Importance of Conserved N-domain Residues Thr441, Glu442, Lys515, Arg560, and Leu562 of Sarcoplasmic Reticulum Ca2+-ATPase for MgATP Binding and Subsequent Catalytic Steps

PLASTICITY OF THE NUCLEOTIDE-BINDING SITE*

Johannes D. Clausen {ddagger}, David B. McIntosh § , Bente Vilsen {ddagger}, David G. Woolley § and Jens Peter Andersen {ddagger} ||

From the {ddagger}Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark and the §Division of Chemical Pathology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa

Received for publication, February 3, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 -> Ala, Glu442 -> Ala, and Leu562 -> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 -> E2P conformational transition, whereas mutations Thr441 -> Ala, Glu442 -> Ala, Lys492 -> Leu, and Lys515 -> Ala inhibited the E1PCa2 -> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 -> Ala, Lys492 -> Leu, Lys515 -> Ala, and Arg560 -> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 -> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sarcoplasmic reticulum Ca2+-ATPase1 is an energy-transducing enzyme responsible for active transport of Ca2+ from the cytosol into the lumen of sarcoplasmic reticulum. It is an integral 110-kDa membrane protein belonging to the family of P-type ATPases, in which autophosphorylation by ATP of a conserved aspartic acid residue is coupled to conformational changes resulting in ion translocation (Scheme 1). The high resolution x-ray structure of the Ca2+-ATPase crystallized in a Ca2+-bound dephospho form (presumably corresponding to E1Ca2 in Scheme 1) has revealed that the enzyme is made up of 10 membrane-spanning helices and a large cytoplasmic head that binds ATP and catalyzes its hydrolysis to yield ADP and Pi. The head consists of three well defined domains: the nucleotide-binding domain ("domain N," supposed to bind the adenosine part of ATP), phosphorylation domain ("domain P" with Asp351, which is phosphorylated), and actuator domain ("domain A," believed to undergo large movements during the reaction cycle) (1). The catalytic site may, at different stages of the transport cycle, consist of residues contributed from all three head domains, and many questions regarding the nature of the nucleotide-binding site(s) are unresolved.



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SCHEME 1.
Ca2+-ATPase reaction cycle. Major conformational changes and ligand binding and dissociation steps are shown. Boxed ATP indicates steps for which the rate is enhanced by additional binding of ATP or MgATP that is not hydrolyzed ("modulatory ATP").

 

In domain N, labeling of Lys515 with FITC (2, 3) or Lys492 with TNP-8N3-ATP (4) is competitive with respect to ATP binding at the catalytic site. The specific photolabeling of Lys492 with TNP-8N3-ATP has permitted measurement of the binding of nucleotide to mutant Ca2+-ATPases in a competition assay and identified Phe487, Arg489, and Lys492 as likely nucleotide-binding residues (5). The location of TNP-AMP in the crystal structure, obtained from a difference Fourier map following soaking of the E1Ca2 crystals in a solution containing TNP-AMP, supported involvement of these residues in the binding of nucleotide, and in addition revealed the proximity of TNP-AMP to several other residues, including Thr441, Glu442, Lys515, Arg560, and Leu562 (1).

A surprising feature of the crystal structure of the Ca2+-bound E1Ca2 dephospho form is that the configuration of the cytoplasmic domains is quite open with the nucleotide-binding site being separated from Asp351 in domain P by more than 25 Å, implying large domain movements during ATP binding and hydrolysis (1). Presumably, the binding of nucleotide leads to closing of the structure. The movements contribute to uncertainties about the nucleotide-binding residues and the orientation of the bound nucleotide. Although the high affinity binding sites for TNP-nucleotide and ATP seem to overlap (6), they are not necessarily identical. Thapsigargin, a potent inhibitor of the Ca2+-ATPase, has no effect on TNP-ATP or TNP-8N3-ATP binding, and yet decreases the affinity of the pump for ATP by at least 100-fold (5, 7). The doubts with respect to the nucleotide site are exacerbated by functional studies showing that ATP in addition to being the phosphorylating substrate exerts modulatory effects on various steps of the pump cycle, see boxed ATP in Scheme 1 (8, 9, 10, 11, 12, 13, 14, 15, 16). In view of the predicted large domain movements and presence of the phosphoryl group at the catalytic site during part of the cycle, it seems unlikely that modulatory ATP binds exactly as substrate ATP does.

In the present study, we have introduced mutations of Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562, and we have also extended the previous study (5) on mutants with alterations to Phe487, Arg489, and Lys492. With the exception of Cys561, these residues show a high degree of conservation throughout the P-type ATPase family (17). A recent study, applying solution nuclear magnetic resonance techniques to a 28-kDa recombinant Ca2+-ATPase fragment corresponding to domain N pinpointed Thr441 and Glu442 as the two residues providing the largest shifts in the backbone15N spectra upon addition of a non-hydrolyzable ATP analogue to the medium (18). Marked spectral shifts were also seen for residues close in the linear sequence to Lys492, Lys515, and Arg560. Site-directed mutagenesis studies have previously shown the importance of Lys515 and Arg560 in the overall ATPase function (19, 20, 21). Mutation of Lys515 to arginine, alanine, glutamine, and glutamate resulted in 65, 30, 25, and 5% Ca2+-transport activity relative to wild type (19, 20). Mutation of Arg560 to alanine abolished phosphorylation from ATP (21). Thr441, Glu442, Cys561, and Leu562, or their homologues in other P-type ATPases, have not been subjected previously to mutational analysis.

This study, presenting the first direct measurements of nucleotide-binding properties of mutants with alterations to Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 of the Ca2+-ATPase, reveals marked effects on nucleotide binding to the substrate site of the dephosphoenzyme, except in the case of the Cys561 mutants. Moreover, our analysis of other steps in the pump cycle (Scheme 1) demonstrates that phosphorylation, the Ca2+-translocating E1PCa2 -> E2P conformational transition, as well as the hydrolysis of the E2P phosphoenzyme intermediate are affected to various extents by the mutations. The low affinity modulatory effects of ATP on the phosphoenzyme-processing steps are, however, not significantly affected. Finally, we provide functional evidence for close interaction between Lys515 and Glu442 in experiments demonstrating direct involvement of Glu442 in the labeling reaction of Lys515 with FITC.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutagenesis, Expression, and Assays of Overall Function—Oligonucleotide-directed mutagenesis of cDNA encoding the rabbit fast twitch muscle Ca2+-ATPase (SERCA1a isoform) was carried out as described previously (22). For expression, the wild-type or mutant cDNA, inserted in the pMT2 vector (23), was transfected into COS-1 cells using the calcium phosphate precipitation method (24). The microsomal fraction containing expressed wild-type or mutant Ca2+-ATPase was isolated by differential centrifugation (19). The concentration of expressed Ca2+-ATPase was quantified by a specific enzyme-linked immunosorbent assay (25) or by determination of the maximum capacity for phosphorylation by inorganic phosphate in the presence of 30% (v/v) dimethyl sulfoxide ("active site concentration," see Ref. 26). Transport of45Ca2+ into the microsomal vesicles was measured by filtration, and the ATPase activity was measured by determining the amount of Pi liberated as described previously (26).

Phosphorylation from [{gamma}-32P]ATP and32PiManual mixing experiments at various buffer and temperature conditions (detailed in the figure legends) were carried out according to the principles described previously (22, 25, 26). Transient kinetic experiments at 25 °C were performed using a Bio-Logic quench-flow module QFM-5 (Bio-Logic Science Instruments, Claix, France) as described (27). In all phosphorylation experiments, acid quenching was performed with 0.5–2 volumes of 25% (w/v) trichloroacetic acid containing 100 mM H3PO4. The acid-precipitated protein was washed by centrifugation and subjected to SDS-PAGE in a 7% polyacrylamide gel at pH 6.0 (28), and the radioactivity associated with the separated Ca2+-ATPase band was quantified by imaging, using a Packard CycloneTM Storage Phosphor System. Background phosphorylation levels, subtracted from all data points, were usually determined in parallel experiments with control microsomes isolated from mock-transfected COS-1 cells. In some of the dephosphorylation experiments, the constant phosphorylation level reached after the exponential decay was taken as background (usually ~5% of the initial phosphorylation).

To remove contaminant ADP and AMP, the non-radioactive ATP added in millimolar concentration in dephosphorylation experiments was purified by ion exchange chromatography on a Sephadex DEAE-A25 column before use. By using an NADH-coupled spectrophotometric assay with phosphoenolpyruvate, lactate dehydrogenase, and pyruvate kinase, the purified ATP preparation was found to contain less ADP than the detection limit of 0.1 mol %.

Assays for Nucleotide Binding—The synthesis of [{gamma}-32P]TNP-8N3-ATP, the photolabeling of COS-1 cell microsomes containing wild-type or mutant Ca2+-ATPase, the inhibition by ATP, and the quantification of labeled bands by electronic autoradiography following SDS-PAGE were carried out as described previously (5, 29). Generally, the concentration of [{gamma}-32P]TNP-8N3-ATP was 3x K0.5 in the inhibition experiments with ATP (where K0.5 is ligand concentration giving half-maximum effect). Details of the buffer composition are given in the legends. CrATP-induced Ca2+ occlusion was measured as described previously (5, 30).

Calculations and Data Analysis—The ion concentrations in the reaction buffers were calculated using the program WEBMAXC, available on the World Wide Web, and the stability constants therein (31). Generally, experiments were conducted at least twice, and the complete set of data was analyzed by nonlinear regression using the SigmaPlot program (SPSS, Inc.). Monoexponential functions were fitted to the phosphorylation and dephosphorylation time courses. The analysis of ligand concentration dependences was based on the Hill equation,

(Eq. 1)
For analysis of the [{gamma}-32P]TNP-8N3-ATP labeling data, a constant or linear component was added to represent nonspecific labeling as described (5), and the Hill coefficient was set to 1. The "true" dissociation constant for ATP and MgATP binding was calculated using the previously validated equation for competitive inhibition of the [{gamma}-32P]TNP-8N3-ATP labeling (5).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutagenesis and Expression—Nine Ca2+-ATPase constructs with mutations of potential nucleotide-binding residues were prepared in this study. Thr441, Glu442, and Lys515 were replaced individually with alanine. Cys561 was replaced with alanine or tryptophan; Arg560 was replaced with leucine, valine, or glutamate; and Leu562 was replaced with phenylalanine. The latter substitution was chosen to test the importance of the leucine for nucleotide binding by either introducing steric hindrance at this position or possibly enhancing the affinity for nucleotide by creating an aromatic "sandwich" for the adenine ring with Phe487. All mutants could be expressed in COS-1 cells at levels similar to that of the wild type (data not shown), allowing studies of the overall function as well as the partial reactions of the enzyme cycle. In addition, some supplementary studies of partial reactions were carried out with previously constructed mutants (5) with alterations to Phe487, Arg489, and Lys492.

Overview of Functional Effects—Fig. 1A shows ATP-driven 45Ca2+ accumulation in microsomal vesicles measured in the presence of oxalate to trap Ca2+ in the lumen. The MgATP concentration was 5 mM, i.e. several hundredfold higher than required to saturate the phosphorylation reaction in wild type. All the mutants were able to transport Ca2+, albeit at reduced rates compared with wild type. The most significant reduction was seen for mutant Arg560 -> Glu with a Ca2+ transport rate corresponding to 17% that of the wild type, whereas ~80% transport was obtained for the Cys561 mutants. The Ca2+ transport rates of the remaining six mutants were roughly half (39–66%) that of wild type.



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FIG. 1.
Initial overview of ATP-driven Ca2+ transport (A), ATPase activity (B), and phosphorylation from ATP (C). A, measurements of ATP-driven Ca2+ transport were performed by filtration following incubation for 5 min at 37 °C in a medium containing 20 mM MOPS/Tris (pH 6.8), 100 mM KCl, 5 mM MgATP, 5 mM potassium oxalate (included to trap Ca2+ inside the microsomal vesicles), 0.5 mM EGTA, and 0.45 mM 45CaCl2. The Ca2+ transport activity of the wild type was taken as 100%, and each mutant was related to this level following correction for the variation in expression level. B, the rate of Ca2+-activated ATP hydrolysis was determined at 37 °C in the presence (gray bars) or absence (black bars) of 1 µM Ca2+ ionophore A23187 [GenBank] in a medium containing 50 mM TES/Tris (pH 7.0), 100 mM KCl, 7 mM MgCl2, 1 mM EGTA, 0.9 mM CaCl2 (giving a free Ca2+ concentration of 3 µM), and 5 mM ATP. Following subtraction of the background activity determined in the absence of Ca2+, the molecular activity (catalytic turnover rate) shown was calculated as the amount of Pi liberated per Ca2+-ATPase molecule/s. C, the phosphorylation with [{gamma}-32P]ATP was carried out for 5 s at 25 °C in a medium containing 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 5 mM MgCl2, 100 µM CaCl2, and either 5 µM [{gamma}-32P]ATP (gray bars) or 50 µM [{gamma}-32P]ATP (black bars). The phosphorylation level of the wild type incubated in a medium containing 100 mM MES/Tris (pH 6.0), 10 mM MgCl2, 2 mM EGTA, 30% (v/v) dimethyl sulfoxide, and 0.5 mM 32Pi for 10 min at 25 °C was taken as 100% ("active site concentration"), and all other values were related to this level following correction for the variation in expression level. Standard errors are indicated by the error bars on the columns.

 

Fig. 1B shows the ATPase activity in the presence of 5 mM MgATP without oxalate, in the absence and presence of the calcium ionophore A23187 [GenBank] . In the absence of ionophore (conditions resulting in net Ca2+ uptake in the vesicles), a similar pattern was obtained for ATP hydrolysis as for Ca2+ transport (Fig. 1B, black columns), Arg560 -> Glu showing the most marked reduction of turnover rate, the Cys561 mutants being wild type like, and the remaining six mutants hydrolyzing ATP at turnover rates of 40–58% as compared with wild type. The good correlation between the turnover rate for ATP hydrolysis and ATP-driven accumulation of Ca2+ in the microsomes demonstrates that the coupling between ATP hydrolysis and Ca2+ transport was retained in the mutants. Addition of calcium ionophore A23187 [GenBank] to the reaction medium increased the rate of ATP hydrolysis in the wild type 2–3-fold because of relief of the "back inhibition" of the rate-limiting E1PCa2 -> E2P transition imposed by Ca2+ accumulated at high concentrations in the microsomal vesicles. A similar increase was seen for Thr441 -> Ala, Glu442 -> Ala, Lys515 -> Ala, and the Cys561 mutants, whereas for Leu562 -> Phe and the three Arg560 mutants the ATPase activity remained essentially unaltered (Fig. 1B, gray columns). The latter result implies that a reaction step other than the E1PCa2 -> E2P transition is rate-limiting in the case of Leu562 Phe and Arg560 mutants.

In the Ca2+-bound E1Ca2 state, the wild-type Ca2+-ATPase forms a phosphoenzyme intermediate by reaction with MgATP (cf. Scheme 1). Fig. 1C shows the results of experiments where phosphorylation of Ca2+-saturated enzyme was carried out for 5 s at 25 °C with either 5 or 50 µM [{gamma}-32P]MgATP. For wild type, the maximum steady-state phosphoenzyme level of ~80% of the total active-site concentration is close to being reached at 5 µM MgATP; also for the Cys561 mutants, there was not much difference between 5 and 50 µM MgATP. By contrast, mutants Thr441 -> Ala, Glu442 -> Ala, Lys515 -> Ala, and in particular Arg560 -> Leu, Arg560 -> Val, and Leu562 -> Phe showed much lower phosphoenzyme levels with 5 µM MgATP than with 50 µM MgATP, indicating a markedly reduced apparent affinity for the nucleotide, relative to wild type. Mutant Arg560 -> Glu was unable to phosphorylate significantly above the background level at either concentration of MgATP (Fig. 1C).

MgATP Dependence and Time Course of Phosphorylation from [{gamma}-32P]ATP—The MgATP concentration dependence of phosphorylation was further investigated at 0 °C, where the ATP hydrolysis rate was sufficiently low to allow the phosphorylation to be studied at submicromolar concentrations of the substrate. As seen in Fig. 2A, a 10–120-fold reduction of apparent affinity for MgATP (increase of K0.5) relative to wild type was found for mutants Thr441 -> Ala, Glu442 -> Ala, Lys515 -> Ala, and Leu562 -> Phe; and for mutants Arg560 -> Leu and Arg560 -> Val, the K0.5 for MgATP was at least 3 orders of magnitude lower than that of wild type.



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FIG. 2.
MgATP concentration dependence (A) and time course (B) of phosphorylation from ATP. A, wild type and mutants were phosphorylated at 0 °C for 15 s in a medium containing 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 5 mM MgCl2, 100 µM CaCl2, and varying concentrations of [{gamma}-32P]ATP as indicated. The lines show the best fits of the Hill equation with the Hill coefficient set to 1, giving the K0.5 values (in µM ± S.E.) indicated in parentheses: open circles, wild type, (0.067 ± 0.005); open squares, Thr441 -> Ala (6.2 ± 0.6); open triangles, Glu442 -> Ala (0.72 ± 0.06); reversed solid triangles, Lys515 -> Ala (1.6 ± 0.2); open diamonds, Arg560 -> Leu (>>100); solid triangles, Arg560 -> Val (~100); and solid circles, Leu562 -> Phe (8.1 ± 1.4). The 100% value corresponds to the phosphorylation level reached at infinite ATP concentration as deduced from the fit. B, phosphorylation was carried out at 25 °C in a medium containing 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 100 µM CaCl2, 5 mM MgCl2, and 5 µM [{gamma}-32P]ATP, and the samples were acid-quenched at serial time intervals, using a Bio-Logic QFM-5 quench-flow module with mixing protocol as described previously (34). In each case, the level of phosphorylation after 5 s was taken as 100% (for a comparison of the specific phosphorylation levels of wild type and mutants after 5 s, see Fig. 1C). The lines show the best fits of a monoexponential function, giving the rate constants (in s-1 ± S.E.) indicated in parentheses: open circles, wild type (59.7 ± 2.0); open squares, Thr441 -> Ala (5.3 ± 0.6); open triangles, Glu442 -> Ala (13.3 ± 0.6); reversed solid triangles, Lys515 -> Ala (11.1 ± 0.8).

 

Fig. 2B shows the time course of phosphorylation at 25 °C of wild type and selected mutants determined by quench-flow methodology in the presence of 5 µM [{gamma}-32P]MgATP under conditions identical to those corresponding to Fig. 1C. Leu562 -> Phe and the three Arg560 mutants were not included in these studies because their phosphorylation levels were too low to allow reliable pre-steady-state measurements (cf. Fig. 1C). The wild type reaches a steady-state level of phosphorylation within a few hundred milliseconds, with a slight initial over-shoot (Fig. 2B, cf. also Ref. 27). For simplicity, a monoexponential function was fitted to the data, giving an apparent rate constant kobs = 60 s-1 for the approach to steady state. In comparison, 5–12-fold lower kobs values were determined for mutants Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala (Fig. 2B). The kobs value decreased in the same order as the apparent MgATP affinity observed in Fig. 2A (Glu442 -> Ala > Lys515 -> Ala > Thr441 -> Ala).

MgATP Dependence of ATP Hydrolysis—For wild-type Ca2+-ATPase, the MgATP activation profile of ATPase activity has a complex appearance, because MgATP (or ATP) in addition to being the phosphorylating substrate exerts modulatory effects on various steps of the pump cycle, cf. Scheme 1 (8, 9, 10, 11, 12, 13, 14, 15, 16). The basal activation to about 20% of the maximum activity occurring at MgATP concentrations below 10 µM in wild type reflects the binding of MgATP to the E1Ca2 form as phosphorylating substrate. It can be seen in Fig. 3 that for mutant Leu562 -> Phe and the three Arg560 mutants, the basal activation was shifted to much higher MgATP concentrations in agreement with the data in Fig. 2A. Less drastic right-shifts of the basal activation were observed for mutants Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala with plateau levels being reached at about 50 µM MgATP, whereas the Cys561 mutants were wild type-like. At least two secondary activation phases occur for the wild-type enzyme, between 10 and 200 µM MgATP and at higher MgATP concentrations, reflecting the accelerating effect of nucleotide on partial reaction steps preceding and subsequent to phosphorylation (9, 10, 12, 13, 14, 15, 16). Fig. 3 shows that the Cys561 mutants displayed a wild type-like secondary activation. For mutant Leu562 -> Phe and the three Arg560 mutants, the severely reduced ATPase activity prevented a clear distinction between the basal and the intermediate modulatory phases (Fig. 3), but a distinct low affinity activation phase similar to that of the wild type is seen above 200 µM MgATP for Arg560 -> Leu, Arg560 -> Val, and Leu562 -> Phe, as well as for Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala, indicating that nucleotide binding with low affinity accelerates at least one partial reaction in these mutants, although in contrast to the wild type the activation profiles level off above 3 mM MgATP for Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala. Furthermore, taking into consideration that the basal activation is shifted to higher MgATP concentrations relative to wild type, the activation occurring with intermediate affinity, between 10 and 200 µM MgATP in wild type, seems to be less pronounced or right-shifted in Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala. This is most obvious for Glu442 -> Ala, where the ATPase activity was nearly constant between 50 and 200 µM MgATP (Fig. 3).



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FIG. 3.
MgATP dependence of ATPase activity. The rate of ATP hydrolysis was measured at 37 °C in a medium containing 50 mM TES/Tris (pH 7.5), 100 mM KCl, 100 µM CaCl2, 1 µM Ca2+ ionophore A23187 [GenBank] , and varying concentrations of Mg2+ and ATP to give the indicated MgATP concentrations and 1 mM free Mg2+. Following subtraction of the background activity determined in the absence of Ca2+ (presence of 1 mM EGTA without CaCl2), the catalytic turnover rate shown was calculated as the amount of Pi liberated per Ca2+-ATPase molecule/s. The symbols are as follows: open circles, wild type; reversed open triangles, Cys561 -> Ala; solid squares, Cys561 -> Trp; solid triangles, Arg560 -> Val; open diamonds, Arg560 -> Leu; solid diamonds, Arg560 -> Glu; open squares, Thr441 -> Ala; open triangles, Glu442 -> Ala; reversed solid triangles, Lys515 -> Ala and solid circles, Leu562 -> Phe.

 

Acetyl Phosphate-driven Ca2+ Uptake—As an alternative to ATP, acetyl phosphate can be used as an energy source to drive Ca2+ transport into the microsomal vesicles (16). Fig. 4 shows the time course for wild type and the mutants. For most mutants, the decrease of the transport rate relative to wild type was rather similar to the decrease seen for ATP-driven transport at 5 mM MgATP (Fig. 1A), as also shown previously for mutant Lys515 -> Ala (20), indicating that at high MgATP concentration (Fig. 1, A and B) the limitation of the overall reaction rate seen for the mutants is not imposed by binding defects specific to the nucleotide, but rather by defects in other partial reaction steps. Interestingly, mutant Leu562 -> Phe is a clear exception to this pattern. This mutant showed a markedly reduced ATP-driven Ca2+ transport (Fig. 1A) and ATPase activity (Fig. 1B), but wild type-like acetyl phosphate-dependent Ca2+ transport (Fig. 4).



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FIG. 4.
Acetyl phosphate-driven Ca2+ transport. Transport activity of COS-1 cell microsomes was measured at 25 °C in 25 mM MOPS/Tris (pH 6.8), 100 mM KCl, 5 mM MgCl2, 0.55 mM 45CaCl2, 0.5 mM EGTA, 5 mM potassium oxalate, and 10 mM acetyl phosphate for the times shown. The ordinate shows the amount of Ca2+ accumulated per mg of Ca2+-ATPase protein. The symbols are as follows: open circles, wild type; open squares Thr441 -> Ala; open triangles, Glu442 -> Ala; reversed solid triangles, Lys515 -> Ala; solid triangles, Arg560 -> Val; open diamonds, Arg560 -> Leu; solid diamonds, Arg560 -> Glu; reversed open triangles, Cys561 -> Ala; and solid circles, Leu562 -> Phe.

 

Ca2+ Dependence—To examine whether a reduced affinity for Ca2+ could be involved in the observed effects of the mutations on Ca2+ transport and ATPase activity, we performed a Ca2+ titration of the ATPase activity. The Ca2+ concentration giving half-maximum activation of ATP hydrolysis was 0.33 ± 0.01 µM for the wild type and 0.64 ± 0.05, 0.41 ± 0.02, 0.35 ± 0.02, 0.33 ± 0.02, 0.54 ± 0.04, 0.38 ± 0.02, 0.47 ± 0.04, and 0.38 ± 0.03 µM for Arg560 -> Leu, Arg560 -> Val, Cys561 -> Ala, Cys561 -> Trp, Leu562 -> Phe, Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala, respectively (the K0.5 value was obtained by fitting the Hill equation, ± S.E., from the regression analysis, data not shown). Thus, although significant alterations to Ca2+ binding are evident for mutants Arg560 -> Leu and Leu562 -> Phe, the mutants were >80% saturated with Ca2+ at the free Ca2+ concentration of 3 µM present during the experiments corresponding to Fig. 1, A and B, and close to 100% saturated at the higher free Ca2+ concentrations present during the experiments corresponding to Fig. 1C, Fig. 2, A and B, Fig. 3, and Fig. 4. Hence, the observed effects of the mutations cannot be ascribed to variable Ca2+ saturation.

Nucleotide Binding—To examine the mutational effects on nucleotide binding directly, we applied a photolabeling assay that takes advantage of the highly specific labeling of Lys492 with [{gamma}-32P]TNP-8N3-ATP (5). In this assay, the affinities for the free and Mg2+-bound forms of [{gamma}-32P]TNP-8N3-ATP are determined from the dependence of photolabeling on the concentration of the label in the absence and presence of Mg2+, respectively. By studying competitive inhibition of photolabeling in the presence of varying concentrations of ATP and MgATP (the latter often being referred to as the "true" substrate (32)), it is also possible to obtain highly accurate values for the KD corresponding to these nucleotides (5, 33, 34). Photolabeling is carried out in the absence of Ca2+ to avoid phosphorylation of the Ca2+-ATPase and hydrolysis of the photolabel, and at pH 8.5 to reduce nonspecific labeling and ensure that the predominant enzyme conformation is E1 (5). Results of photolabeling experiments with the nine mutants in the absence and presence of Mg2+ are summarized in Table I. Also presented here for the first time are nucleotide-binding parameters in the absence of Mg2+ for mutants Phe487 -> Leu, Arg489 -> Leu, and Lys492 -> Tyr of the487FSRDRK loop in domain N. These latter mutants have only been analyzed previously in the presence of Mg2+ (5). We have shown before (33, 34), and reproduced in Table I, that wild type exhibits a striking Mg2+ dependence for both TNP-8N3-ATP and ATP binding; Mg2+ lowers the apparent affinity for the TNP-nucleotide and increases (as much as 40-fold) that for ATP.


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TABLE I
Nucleotide binding parameters in the presence and absence of Mg2+

Photolabeling with [{gamma}-32P]TNP-8N3-ATP was carried out as described in Ref. 5 and competition with ATP/MgATP as described in Ref. 5 and illustrated in Fig. 5. The parameters shown were derived from analysis of the data as described for Fig. 5 and under "Experimental Procedures."

 



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FIG. 5.
Binding of ATP determined by inhibition of TNP-8N3-ATP photolabeling in the presence (closed symbols) or absence (open symbols) of Mg2+ Examples of data for Thr441 -> Ala (squares), Glu442 -> Ala (triangles), Arg560 -> Leu (diamonds), and Leu562 -> Phe (circles) are shown together with the best fits to the data for the expressed wild type obtained in the presence and absence of Mg2+ (short dashed and long dashed lines, respectively) (5, 33). Photolabeling was performed in 25 mM EPPS/TMAH (pH 8.5), 20% (v/v) glycerol at a [{gamma}-32P]TNP-8N3-ATP concentration of 3x K0.5 and the indicated concentrations of ATP with either 1 mM MgCl2 and 0.5 mM EGTA (presence of Mg2+) or 2 mM EDTA (absence of Mg2+). The lines show the best fits to the data of the equations described under "Experimental Procedures." Table I lists KD values derived in this way.

 
The two Cys561 mutants differed little from wild type in the binding assays, whereas the remaining mutants showed interesting differences from wild type (Table I). Examining the interaction with TNP-8N3-ATP and TNP-8N3-MgATP first, there were major changes relative to wild type in both labeling efficiency and in the apparent affinity for the photolabel. Lys515 -> Ala and Arg560 -> Glu showed no specific photolabeling above the background level either in the absence or presence of Mg2+, and Arg560 -> Val could not be labeled in the absence of Mg2+. Arg560 -> Leu and Arg560 -> Val showed conspicuous ~10-fold decreases, relative to wild type, of the apparent affinity for the photolabel (i.e. increased K0.5) in the presence of Mg2, whereas Thr441 -> Ala, Glu442 -> Ala, and Leu562 -> Phe showed slight increases of the apparent affinity (i.e. reduced K0.5). It is evident that Arg560 plays a major role in binding the TNP-nucleotide in the presence of Mg2+. On the other hand, Arg560 -> Leu showed no significant deviation from wild type in the absence of Mg2+. In Thr441 -> Ala, the ability to respond to Mg2+ with a reduced affinity for the TNP-nucleotide was lost; in the presence of Mg2+ this mutant displayed an affinity for the photolabel similar to or slightly higher than that displayed in the absence of Mg2+, whereas in the wild type Mg2+ reduces the affinity for the photolabel. With respect to the three mutants with alterations to residues of the 487FSRDRK loop, Arg489 -> Leu and Lys492 -> Tyr showed a large decrease in affinity for the photolabel relative to wild type in the absence of Mg2+, corresponding to 31- and 17-fold, respectively, whereas Phe487 -> Leu exhibited an increased affinity. Evidently the two basic amino acid residues are critical for binding the nucleotide in the absence of Mg2+.

Although the binding sites for ATP and TNP-8N3-ATP may not be identical, there is sufficient overlap to ensure efficient competition when ATP or MgATP is added at varying concentrations during photolabeling, and we have previously demonstrated that the competition assay provides highly accurate values for the ATP and MgATP binding affinities of wild type and mutant Ca2+-ATPases expressed in COS-1 cells (5). For wild type, the true affinities (KD values) for MgATP and ATP calculated under the assumption of competitive inhibition as described previously (5) were found to be 0.51 and 21 µM, respectively (Table I). Fig. 5 shows examples of the experimental data from which the KD values for ATP and MgATP in Table I were derived, corresponding to Thr441 -> Ala, Glu442 -> Ala, Arg560 -> Leu, and Leu562 -> Phe, with data for wild type (see also Refs. 5 and 33) indicated by dashed lines without data points. Because of the deficient photolabeling described above, competition experiments could not be carried out for Lys515 -> Ala and Arg560 -> Glu. Mutation Arg560 -> Leu decreased the affinity (i.e. increased KD) for MgATP more than 1000-fold, whereas mutations Cys561 -> Ala and Cys561 -> Trp had little effect, as mentioned above. A decrease of affinity for MgATP of 40-fold relative to wild type was seen for Arg560 -> Val, and as much as 70–180-fold for Thr441 -> Ala, Glu442 -> Ala, and Leu562 -> Phe. In the absence of Mg2+, Arg560 -> Leu still showed a conspicuous 30-fold decrease of affinity for ATP, whereas for Thr441 -> Ala, Glu442 -> Ala, and Leu562 -> Phe, the affinity for ATP was only 2–5-fold reduced relative to wild type.

In the case of the487FSRDRK mutants, Arg489 -> Leu and Lys492 -> Tyr showed extremely poor binding of ATP in the absence of Mg2+, reproducing what was found for the TNP-nucleotide. For Phe487 -> Leu the changes in the presence and absence of Mg2+ were rather similar, 10–20-fold reduced affinity compared with wild type.

For Thr441 -> Ala and Arg560 -> Leu, competition studies were also carried out with ADP. For wild type, the KD values for ADP in the presence and absence of Mg2+ were 4 and 28 µM, respectively. For both Thr441 -> Ala and Arg560 -> Leu, there were pronounced decreases in affinity for ADP as well as MgADP (8- and >100-fold, respectively, for ADP, and ~30- and ~200-fold, respectively, for MgADP).

CrATP-induced Ca2+ Occlusion—The conspicuous effects found in the above-described experiments testing the nucleotide-binding properties of the mutants, and the uncertainty with respect to Lys515 -> Ala, because it could not be photolabeled with TNP-8N3-ATP, encouraged us to study, as an alternative, the binding of another nucleotide, the non-phosphorylating {beta},{gamma}-bidentate chromium(III) complex of ATP ("CrATP"). In the wild type, CrATP binds with relatively low affinity, forming a very stable complex with the enzyme, in which Ca2+ is "occluded" at the transmembranous high affinity Ca2+ sites (30, 35). The high stability of the Ca2+-occluded CrATP-enzyme complex upon removal of free CrATP from the medium (35) makes it feasible to quantify the complex by size-exclusion chromatography of detergent-solubilized microsomal protein following incubation with CrATP in the presence of radioactive 45Ca2+, and we have previously demonstrated a correlation between deficient CrATP-induced 45Ca2+ occlusion and defective ATP binding in mutants of the Ca2+-ATPase (5, 33). Because the Ca2+ titration of ATPase activity described above indicated that all the mutant enzymes bind Ca2+ with an affinity similar or close to that of wild type, the Ca2+ sites are saturated at the 45Ca2+ concentration of 40 µM used here, and the amount of 45Ca2+ occluded should, therefore, reflect the ability to bind CrATP. Fig. 6 shows 45Ca2+ elution profiles obtained for wild type and selected mutants, as well as for control microsomes harvested from cells transfected with the expression vector without insert. For wild type, a distinct peak is seen at 14–15 min of retention time, corresponding to the elution of Ca2+-ATPase, whereas the control only shows a rather broad peak extending between 10 and 16 min of retention time. The difference between the test and control curves at 14–15 min of retention time provides a measure of 45Ca2+ bound in the occluded state in the Ca2+-ATPase. For mutant Thr441 -> Ala, a minor peak of radioactivity was evident at the position corresponding to Ca2+-ATPase, indicating partial, but extremely reduced, formation of the Ca2+-occluded CrATP-enzyme complex in the present incubation conditions. No such peak was obtained in experiments performed with mutants Glu442 -> Ala, Lys515 -> Ala, Arg560 -> Leu, and Leu562 -> Phe (Fig. 6), or with Arg560 -> Val and Arg560 -> Glu (data not shown). For the Cys561 mutants, the 45Ca2+ peak corresponding to Ca2+-ATPase was similar to that seen for wild type (data not shown). Thus, the effects of the mutations on formation of the Ca2+-occluded CrATP-enzyme complex are in accordance with the effects on MgATP binding described above, and Lys515 -> Ala is clearly included among the mutants showing defective nucleotide binding.



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FIG. 6.
CrATP-dependent Ca2+ occlusion. Wild-type or mutant Ca2+-ATPase expressed in COS-1 cell microsomes was incubated for 1 h at 37 °C in 50 mM TES/Tris (pH 7.0), 100 mM NaCl, 5 mM MgCl2, 40 µM 45CaCl2, and 1 mM CrATP. At the end of the incubation period, the membranes were solubilized by addition of 5 mg/ml of the non-ionic detergent C12E8. Following centrifugation, the supernatant was subjected to size-exclusion high pressure liquid chromatography as described previously (5). The elution buffer contained 50 mM TES/Tris (pH 7.0), 100 mM NaCl, 10 mM MgCl2, 1.5 mM CaCl2, 1 mM EGTA, and 2 mg/ml C12E8. The solid circles show the radioactivity in collected fractions. The result of a control experiment with microsomes harvested from cells transfected with the expression vector without insert is indicated by the thin line without data points. The difference between the test and control curves at 14–15 min of retention time, corresponding to the elution of Ca2+-ATPase, provides a measure of the occluded 45Ca2+. The amount of mutant Ca2+-ATPase protein applied to the column was roughly equivalent to the amount of wild type (±30%, due to variation of the expression level, as determined by enzyme-linked immunosorbent assay).

 

The E1PCa2 to E2P Conformational Transition—To examine the effects of the mutations on the E1PCa2 -> E2P transition, we determined the rate of decay of phosphoenzyme under conditions (0 °C, presence of K+, and neutral pH) where the E1PCa2 -> E2P transition is rather slow and, thus, rate-limiting for the dephosphorylation, whereas the subsequent hydrolysis of E2P (Scheme 1) is relatively rapid. The phosphorylation by [{gamma}-32P]MgATP was terminated by addition of an excess of EGTA (to remove Ca2+). As seen in Fig. 7 and Table II (column marked "EGTA," i.e. no addition of non-labeled MgATP), the rate of phosphoenzyme decay was reduced as much as 6–8-fold relative to wild type, for mutants Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala. Leu562 -> Phe deviated much less from wild type. Among the Arg560 mutants, only Arg560 -> Val could be examined, because of the very low phosphorylation levels obtained with the other two Arg560 mutants at 0 °C. It appears from Fig. 7 and Table II that Arg560 -> Val showed a unique 2-fold enhancement of the rate of phosphoenzyme decay, relative to wild type.



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FIG. 7.
Dephosphorylation at 0 °C of phosphoenzyme formed from ATP. Phosphorylation was performed for 15 s at 0 °C in a medium containing 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 during phosphorylation of 10 µM), 10 µM calcium ionophore A23187 [GenBank] , and 5 µM [{gamma}-32P]ATP (50 µM in case of Arg560 -> Val, because of its low affinity, cf. Fig. 2A). To measure dephosphorylation, the phosphoenzyme was chased by addition of 10 mM EGTA (solid triangles), 10 mM EGTA with 1 mM non-radioactive MgATP (open circles), or 10 mM EGTA with 5 mM non-radioactive MgATP (solid circles), and acid quenching was performed at the indicated time intervals. The lines show the best fits of a monoexponential decay function; the rate constants are listed in Table II.

 

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TABLE II
Dephosphorylation of phosphoenzyme formed from [{gamma}-32P]ATP or 32Pi

Formation of E1PCa2 from [{gamma}-32P]ATP at 0 °C or of E2P from 32Pi at 25 °C, and subsequent chase of the phosphoenzyme with dephosphorylation buffer containing excess EGTA (to terminate phosphorylation from [{gamma}-32P]ATP by removal of Ca2+) or excess EDTA (to terminate phosphorylation from 32Pi by removal of Mg2+) and varying amounts of non-radioactive MgATP or ATP as indicated, was carried out as described in the legends to Figs. 7 and 9. A monoexponential decay function was fitted to the data, and the rate constant (kdephos) is shown. ADP sensitivity was tested on phosphoenzyme formed from [{gamma}-32P]ATP by addition of 1 mM ADP with 10 mM EGTA (final concentrations), and the fraction of the phosphoenzyme remaining after 5 s ("EP") is shown.

 



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FIG. 9.
Dephosphorylation of phosphoenzyme formed from Pi. Wild type and mutants were phosphorylated at 25 °C for 10 min in a medium containing 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 30% (v/v) dimethyl sulfoxide, 10 mM MgCl2, and 0.5 mM 32Pi. Dephosphorylation was studied at 25 °C by a 19-fold dilution into a medium containing EDTA (to remove free Mg2+) corresponding to a final concentration of 10 mM, 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 15% (v/v) dimethyl sulfoxide, 0.5 mM non-radioactive Pi, without (open circles) or with (solid circles) 1 mM ATP, followed by acid quenching at serial time intervals. The lines show the best fits of a monoexponential decay function; the rate constants are listed in Table II.

 
To test the low affinity modulatory effect of the nucleotide on the E1PCa2 -> E2P transition (cf. Scheme 1, boxed ATP), the dephosphorylation was also examined at 1 and 5 mM non-labeled MgATP added with EGTA. It can be seen in Fig. 7 and Table II that the nucleotide induced a significant enhancement of the dephosphorylation rate in the wild type as well as the mutants. Both in wild type and in Thr441 -> Ala, Glu442 -> Ala, Lys515 -> Ala, Arg560 -> Val, and Leu562 -> Phe the rate constant observed at 5 mM MgATP was roughly 2-fold higher than that observed at 1 mM MgATP, which is surprisingly consistent with the increase of ATPase activity in this concentration range (Fig. 3), taking into account the different temperatures under which these experiments were carried out.

Table II also shows the results of similar measurements performed with mutants Phe487 -> Leu, Arg489 -> Leu, and Lys492 -> Leu, whose MgATP and ATP affinities at the substrate site were reported in the previous paper (5) and in Table I of the present paper, respectively. Lys492 -> Leu showed a conspicuous 6-fold slowing of the phosphoenzyme decay observed upon EGTA addition, whereas only small differences from wild type were seen for Phe487 -> Leu and Arg489 -> Leu. For all 3 mutants, there was a substantial modulatory effect of millimolar MgATP, although slightly less pronounced than for the wild type and the above-described mutants (1.4–1.6-fold enhancement from 1 to 5 mM MgATP).

ADP Sensitivity of the Phosphoenzyme—The two phosphoenzyme intermediates E1PCa2 and E2P can normally be distinguished by their different sensitivity to ADP. E1PCa2 is ADP-sensitive, i.e. able to donate the phosphoryl group back to ADP, forming ATP, whereas E2P is insensitive to ADP and dephosphorylates only by hydrolysis. To test the ADP sensitivity, 1 mM ADP was added following phosphorylation under the same conditions as described for Fig. 7. This resulted in almost complete dephosphorylation of wild type within 5 s, demonstrating accumulation of the E1PCa2 form of the phosphoenzyme, and similar effects of ADP were seen for all the mutants studied except Arg560 -> Val, for which as much as 24% phosphoenzyme remained after the 5-s incubation with ADP (Table II), indicating a significantly reduced sensitivity to ADP. This could be the result of accumulation of E2P, as a consequence of the enhancement of the E1PCa2 -> E2P transition reported above. Alternatively, the reduced ADP sensitivity reflects a lowered affinity of the E1PCa2 form for ADP. This would be in keeping with the very pronounced decrease in affinity for MgADP and ADP shown above for Arg560 -> Leu (Table I).

Properties of the E2P Phosphoenzyme—The E2P phosphoenzyme can be formed by "backward" phosphorylation with inorganic phosphate of the E2 state in the absence of Ca2+ and presence of Mg2+ (cf. Scheme 1). Fig. 8 shows the phosphorylation at varying concentrations of 32Pi under conditions optimal for stabilization of the E2P phosphoenzyme (acidic pH, presence of the organic solvent dimethyl sulfoxide, and absence of alkali metal ions). The concentration of Pi giving half-maximum phosphorylation is close to 10 µM for wild type. Significantly reduced apparent affinity (increased K0.5) for Pi was seen for Glu442 -> Ala and Lys515 -> Ala (5- and 2.5-fold, respectively), whereas Arg560 -> Leu and Arg560 -> Val showed 2-fold increased apparent affinity for Pi. Minor shifts were also seen for mutants Thr441 -> Ala and Cys561 -> Ala (K0.5 1.5-fold increased and 1.5-fold decreased, respectively), whereas mutants Arg560 -> Glu and Leu562 -> Phe (Fig. 8), as well as Cys561 -> Trp (not shown), were indistinguishable from wild type. It is important to stress here that because the K0.5 is a function of several kinetic constants, it should not necessarily correlate with the true dissociation constant, KD, describing the non-covalent enzyme-Pi complex (E2·Pi in Scheme 1). As discussed previously (34), the respective rate constants, k2 and k-2, for formation and hydrolysis of the covalent bond in E2P may be important as well. To study k-2, the wild type and selected mutants were phosphorylated under optimal conditions for formation of the E2P phosphoenzyme, followed by dilution at 25 °C into a medium of the same composition except for the absence of radioactively labeled Pi, a reduction of the dimethyl sulfoxide concentration from 30 to 15%, and the presence of excess EDTA to remove free Mg2+ and thus terminate phosphorylation (Fig. 9 and Table II). It should be noted that because of the acidic pH, absence of K+, and presence of 15% dimethyl sulfoxide, the rate of dephosphorylation of E2P is much slower than under physiological conditions. For mutants Glu442 -> Ala and Lys515 -> Ala, the reduced apparent affinities for Pi (Fig. 8) were well accounted for by significantly increased rates of E2P hydrolysis (5- and 3.5-fold, respectively). A similar increase was noted for Lys492 -> Leu (Table II). For mutant Thr441 -> Ala, a slightly (1.4-fold) increased rate of E2P hydrolysis (Fig. 9) explains the 1.5-fold increased K0.5 (Fig. 8). Leu562 -> Phe, Phe487 -> Ser, Phe487 -> Leu, and Arg489 -> Leu also showed slight increases of the rate of E2P hydrolysis, and the Cys561 mutants (data not shown) were indistinguishable from wild type. Interestingly, mutant Arg560 -> Glu showed a conspicuous 5-fold increase of the rate of E2P hydrolysis (Fig. 9), despite its wild type-like apparent affinity for Pi (Fig. 8). In analogy with this discrepancy, mutants Arg560 -> Leu and Arg560 -> Val showed wild type-like rates of E2P hydrolysis (Fig. 9), but 2-fold increased apparent affinities for Pi (Fig. 8). Thus, for the Arg560 mutants, an increase of the true affinity for Pi (KD) or an increased rate constant for formation of the covalent bond in E2P (k2) must contribute to the change (and lack thereof) in the apparent affinity for Pi.



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FIG. 8.
Phosphorylation from Pi. Wild type and mutants were phosphorylated at 25 °C for 10 min in a medium containing 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 30% (v/v) dimethyl sulfoxide, 10 mM MgCl2, and varying concentrations of 32Pi as indicated. The lines show the best fits of the Hill equation with the Hill coefficient set to 1, giving the K0.5 values (in µM ± S.E.) indicated in parentheses: open circles, wild type (10.5 ± 0.6); open squares, Thr441 -> Ala (16.4 ± 1.5); open triangles, Glu442 -> Ala (52.8 ± 8.2); reversed solid triangles, Lys515 -> Ala (27.5 ± 5.2); open diamonds, Arg560 -> Leu (4.8 ± 0.4); solid triangles, Arg560 -> Val (5.0 ± 0.4); solid diamonds, Arg560 -> Glu (9.0 ± 1.0); reversed open triangles, Cys561 -> Ala (6.9 ± 0.4); and solid circles, Leu562 -> Phe (10.6 ± 0.9). The 100% value corresponds to the phosphorylation level reached at infinite Pi concentration as deduced from the fit.

 

Fig. 9 and Table II furthermore show that inclusion of 1 mM ATP enhanced the dephosphorylation of E2P close to 2-fold in the wild type and all the mutants studied, thus indicating that the modulatory effect of ATP on this partial reaction step (cf. boxed ATP in Scheme 1) is as unaffected by the mutations as the low affinity modulatory effect on the E1PCa2 -> E2P transition described above. Because the modulation of E2P -> E2 has been ascribed to metal-free ATP rather than the MgATP complex (14), it is important to note here that when ATP was included in the assay for dephosphorylation of E2P, the ATP was present in the metal-free form as a consequence of the co-addition of excess EDTA, unlike the assay for the E1PCa2 -> E2P transition described above, where the major fraction of the ATP was present as MgATP.

Reaction of Lys515 with FITC—FITC is a potent inhibitor of Ca2+-ATPase activity (2) and acts by specific covalent attachment to Lys515 (3), blocking high affinity ATP binding (2). The specificity of the reaction is mainly due to a high affinity of FITC for the nucleotide site rather than an unusual high reactivity of Lys515 (36). Because the side chains of Lys515 and Glu442 are in close proximity, facing each other in the crystal structure of the Ca2+-ATPase (1), we considered that functionally important interaction between them might occur in the native enzyme and that this might be revealed by testing the effect of the Glu442 -> Ala mutation on the reactivity of Lys515 toward FITC. The kinetics of the reaction of FITC with wild type and mutant Glu442 -> Ala is shown in Fig. 10A. The reaction was monitored by taking advantage of the inhibition of [{gamma}-32P]TNP-8N3-ATP photolabeling by FITC binding. At 2 µM FITC, the half-time for the reaction of wild type with FITC was ~2 min. In contrast, the reaction of mutant Glu442 -> Ala was too fast to measure, being completed in less than 15 s. At 0.2 µM FITC, the kinetics of the mutant resembled that of the wild type at 2 µM (Fig. 10A). The rapid reaction with FITC seen for the mutant could either be due to an increased binding affinity of the mutant enzyme for FITC or a true increase in the rate constant for the covalent reaction. Note that the concentration of FITC during the reaction was much lower than the KD for FITC binding (70 µM according to Ref. 36, also see below for the value of fluorescein), meaning that an increase in affinity, and proportion of bound FITC, could have a significant effect on the kinetics. To distinguish between these two possibilities, we determined whether the mutation altered the affinity for fluorescein, which is similar to FITC except it lacks the reactive isothiocyanate group. The isothiocyanate group, however, is unlikely to contribute significantly to the binding energy as it is involved in the chemical reaction. As for FITC, the binding of fluorescein was measured through the competitive inhibition of photolabeling, and the results are shown in Fig 10B. At two different concentrations of [{gamma}-32P]TNP-8N3-ATP, the KD values for fluorescein binding to wild type and mutant Glu442 -> Ala were similar, the average values being 47 and 34 µM, respectively (see figure legend). Hence, the effect of the mutation on the reaction with FITC must be largely due to an increase in the rate constant corresponding to the covalent reaction.



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FIG. 10.
Kinetics of FITC reaction (A) and concentration dependence of fluorescein binding (B) to wild type and mutant Glu442 -> Ala as determined by inhibition of TNP-8N3-ATP photolabeling. A, the FITC reaction (open symbols) with wild type (diamonds) or mutant Glu442 -> Ala (triangles and squares) was performed in 25 mM ammonium bicarbonate, pH 7.8, 1 mM MgCl2, 0.5 mM EDTA, 0.3 M sucrose, 0.2 (squares) or 2 (diamonds and triangles) µM FITC, and 10–15 pmol/ml Ca2+-ATPase. Controls (solid symbols) did not contain FITC. The reaction was terminated at the time intervals shown, by diluting aliquots 4.2-fold into ice-cold irradiation medium to yield final concentrations of 25 mM EPPS/TMAH, pH 8.5, 1.24 mM MgCl2, 0.5 mM EGTA, 0.12 mM EDTA, 20% (v/v) glycerol, 2 µM [{gamma}-32P]TNP-8N3-ATP, and 2.4–3.6 pmol/ml Ca2+-ATPase. The suspensions were irradiated for 1 min, the protein solubilized and subjected to SDS-PAGE, and the relevant bands quantified. B, inhibition of [{gamma}-32P]TNP-8N3-ATP photolabeling by fluorescein in wild type (circles) and mutant Glu442 -> Ala (triangles) as described for MgATP in the legend to Fig. 5. The concentrations of [{gamma}-32P]TNP-8N3-ATP were close to the K0.5 (0.79 µM for wild type, 0.46 µM for mutant Glu442 -> Ala, cf. Table I) (solid symbols) or to 0.25x K0.5 (open symbols). The lines show the best fits using the Hill equation. Average true KD values for fluorescein binding of 47 µM and 34 µM for wild type and Glu442 -> Ala, respectively, were derived from the K0.5 values at the two concentrations of photolabel using the competitive binding equation provided in Ref. 5.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we show for the first time, using a direct binding assay, that amino acids Thr441 and Glu442 of the 441TETAL helix and Lys515, Arg560, and Leu562 of two central {beta}-strands, all in domain N, are crucial to MgATP binding at the substrate site. Subsequent catalytic steps were also affected by the mutations. Arg560 seems to play a critical role in optimizing the rate of phosphoryl transfer from the substrate to Asp351, and the Arg560 -> Val mutation furthermore enhanced the rate of the Ca2+-translocating E1PCa2 -> E2P conformational transition, whereas this transition was slowed substantially by alteration to Thr441, Glu442, Lys515, or Lys492. Moreover, some of the mutations influenced the hydrolysis of E2P. On the other hand, none of the residues examined seems very important for the binding of modulatory nucleotide to the phosphoenzyme.

The crystal structure of the Ca2+-ATPase has revealed that domain N is composed of a seven-stranded anti-parallel {beta}-sheet arranged as a half-barrel wrapped around two central helices, with sequences 407FDGLVELATICALCN421 and 441TETALTTLVEKMN453 (helices labeled A and B, respectively, in Fig. 11). The {beta}-sheet is made up of two sections; one is linked to two hinge segments, incorporating Asp601 and Asn359 leading to and from domain P. The inner edge/strand of this portion of the sheet is made up of segment 560RCLALA. The second part of the sheet, incorporating segments 487FSRDRK and 515KGAPE, protrudes from the domain. Our results here, in conjunction with a previous study (5), support the hypothesis that the MgATP high affinity binding site is composed of elements of 441TETAL, 487FSRDRK, 515KGAPE, and 560RCLALA. The adenine five-membered ring could fit in the gap between the side chains of Phe487 and Leu562 (cf. Fig. 11), such that, depending on the orientation, the 8- or 2-position of the base faces Lys492, in the former case accounting for the photolabeling of this residue with TNP-8N3-ATP. Glu442 and Lys515 are buried deep in the binding pocket and may interact with the six-membered ring of the adenine moiety. The fact that even the bulky tryptophan replacement of Cys561 was without much influence on nucleotide binding supports these ideas, as Cys561 points away from the putative binding pocket formed by the critical residues. The bound nucleotide would link the structural elements of the nucleotide-binding site in a manner that could stabilize a closer interaction, as part of the nucleotide-induced conformational change previously detected (37, 38, 39, 40).



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FIG. 11.
Positions of key residues discussed, in the E1Ca2 crystal structure of the Ca2+-ATPase. The view is down toward the membrane from the cytoplasmic side. Carbon atoms are shown in gray, nitrogen in blue, oxygen in red, sulfur in yellow, and phosphorus in orange. Schematic representations of {alpha}-helices and {beta}-strands are shown in red and cyan, respectively. Residues discussed in the text are labeled. An ATP molecule in a conformation that could fit into the nucleotide-binding site is depicted in the top-left corner. N, nucleotide-binding domain; P, phosphorylation domain. Helices labeled A and B indicate the two central N-domain helices with sequences 407FDGLVELATICALCN421 and 441TETALTTLVEKMN453, respectively. Prepared by use of WebLab Viewer Pro (Molecular Simulations Ltd., Cambridge, England), using the atomic coordinates obtained from the Protein Data Bank under accession code 1EUL [PDB] (1).

 

Thr441, Glu442, and Leu562 cluster together in the binding pocket, and the consequences of mutations here for nucleotide binding were rather similar (Table I). MgATP binding was strongly disturbed (70–180-fold increase of KD), whereas the affinity for uncomplexed ATP was much less affected (only 2–5-fold increase of KD). The resulting affinities for ATP and MgATP were very similar, thus demonstrating that these mutants are rather insensitive to Mg2+. For Thr441 -> Ala, the KD values for ADP and MgADP also differed significantly less (~2-fold) as compared with wild type (~7-fold). Furthermore, the ability to respond to Mg2+ with a reduced affinity for the TNP-nucleotide was lost in Thr441 -> Ala. One possible cause of these effects is interference with the interaction between the protein and the Mg2+ ion complexed by the nucleotide, implying that at least Thr441 could be a Mg2+-ligating residue in the initial encounter of the nucleotide with the binding site. This possibility is consistent with recent evidence from Fe2+-catalyzed oxidation, suggesting that the metal ion of FeATP interacts close to Thr441 in the Ca2+-ATPase and in the Na+, K+-ATPase close to the residue equivalent to Glu439 (41, 42). Interestingly, mutations of Thr353 in domain P are also more detrimental to MgATP binding as compared with ATP binding, and in some non-ATPase members of the family of aspartyl phosphate-utilizing phosphohydrolases and phosphotransferases the residue equivalent to Thr353 of the Ca2+-ATPase has been shown to be involved in Mg2+ ligation (see Ref. 34 and references therein). On the basis of the crystal structure (Fig. 11), it is not unrealistic to think that upon nucleotide binding the Thr441/Glu442/Leu562 cluster could come close to Thr353 or that the Mg2+ ion initially complexed with ATP could move from a site near Thr441 to a site involving Thr353. As an alternative, the preferential loss of affinity for the Mg2+-complexed forms of ATP and ADP seen for Thr441 -> Ala, Glu442 -> Ala, and Leu562 -> Phe could be explained without having to assume a direct interaction with Mg2+, if these residues interact only with the nucleotide per se, and the Mg2+ ion complexed by the nucleotide assists the docking of the phosphoryl part in domain P in such a way that it changes the position of the rest of the nucleotide and, thus, its interaction with the mutated residues. Thereby, the contributions of these residues to the binding energy would depend on Mg2+. In other words, the site binding ATP differs somewhat from the binding site for the MgATP complex.

In contrast to the above-mentioned mutants, pronounced effects on nucleotide binding in the absence of Mg2+ were seen for mutants Arg489 -> Leu and Lys492 -> Tyr (Table I). In this connection, it is interesting to note that Lys492 is cross-linked to Arg678 in domain P by glutaraldehyde in what is likely to be a zero distance linkage (43), and the clustering of the three positive charges of Arg489, Lys492, and Arg678 could make interaction with phosphates energetically very favorable. This is also where decavanadate binds (1, 44). Thus, at least in the absence of Mg2+, parts of the phosphate chain could interact with these basic residues.

Mutation of Arg560 to leucine was shown to be detrimental both to ATP and ADP binding, in the absence as well as in the presence of Mg2+ (Table I). The very large effects of the Arg560 -> Leu mutation on ADP and MgADP binding support a role for the positive charge of the guanidino group of Arg560 in interaction with the {beta}-phosphoryl, the {alpha}-phosphoryl, ribose, or adenine. Neither of these possibilities can be excluded on the basis of the two published crystal structures of the enzyme. Modeling of MgATP into the structure of the E2 form with bound thapsigargin, bringing the Mg2+ ion complexed between the {beta}- and {gamma}-phosphates of ATP close to the side-chain oxygens of Thr441 and Glu442, suggested contribution of Arg560 to ligation of adenine (41). In E1Ca2 crystals soaked with TNP-AMP (1), Arg560 is pointing toward the TNP moiety, suggesting that Arg560 could be interacting with the 2'- and/or 3'-hydroxyl of the ribose in ATP. Our data on TNP-nucleotide binding to Arg560 mutants in the presence of Mg2+ are compatible with the interaction between the TNP moiety and Arg560 observed in the crystals, in the presence of Mg2+. Rather surprisingly, in Arg560 -> Leu, the binding of TNP-8N3-ATP was hardly disturbed in the absence of Mg2+. Hence, Arg560 is not essential to the binding of the TNP moiety in this condition, lending further support to the hypothesis that Mg2+ changes the position of bound nucleotide.

The role of Arg560 does not seem to be confined to merely binding the nucleotide. The Arg560 mutants showed substantially reduced steady-state levels of phosphoenzyme (Figs. 1 and 2A) and of acetyl phosphate-supported Ca2+ transport (Fig. 4). It has been demonstrated for the wild type that in addition to the rate of the E1PCa2 -> E2P transition (cf. Scheme 1), the rate of formation of E1PCa2 is limiting for the Ca2+ transport rate attainable with acetyl phosphate as substrate (16). We were able to demonstrate that the E1PCa2 -> E2P transition rate is, in fact, enhanced in Arg560 -> Val (Fig. 7). Thus, it seems likely that the rate of formation of E1PCa2 is reduced in this mutant. Furthermore, mutation Arg560 -> Val was less detrimental to MgATP binding (40-fold decrease of affinity) than the mutations of Thr441 and Glu442 (>120-fold decrease of affinity, Table I), and yet the K0.5 for MgATP dependence of phosphorylation was much higher for Arg560 -> Val than for the latter mutants (Fig. 2A). The K0.5 depends on the intrinsic (true) affinity for MgATP, determined in the binding assay, as well as the rate constant for transfer of the phosphoryl group from ATP to the enzyme. Therefore, the high K0.5 observed for Arg560 -> Val may reflect a role of Arg560 in the latter reaction. The low ATPase activity, even at saturating MgATP concentrations, and lack of activation by calcium ionophore, seen in all the Arg560 mutants (Fig. 1), is also consistent with a slow, rate-limiting phosphorylation step, because activation by ionophore requires that the E1PCa2 -> E2P transition, sensitive to inhibition by lumenal Ca2+, is rate-limiting (26). It all points to mutations of Arg560 as affecting not only nucleotide binding but also phosphoryl transfer. This could mean that in the transition state existing during phosphoryl transfer, Arg560 has important interaction with the {gamma}-phosphoryl group of ATP in addition to the {beta}-phosphoryl, in line with a suggestion for the corresponding arginine of the Na+,K+-ATPase (45). There are, however, alternative possibilities. On the basis of the crystal structure of E1Ca2, one can predict that Arg560 could come very close to Asp627 when domain N approaches domain P for phosphorylation. Because Asp627 points toward Lys352 (cf. Fig. 11), a critical ATP binding and catalytic residue (33), the Arg560–Asp627 interaction may be necessary to allow Lys352 to interact with the substrate. It is understandable in this scenario how mutation of the arginine could affect phosphorylation.

Unlike the Arg560 mutants, Leu562 -> Phe showed a normal rate of acetyl phosphate-supported Ca2+ transport (Fig. 4), despite markedly reduced rates of ATP-driven Ca2+ transport and ATPase activity, and lack of ionophore activation of the latter reaction (Fig. 1). This means that the role of Leu562 to a large extent is nucleotide-specific. In Leu562 -> Phe, the rate of phosphoryl transfer may very well be reduced relative to wild type with MgATP as substrate but not with acetyl phosphate as substrate. A reduced rate constant for phosphoryl transfer from MgATP in Leu562 -> Phe would be consistent with the finding that the K0.5 for MgATP dependence of phosphorylation was higher for Leu562 -> Phe than for Glu442 -> Ala (Fig. 2A), whereas the KD value for MgATP determined in the binding assay was highest for Glu442 -> Ala (Table I).

Lys515 lies deep in the binding cleft and is the residue that reacts with FITC (3). Mutation of Lys515 to alanine prevented TNP-8N3-ATP photolabeling of Lys492, showing that the adenine positioning in relation to the latter residue was disturbed. CrATP binding was also eliminated in Lys515 -> Ala (Fig. 6), indicating that the binding deficiency is not confined to the TNP-nucleotide. Glu442 points toward Lys515 in the crystal structure (Fig. 11), and we found that the reactivity of the amino group of Lys515 was profoundly increased by mutation Glu442 -> Ala, indicating that the amino group was changed into the more reactive uncharged form as a consequence of the elimination of the charge on residue 442. This suggests that in the native wild-type enzyme the two residues are ion-paired, forming a salt link, in accordance with the structure of the crystallized enzyme (1). Ion pairs are not favored over the neutral forms in a low dielectric medium, unless strengthened by additional hydrogen bonding (46). It is evident from the crystal structure that these two residues are both likely to hydrogen-bond to backbone amides and/or carbonyls at the end of the 407FDGLVELATICALCN421 helix (Fig. 11). Strengthening the salt link may be important for the nucleotide-induced conformational change alluded to above, thus aiding the interaction of the secondary flange, where Lys515 resides, with the 441TETAL helix. Thus the lysine and the glutamate may have dual roles, one in nucleotide ligation and the other as a clasp linking two key structural elements in the binding site.

For Glu442 -> Ala and Lys515 -> Ala, the reduced kobs values for phosphorylation seen in the presence of 5 µM MgATP (Fig. 2B) seem to be well explained by the poor nucleotide affinity, resulting in a low fraction of enzyme with bound nucleotide. In Thr441 -> Ala, on the other hand, the rate constant for phosphoryl transfer from MgATP may be somewhat reduced, relative to Glu442 -> Ala, because the K0.5 for MgATP dependence of phosphorylation (Fig. 2A) and the kobs for phosphorylation (Fig. 2B) were more strongly affected in Thr441 -> Ala than in Glu442 -> Ala, whereas the opposite was the case for the KD for MgATP determined in the binding assay (Table I) and the ability to bind CrATP (Fig. 6).

As demonstrated by the data in Fig. 7 and Table II, Thr441 -> Ala, Glu442 -> Ala, Lys492 -> Leu, and Lys515 -> Ala showed a conspicuous 6–8-fold reduction of the rate of the E1PCa2 -> E2P conversion. This contrasts with the 2-fold enhancement seen for Arg560 -> Val (for Arg560 -> Glu and Arg560 -> Leu, the E1PCa2 -> E2P transition could not be studied, because of the low phosphorylation level with nucleotide substrate). The strongly reduced rate of the E1PCa2 -> E2P conversion explains the decrease in acetyl phosphate-supported Ca2+ transport observed for Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala (Fig. 4), without necessarily having to assume a reduced rate of the phosphorylation step in these cases. Moreover, the reduced maximum rate of ATP hydrolysis seen with these mutants (Fig. 3) can be explained by the reduced rate of the E1PCa2 -> E2P conversion, and the enhancement seen upon addition of ionophore (Fig. 1) is consistent with a rate-limiting role of the E1PCa2 -> E2P conversion (26), in contrast to the lack of ionophore effect seen for the mutants with alteration to Arg560 or Leu562.

Previously, a number of point mutations, mainly located in the cytoplasmic domains A and P and in the segment connecting transmembrane segment M4 with domain P, have been shown to inhibit the E1PCa2 -> E2P transition (22, 25, 28, 34, 47, 48, 49). Thr441 -> Ala, Glu442 -> Ala, Lys492 -> Leu, and Lys515 -> Ala are the first N-domain mutants for which this characteristic is reported. The reduced rate of the E1PCa2 -> E2P transition could reflect a general destabilization of interor intra-domain interaction specific to the E2P conformation. In the wild-type enzyme, the E1PCa2 -> E2P transition seems to lead to tight and specific interaction of the conserved 181TGES motif of domain A with the phosphorylation site, thereby changing the interactions of domain N (1, 50). A glutaraldehyde cross-link between Lys492 and Arg678, tying the N and P domains together, prevents this docking of domain A in domain P (43). The recently published crystal structure of the Ca2+-ATPase dephosphoenzyme in E2 form with bound thapsigargin (51) shows that the end of the 441TETAL helix is only 7 Å away from the glutamate of the 181TGES motif of domain A, and a further approach might be expected in E2P. Alteration to the structure of domain N induced by the mutations could conceivably interfere with the change of its interaction with domain P and domain A in relation to the E1PCa2 -> E2P transition and, possibly, also with the docking of domain A in domain P. For Glu442 -> Ala and Lys515 -> Ala, disruption of the Glu442–Lys515 salt link may be an important contributing factor, whereas Thr441 -> Ala might affect more directly the position of the 441TETAL helix. Moreover, a defective Mg2+ coordination, as suggested above for Thr441 -> Ala, could contribute to the inhibition of the E1PCa2 -> E2P transition, as a normal rate of this step seems to require the binding of Mg2+ (52).

For Glu442 -> Ala, Lys492 -> Leu, Lys515 -> Ala, and Arg560 -> Glu, we also observed a significant enhancement of the hydrolysis of E2P (Fig. 9 and Table II). The location of Arg560 rather close to domain P is likely important for the effect of the Arg560 -> Glu mutation on the hydrolysis of E2P, as well as for the increased true affinity for Pi (KD) or increased rate of phosphorylation from Pi (k2) deduced for the Arg560 mutants on the basis of the E2P hydrolysis rate in conjunction with apparent Pi affinity (see "Results"). For the mutants with a slowed E1PCa2 -> E2P transition, the enhanced hydrolysis of E2P may on the other hand result from the more general destabilization of the E2P conformation discussed above.

In addition to being the phosphorylating substrate, MgATP and/or ATP exert modulatory effects on various steps of the pump cycle (boxed ATP in Scheme 1). Hence, in the wild-type Ca2+-ATPase, the MgATP dependence of ATPase activity exhibits at least three activation phases, corresponding to high affinity (0–10 µM), intermediate affinity (10–200 µM), and low affinity (>200 µM). The high affinity phase reflects MgATP binding to the E1Ca2 form as phosphorylating substrate. The low affinity activation phase reflects a modulatory effect of nucleotide on the E1PCa2 -> E2P transition (13, 15, 16), whereas the intermediate phase most likely is due to modulation of the Ca2+-binding E2 -> E1Ca2 conformational transition of the dephosphoenzyme (10, 26, 53, 54), cf. Scheme 1. In addition, ATP in the submillimolar concentration range enhances E2P hydrolysis (9, 12, 14). There is evidence that the latter effect is exerted only by free ATP and not MgATP (14), which implies a rather low apparent affinity in the presence of Mg2+. Furthermore, E2P hydrolysis is so rapid in the wild type, at pH values close to neutral and in the presence of K+, that it only contributes to rate limitation of the overall reaction at MgATP concentrations where the modulatory effect on the E1PCa2 -> E2P transition is fully implemented (13). In a titration curve such as that shown in Fig. 3, the modulatory effect on E2P hydrolysis should, therefore, be apparent only for the highest MgATP concentrations. A subject of much controversy is the question whether the phosphorylating and modulatory MgATP/ATP molecules are at the same locus, exhibiting variable affinity during the transport cycle depending on conformational state, or a separate low affinity allosteric site exists on the same Ca2+-ATPase polypeptide chain (14, 29, 55, 56, 57, 58). A similar discussion exists for the closely related Na+, K+-ATPase, although in this case it seems that only the E2(K2) -> E1(Na3) transition and not phosphoenzyme processing is modulated by nucleotide (59, 60, 61, 62).

To the extent it could be studied, the intermediate activation phase, reflecting modulation of the E2 -> E1Ca2 conformational transition of the unphosphorylated Ca2+-ATPase, seemed less pronounced or right-shifted in the mutants that showed reduced MgATP binding affinity at the substrate site (Fig. 3), consistent with the evidence that only one site per polypeptide chain is available for binding of nucleotide within the high and intermediate affinity ranges (14, 29). On the other hand, mutants Arg560 -> Leu, Arg560 -> Val, Leu562 -> Phe, Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala all showed a distinct low affinity modulatory effect of nucleotide on the overall ATPase reaction and on the rate-limiting E1PCa2 -> E2P transition, as well as a modulatory effect on the hydrolysis of E2P, despite the fact that a conspicuous lowering of nucleotide affinity was seen for the substrate site of the dephosphoenzyme in these mutants, as discussed above. There was no evidence for a significant lowering of the apparent affinity with which MgATP or ATP modulates the E1PCa2 -> E2P transition in any of these mutants, as the increase of MgATP concentration from 1 to 5 mM induced a wild type-like 2-fold enhancement in all cases (Fig. 7 and Table II). The tendency for the ATPase activity of Thr441 -> Ala, Glu442 -> Ala, and Lys515 -> Ala to level off more abruptly than that of the wild type at the highest MgATP concentrations (Fig. 3) is likely due to the very strong rate limitation by the E1PCa2 -> E2P transition in these mutants, which prevents the ATP modulation of E2P hydrolysis from contributing significantly to the overall activity, because E2P hydrolysis is already much faster than the E1PCa2 -> E2P transition without ATP modulation. When the nucleotide dependence of phosphoenzyme processing was examined for the mutants with alterations to Phe487, Arg489, and Lys492 (Table II), we also found a significant modulatory effect, although slightly less pronounced than for wild type. Hence, our results seem to imply that none of the residues studied here are critical to the ligation of modulatory nucleotide in the phosphoenzyme. It is interesting to note the resemblance of the MgATP dependences of ATPase activity of the mutants to data obtained for wild type by titration with MgATP deoxy analogues lacking the 2'- or 3'-hydroxyl group on the ribose (63). Also, in these cases, the affinities corresponding to the substrate site of the dephosphoenzyme were lowered, whereas the activation phase above 200 µM was virtually unaltered (63). These data, as well as our results, seem to support the hypothesis that the modulatory site on the phosphoenzyme differs substantially from the substrate site. On the other hand, the "plasticity" exhibited by the substrate site, as seen by the dependence of the mutational effects on the nucleotide studied, calls for some caution of interpretation. We have discussed above that ATP may bind differently from MgATP. Furthermore, it is noteworthy that TNP-8N3-MgATP can function as a substrate supporting efficient Ca2+ transport and is hydrolyzed even when tethered at Lys492 (64), although the data in Table I clearly indicate that TNP-8N3-MgATP is not bound exactly as MgATP. Thus, the adenosine portion of the substrate may not need to occupy a very precise locus, and the same could be even more applicable to the modulatory nucleotide bound with low affinity. It is possible that the modulation of the kinetics of the conformational change and hydrolysis of the phosphoenzyme arise primarily from electrostatic interaction between the covalently attached phosphoryl group and the phosphates of the modulatory ATP. The principal requirement might, thus, be that the {gamma}-phosphate or {beta}- and {gamma}-phosphates is/are near the phosphoryl group covalently attached at Asp351. This notion is supported by the finding that ATP, AMP(CH2)PP, or PPi, but not ADP or Pi, exert modulatory effects on ATPase activity (65). Because ATP and not MgATP seems to bind to E2P, which contains Mg2+ tightly bound together with the covalently attached phosphoryl group (14), it may be speculated that one of the major interactions responsible for the low affinity binding of nucleotide to the phosphoenzyme occurs between ATP and the Mg2+ already bound. In this scenario, there may not be very specific requirements for amino acid side chains in domain N to ligate the adenosine part of the modulatory nucleotide. At the moment this seems an attractive working hypothesis as an alternative to invoking a second site at a location completely distinct from that of the substrate site.


    FOOTNOTES
 
* This work was supported in part by grants from the Danish Medical Research Council, the Novo Nordisk Foundation, Denmark, the Lundbeck Foundation, Denmark, the Research Foundation of Aarhus University (to J. P. A. and B. V.), and the National Research Foundation of South Africa (to D. B. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

¶ || ¶ To whom correspondence may be addressed: Division of Chemical Pathology, Dept. of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa. Fax: 27-21-4488150; E-mail: davidmci{at}chempath.uct.ac.za.|| To whom correspondence may be addressed: Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark. Fax: 45-86129065; E-mail: jpa{at}fi.au.dk.

1 The abbreviations used are: Ca2+-ATPase, the sarco(endo)plasmic reticulum Ca2+-transporting adenosine triphosphatase (EC 3.6.1.38 [EC] ); CrATP, {beta},{gamma}-bidentate chromium(III) complex of ATP; E1, enzyme form with cytoplasmically facing high affinity Ca2+-binding sites; E2, enzyme form with low affinity for Ca2+; E1PCa2, phosphoenzyme intermediate containing Ca2+ in the occluded state; E2P, phosphoenzyme intermediate with luminally facing low affinity Ca2+-binding sites; EPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid; FITC, fluorescein isothiocyanate; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; TES, N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid; TMAH, tetramethylammonium hydroxide; TNP-AMP, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-monophosphate; TNP-8N3-ATP, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate. Back


    ACKNOWLEDGMENTS
 
We thank Lene Jacobsen, Karin Kracht (Aarhus, Denmark), and Irene Mardarowicz (Cape Town, South Africa) for expert technical assistance. We also thank Dr. R. J. Kaufmann (Genetics Institute, Boston) for the gift of the expression vector pMT2.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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