lumen. Because the present data do not allow a distinction between the two Ca²⁺ ions, it still remains to be clarified how the other Ca²⁺ ion is translocated; that is, whether it also uses Glu⁹⁰ or takes a different exit pathway from the binding pocket?