The Role of the M6-M7 Loop (L67) in Stabilization of the Phosphorylation and Ca2+ Binding Domains of the Sarcoplasmic Reticulum Ca2+-ATPase (SERCA)*

Zhongsen Zhang, David Lewis, Carlota Sumbilla, and Giuseppe InesiDagger

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, November 30, 2000, and in revised form, February 2, 2001

Chikashi Toyoshima

From the Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Received for publication, November 30, 2000, and in revised form, February 2, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca2+ transport ATPase was subjected to mutational analysis. Mutation of Pro820 to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys819 or Arg822 to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with enzyme phosphorylation by ATP in the presence of Ca2+, and also by Pi in the absence of Ca2+. The Lys819 and Arg822 mutants undergo slight and moderate reduction of Ca2+ binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp813, Asp815, and Asp818 to Asn interferes strongly with enzyme activation by Ca2+ binding and formation of phosphorylated enzyme intermediate by utilization of ATP. Enzyme phosphorylation by Pi in the absence of Ca2+ undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca2+ binding domain through a network of hydrogen bonds.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The sarcoplasmic reticulum (SR)1 ATPase is a membrane-bound enzyme that plays a crucial role in sequestration of cytosolic Ca2+ in muscle fibers (1-3). The catalytic and transport cycle begins with high affinity binding of two Ca2+ per ATPase, whereby the enzyme is activated and proceeds to utilization of ATP. Enzyme phosphorylation by ATP induces vectorial translocation of bound Ca2+, and the cycle is then completed by hydrolytic cleavage of the phosphoenzyme. The SR ATPase contains 994 amino acids (4), folded to form 10 transmembrane segments (M1-M10) and a large extramembranous (cytosolic) region (5, 6). Mutational (7) and structural studies (8) have shown that the Ca2+ binding domain resides within the membrane-bound region of the ATPase, at a 50-Å distance from the phosphorylation domain in the cytosolic region of the enzyme. Functional interdependence of Ca2+ and phosphorylation domains occurs through long range intramolecular linkage (9).

The cytosolic region of the ATPase includes a short N-terminal segment (Met1-Glu58) and the loop (Trp107-Ser261) between the M2 and M3 transmembrane segments, folded in the A domain. Furthermore, the cytosolic region includes the large loop between the M4 and M5 transmembrane segments, folded in separate P (phosphorylation) and N (nucleotide binding) domains (8). Although the loops between the remaining transmembrane segments are relatively small, attention has been brought to L67 (Phe809-Gly831) by Falson et al. (10) and Menguy et al. (11), who found that cluster mutations of aspartic residues to alanine raise the Ca2+ concentration required for ATPase activation. Our interest on L67 was heightened by its close relationship with the P domain (8), and the major role played by the contiguous M6 segment in Ca2+ binding (12). Therefore, we performed a detailed mutational analysis of L67.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DNA Constructs and Vectors-- The chicken fast muscle SR ATPase (SERCA-1) cDNA (13) was inserted into the pUC19 plasmid for amplification, and then subcloned into the pSELECT-1 vector for site directed mutagenesis. Mutations were carried out by the Altered Sites in vitro mutagenesis system made available by Promega (Madison, WI), or by overlap extension using the polymerase chain reaction.

Wild type and mutated cDNA was subcloned into the shuttle plasmid pDelta E1sp1A (Microbix BioSystems). In the final constructs, the cDNA was preceded by the SV40 or the cytomegalovirus promoter, and followed by the SV40 polyadenylation signal. The shuttle plasmids were either used directly for transfection of COS-1 cells by the DEAE-dextran method, or for cotransfection of HEK293 cells in conjunction with the replication defective adenovirus plasmid pJM17 (Microbix BioSystems) to obtain recombinant adenovirus vectors (14). Alternatively, cDNA constructs were subcloned into pAd-lox shuttle vector and cotransfected with purified Psi 5 adenovirus genome in CRE8 cells derived from the HEK293 line. CRE8 cells constitutively express the Cre recombinase, which catalyzes efficient recombination between loxP sites in the Psi 5 genome and in the pAd-lox to yield recombinant adenovirus (15). The recombinant products were plaque-purified and cesium-banded, yielding concentrations on the order of 1010 plaque-forming units/ml.

Cell Cultures and Transfections-- Cultures of HEK293, CRE8, and COS-1 cells were maintained as described by Graham and Prevec (14), Hardy et al. (15) and Sumbilla et al. (16), respectively. The growth medium for COS-1 cells was Life Technologies, Inc. Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and containing Penn-Strep (100 units/ml) and Fungizone (1 µg/ml).

Transfections of COS-1 cells with wild type or mutated SERCA-1 cDNA, subcloned into the shuttle vector pDelta E1sp1A or pAd-lox, were conducted by the DEAE-dextran method (16).

Recombinant adenovirus vectors were used for infection of COS-1 cells as described by Zhang et al. (12).

Northern blots were performed with total RNA obtained by aspirating the medium from cell culture dishes, adding 0.3-0.4 ml of Trizol-LS (Life Technologies, Inc.)/10-cm2 cell lawn, and following the Life Technologies procedure. The final mRNA pellet was dissolved in diethyl pyrocarbonate-water (Quality Biological) to yield ~1 mg of RNA from one 150-mm dish.

Electrophoresis and blotting were performed by the use of a Northern Max-Gly blotting kit from Ambion, and mRNA detection was obtained with DIG High Prime labeling and detection kit from Roche Molecular Biochemicals. Alternatively, a 32P-labeled probe was used. A SERCA-1 cDNA cassette was used as a template for the probe.

Microsome Preparation and Immunodetection of Expressed Protein-- The procedure for microsome preparation from infected COS-1 cells was as described by Autry and Jones (17), and the final product was stored in small aliquots at -70 °C. The total microsomal protein was determined using bicinchoninic acid with the Biuret reaction (Pierce). The expressed SERCA-1 ATPase was detected by Western blotting (12). Quantitation of immunoreactivity was obtained by densitometry, and standardized with samples of wild type ATPase to be used as controls for the functional studies.

Functional Studies-- ATPase hydrolytic activity was assessed by measuring Pi production (18). The reaction mixture contained 20 mM MOPS, pH 7.0, 80 mM KCl, 10 mM MgCl2, 5 mM sodium azide, 1.0 mM EGTA, and CaCl2 to yield various free Ca2+ concentrations, 10-30 µg of microsomal protein, and 3 µM A23187 Ca2+ ionophore. The reaction was started by addition of 3.0 mM ATP and run at 25 °C temperature. Serial samples were taken every 2 min for 12 min. Due to the presence of the Ca2+ ionophore, the ATPase reaction proceeded at constant velocity, yielding linear plots of Pi production.

Steady state levels of phosphorylated intermediate by utilization of ATP was obtained in the presence of 20 mM MOPS, pH 7.0, 80 mM KCl, 10 mM MgCl2, 30 µg of microsomal protein, 5 µM Ca2+, and 5 µM [gamma -32P]ATP, in a total volume of 50 µl. The reagents were pre-cooled in ice. The reaction was started by the addition of ATP, run for 10 s at 2-3 °C temperature, and quenched by the addition of 1.0 ml of cold 1 M PCA. Rapid mixing upon addition of ATP and PCA was obtained by vortexing. The quenched samples were transferred into a 1.7-ml Eppendorf tube containing 100 µg of bovine serum albumin as carrier protein, and placed in ice. The samples were then spun in a refrigerated clinical centrifuge at 5000 rpm for 5 min, and the sediments ware washed three times with 1.0 ml of cold 0.125 MPCA and once with cold water. The final pellets were dissolved in 40 µl of denaturing buffer (50 mg of lithium dodecyl sulfate, 0.01 ml of 2-mercaptoethanol, and 0.05 ml of Weber-Osborn buffer per ml) and 10 µl of tracking dye solution (1 mg of bromphenol blue and 0.3 g of sucrose per ml). The entire samples were then run on 6.5% acrylamide gels at pH 6.2, with a current limit of 100 milliamperes, at 15 °C temperature. The radioactive phosphoenzyme was detected both by autoradiography and phosphoimaging.

The time course of phosphoenzyme formation following addition of ATP and Ca2+, or ATP to enzyme preincubated with Ca2+, was determined by preincubating 30 µg of WT protein, or corresponding aliquots of mutant ATPase (as indicated by Western blot analysis) at 3 °C. The reaction medium contained 20 mM MOPS, pH 7.0, 80 mM KCl, 10 mM MgCl2, and 1 mM EGTA in the absence or in the presence of 1 mM CaCl2. The reaction was started by the addition of 1 µM [gamma -32P]ATP and 1 mM CaCl2, or 1 µM [gamma -32P]ATP. Acid quenching at serial times, washing, and detection of phosphorylated intermediate was obtained by electrophoresis, autoradiography, and phosphoimaging. The time of autoradiographic exposure was the same for all samples, so as to reveal differences in phosphoenzyme levels, if any.

The time course of radioactive phosphoenzyme decay was determined by adding a chase pulse of 0.5 ml of 1.0 mM non-radioactive ATP, 10 s following the initial addition of radioactive ATP. Several samples were obtained by quenching the reaction at serial time with PCA. Radioactive phosphoenzyme was then determined by electrophoresis, autoradiography, and phosphoimaging.

Reverse enzyme phosphorylation by Pi was obtained by adding 30 µg of microsomal protein to 0.2 ml of reaction mixture containing 50.0 mM MesTris, pH 6.2, 10 mM MgCl2, 20.0% Me2SO, and 2.0 mM EGTA. Alternatively, the EGTA was omitted and 10, 50, or 100 µM CaCl2 was added. Following a 10-min incubation at 25 °C, 1.0 ml of 1.0 M ice-cold PCA was added, the samples were transferred into a 1.7-ml Eppendorf tube containing 100 µg of bovine serum albumin as a carrier protein, and placed in ice. Centrifugation, washing, electrophoresis, and detection of phosphoenzyme were then conducted as described above for enzyme phosphorylation with ATP.

Measurements of Ca2+ binding to recombinant ATPase in the absence of ATP were performed in microsomes obtained from COS-1 cells infected with adenovirus vectors (as opposed to simple transfections), taking advantage of the higher concentration of recombinant ATPase in these samples. The measurements were performed exactly as described by Zhang et al. (12), in the presence of 3 µM free Ca2+, as determined by an EGTA-Ca buffer. The measured Ca2+ binding levels were adjusted to compensate for variations of recombinant ATPase expression in various preparations, with reference to a wild type preparation as indicated by Western blots. The difference between samples incubated in the absence and in the presence of thapsigargin was considered to be specific Ca2+ binding.

Computations-- Calculation of free Ca2+ in various reaction mixtures were based on the concentrations of total calcium and EGTA as originally described by Fabiato and Fabiato (19).

Simulations of steady state kinetics, yielding overall ATPase velocity and levels of intermediate species, were based on user entered rate constants and concentrations of substrate, ligands, and products, as described by Inesi et al. (20). Computations were performed on a PC microcomputer, using a 14-digit precision MEGABASIC with BCD coding (American Planning Corp., Alexandria, VA).

Copies of the computational programs can be obtained from M. Kurzmack (La Porte, CO).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of Mutants-- L67 includes the Gly808-Gly832 segment of the ATPase sequence, in which we produced several site directed mutations (Table I). As a comparison, we also mutated residues in the small M8/M9 loop (Ser917-Glu920). Expression of all mutants in COS-1 cells was similar to, or somewhat (10-30%) lower than, that of WT ATPase. An exception was the Pro820 right-arrow Ala mutant, whose protein recovery was negligible, even though its mRNA level was comparable to that produced by WT cDNA (Fig. 1). It is of interest that Menguy et al. (11) obtained expression of a totally inactive Pro820 right-arrow Ala mutant. This suggests that mutation of Pro820 interferes profoundly with protein folding, resulting in degradation of product by the COS-1 cells used for expression in our experiments, and production of inactive protein by the yeast cells used by Menguy et al. (11).

                              
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Table I
List of mutants, ATPase activity (3 µM free Ca2+), steady state levels of phosphoenzyme obtained in the presence of ATP and Ca2+, and equilibrium levels of phosphoenzyme obtained in the presence of Pi and no Ca2+
The values are given in percentage of WT.


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Fig. 1.   Western and Northern blots showing protein and mRNA levels obtained in COS-1 cells transfected with WT or P820A cDNA. Identical amounts of microsomal protein (30 µg) and total RNA (30 µg), derived from COS-1 cells transfected with WT SERCA-1A cDNA or P820A mutant, were subjected to Western or Northern blot analysis, as described under "Materials and Methods."

Functional Characterization of Aspartate Mutants-- Falson et al. (10) and Menguy et al. (11) reported that cluster mutations of Asp813, Asp815, and Asp818 to Ala reduce the affinity of the ATPase for Ca2+, an effect that was confirmed by direct measurements of Ca2+ binding by equilibrium experimentation (12). We now find that even conservative mutation of the Asp813, Asp815, and Asp818 cluster to Asn produces strong inhibition of ATPase steady state velocity, and maximal activity cannot be reached even by raising the Ca2+ concentration to the 0.1 mM level (Fig. 2A). The effect of the triple mutant can be to a large extent reproduced (data not shown) by single mutation of Asp813 (but not Asp815 or Asp818) to Asn.


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Fig. 2.   Plots of ATPase velocity as a function of Ca2+ concentration. ATPase velocities of WT enzyme and mutants were obtained by serial determination of Pi production, as described under "Materials and Methods." The ATP concentration was 3 mM, and the free Ca2+ concentration was obtained with EGTA-Ca buffers. The ATPase velocities were obtained from linear plots of several time points, and then corrected to reflect the concentrations of mutant and WT enzyme in different microsomal preparations. Average values obtained from three different measurements are listed in Table I.

The triple aspartate mutation strongly reduces the steady state levels of phosphorylated intermediate formed with ATP in the presence of Ca2+ (Fig. 3 and Table I). However, it affects much less prominently enzyme phosphorylation by Pi in the absence of Ca2+ (Fig. 4). In agreement with previous findings (12), direct measurements with radioactive tracer show that the cluster mutation of Asp813, Asp815, and Asp818 to Asn displays very low levels of high affinity Ca2+ binding in the presence of 3 µM Ca2+ (Table II). Direct measurements of Ca2+ binding cannot be performed at higher Ca2+ concentrations due to unfavorable signal to noise ratio. Nevertheless, we found that phosphorylation of the cluster mutant with Pi is only moderately inhibited by 10-100 µM Ca2+ (Fig. 5), indicating that Ca2+ binding occurs at lower levels than in the WT enzyme.


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Fig. 3.   Phosphoenzyme formed by utilization of ATP in the presence of Ca2+. Steady state levels of phosphoenzyme were obtained by incubation of COS-1 cell microsomes with 5 µM [gamma -32P]ATP and 5 µM Ca2+ at 3 °C, as described under "Materials and Methods." The concentrations of protein in the samples were adjusted to yield the same concentration of WT or mutant ATPase, as indicated by Western blot analysis. The time of autoradiographic exposure was the same for all samples in order to reveal differences in steady state levels of phosphoenzyme, if any. Average values obtained from three experiments are given in Table I.


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Fig. 4.   Equilibrium levels of phosphoenzyme obtained by incubation with Pi in the absence of Ca2+. Phosphoenzyme was obtained by incubating COS-1 cell microsomes with [32P]Pi at 25 °C as described under "Materials and Methods." The concentration of microsomal protein was adjusted to yield the same concentration of WT or mutant ATPase, as indicated by Western blot analysis. The time of radiographic exposure was the same for all samples, in order to reveal differences in the equilibrium levels of phosphoenzyme, if any. The greater width of some bands (e.g. P811A) is due to spread of larger amounts of total microsomal protein in the sample, as required to yield the same amount of recombinant ATPase. Average values obtained from three different experiments are listed in Table I.

                              
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Table II
Effects of mutations on Ca2+ binding in the presence of 3 µM free Ca2+ and no ATP
The values are in percentage of WT. ND, none detected.


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Fig. 5.   Ca2+ inhibition of enzyme phosphorylation with Pi. Equilibrium levels of phosphoenzyme were obtained by incubation of WT or mutant enzyme with [32P]Pi at 25 °C, as described under "Materials and Methods," in the absence of Ca2+ (EGTA present) or in the presence of Ca2+ as indicated. The acid-quenched samples were washed and subjected to electrophoresis, and the radioactive phosphoenzyme was detected by autoradiography. As the phosphoenzyme levels were different in WT and mutant proteins (see Fig. 7), autoradiographic exposure was adjusted to yield signals of similar intensity at 0 Ca2+, in order to render possible a comparison of the effects of Ca2+. Average values (relative to EP levels in the absence of Ca2+) obtained from three different experiments are plotted in the right panels. Standard deviations ranged from 1 to 13.

Time resolution of enzyme phosphorylation with ATP at low temperature show again significant inhibition, especially when the reaction is started by addition of ATP and Ca2+ to enzyme deprived of Ca2+, as compared with addition of ATP to enzyme already activated by Ca2+ (Fig. 6). On the other hand, pulse-chase experiments indicate that decay of (already formed) phosphoenzyme is not significant delayed by the triple mutation (Fig. 7).


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Fig. 6.   Time course of phosphoenzyme formation following addition of ATP and Ca2+ (upper panels), or ATP to enzyme preincubated with Ca2+ (lower panels). 30 µg of WT protein, or corresponding aliquots of mutant ATPase (as indicated by Western blot analysis), was preincubated at 3 °C in a medium containing 20 mM MOPS, pH 7.0, 80 mM KCl, 10 mM MgCl2 and 1 mM EGTA in the absence (upper panels) or in the presence of 1 mM CaCl2 (lower panels). The reaction was started by the addition of 1 µM [gamma -32P]ATP and 1 mM CaCl2, or 1 µM [gamma -32P]ATP. Acid quenching at serial times, washing, and detection of phosphorylated intermediate was carried out as described under "Materials and Methods." The time of autoradiographic exposure was the same for all samples, so as to reveal differences in phosphoenzyme levels, if any. Average values (relative to maximal EP levels) obtained from three different experiments are given in the right panels. Standard deviations ranged from 0.2 to 6.6.


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Fig. 7.   Kinetics of phosphoenzyme decay. Steady state levels of phosphoenzyme were obtained by incubating WT or mutant enzyme with radioactive [gamma -32P]ATP (5 µM) and Ca2+ (5 µM), for 10 s at 3 °C. Decay of radioactive phosphoenzyme was started with a chase with 0.5 mM non radioactive ATP. Samples were quenched at serial times, and the residual radioactive phosphoenzyme was determined by electrophoresis and autoradiography. Autoradiographic exposure was adjusted to yield a signal of similar intensity for all samples at zero time. Average values obtained in three different experiments are plotted in the right panel. Standard deviation ranged from 0.1 to 6.4.

Single mutations of Asp815 to Asn or Ala do not produce significant ATPase inhibition, although slight displacements of the Ca2+ concentration dependence of ATPase activation (Fig. 2) and of the inhibitory effect of Ca2+ on the Pi reaction are apparent in Figs. 2 and 5, respectively. Ca2+ binding in the absence of ATP (3 µM free Ca2+) is not significantly reduced by single Asp815 mutations (Table II). Mutation of Leu814 to Ala does not produce significantly effects.

Functional Characterization of Lysine and Arginine Mutants-- Single mutations of Lys819 to Ala or Glu, and of Arg822 to Phe, Glu, or Ala produce very strong inhibition of the steady state ATPase velocity even at relatively high Ca2+ concentrations (Fig. 2B). WT enzyme and mutants reach their own maximal activity within the same ATP concentration range (data not shown), indicating that the steady state ATPase dependence on the ATP concentration is not significantly changed by the Lys and Arg mutations. Direct measurements of Ca2+ binding in the absence of ATP show slight and moderate reduction in the Lys819 and Arg822 mutants, respectively (Table II). This finding is in agreement with the interference with the inhibitory effect of Ca2+ on the Pi reaction (Fig. 5).

A marked effect of the Lys819 and Arg822 mutations is strong reduction of the steady state levels of phosphorylated intermediate formed by utilization of ATP. This reduction is only slightly compensated for by raising the Ca2+ concentration in the medium (Fig. 3). Low levels of phosphoenzyme are already apparent in the initial phase of its formation upon simultaneous addition of ATP and Ca2+ to enzyme pre-incubated with EGTA, or even when ATP alone is added to enzyme preincubated with Ca2+. In the former case the phosphoenzyme level rises slowly due to slow activation by Ca2+ (Fig. 6A), while in the latter case the steady state level of phosphoenzyme is reached faster since the enzyme is already activated by Ca2+ (Fig. 6B). It is clear that, in either case, net formation of phosphoenzyme is much lower in the mutants than in the WT samples.

We also conducted pulse-chase experiments to test whether hydrolytic cleavage of the phosphorylated intermediate (once formed) is influenced by the Lys819 and Arg822 mutations (Fig. 7). We found that the time course of phosphoenzyme cleavage is not significantly affected by the Lys and Arg mutations (Fig. 7).

It should be pointed out that the experiments on phosphoenzyme formation and cleavage were conducted at low temperature and non-saturating ATP concentrations in order to obtain kinetic resolution. The relevance of these findings to the steady state behavior of the enzyme at 25 °C is considered under "Discussion."

It is of interest that the equilibrium levels of phosphoenzyme formed with Pi in the absence of Ca2+ and ATP, are also very much reduced (Fig. 4). These experiments were conducted at 25 °C and, consistent with the ATP experiments conducted at low temperature, indicate that Lys819 and Arg822 play an important role in determining the functional integrity of the phosphorylation domain. It is then noteworthy that the phosphorylation defect resulting from the Lys819 and Arg822 mutations can be observed in the presence and in the absence of Ca2+, and at low and high temperature.

Functional Characterization of Proline Mutants-- We found no inhibition of ATPase activity in the Pro811 right-arrow Ala and Pro812 right-arrow Ala mutants, and moderate inhibition in the Pro821 right-arrow Ala, Pro824 right-arrow Ala, and Pro827 right-arrow Ala mutants (Fig. 2). As noted above, however, the Pro820 mutation resulted in negligible protein recovery, despite normal mRNA levels. All tested proline mutants exhibited a Ca2+ concentration dependence nearly identical to that of the WT ATPase, independent of whether the ATPase velocity was reduced or not (Fig. 2). The proline mutants yielded phosphoenzyme levels similar to those obtained with WT ATPase, by utilization of either ATP in the presence of Ca2+ (Fig. 3) or Pi in the absence of Ca2+ (Fig. 4). In agreement with previous reports (11), we found that the proline mutations did not interfere significantly with Ca2+ inhibition of enzyme phosphorylation with Pi (Fig. 5).

Functional Characterization of Mutants in the L89-- A set of single mutations were produced in the loop intervening between the M8 and M9 transmembrane segments (L89), to obtain a comparative evaluation with the effects obtained with mutations in L67. We found that the Ser917 right-arrow Ala and Gln920 right-arrow Ala mutants sustained ATPase activity at rates equal to the WT enzyme, while the Glu918 right-arrow Ala and Asn919 right-arrow Ala exhibited slightly lower rates. The Ca2+ concentration dependence of the M8/M9 loop mutants was nearly identical to that of the WT ATPase (Fig. 2).

    DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
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Functional Analysis-- Our experiments demonstrate that single or cluster mutations within L67 interfere profoundly with the ATPase function. It is remarkable that L67 can affect the molecular events occurring at both phosphorylation site (Asp351) and Ca2+ binding sites, which are separated by a more than 50-Å distance. L67 plays a crucial role even in protein folding. In fact, mutation of Pro820 (unique among six L67 prolines) interferes with the appearance of significant levels of expressed protein. By comparison, mutations within L89 produce little or no interference. We want to clarify here how the twenty five residues of the L67 loop can interfere with such various aspects of the enzyme structure and function.

Particularly interesting are single mutations of Arg822 and Lys819. Mutations of these residues inhibit strongly ATPase activity, due to interference with formation of phosphorylated enzyme intermediate both by utilization of ATP in the presence of Ca2+, and of Pi in the absence of Ca2+. While these mutations affect events that occur at the phosphorylation site, they interfere only to a lower degree with Ca2+ binding. Yet another type of functional defect is produced by mutations of L67 aspartate residues, as originally pointed out by Falson et al. (10) and Menguy et al. (11) who mutated these residues to Ala. We find that removal of negative charge by conservative mutations of aspartate residues interferes with high affinity Ca2+ binding and Ca2+ dependent enzyme phosphorylation by ATP. Inhibition of steady state velocity is observed even at saturating Ca2+. No significant inhibition of enzyme phosphorylation with Pi in the absence of Ca2+ is produced by the aspartate mutations.

Considering the heterogeneity of effects produced by mutations within the L67 loop, we sought to verify by kinetic analysis whether the observed functional behavior of various mutants could be accounted quantitatively for by perturbations of the phosphorylation reaction, or rather by interference with Ca2+ binding and enzyme activation. For this purpose we utilized a complete reaction sequence (Fig. 8) to simulate the steady state ATPase behavior. The rate constants listed in Fig. 8, obtained at 25 °C as explained previously (20), generate a simulated pattern of ATPase Ca2+ activation that is identical to that obtained experimentally with the WT enzyme (Figs. 2 and 9). Any of the rate constants can then be changed to explore the effects of specific perturbations of partial reactions on the overall steady state behavior of the ATPase. A change of the forward rate constant for any particular reaction must be accompanied by a proportional change of the reverse rate constant of the same or of another appropriate reaction so that the overall equilibrium constant (corresponding of that ATP hydrolysis to ADP and Pi) remains unchanged.


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Fig. 8.   ATPase reaction scheme and bidirectional rate constants for the partial reactions. The prime (') refers to the occurrence of the Ca2+-induced conformational transition involved in cooperative binding and enzyme activation. The asterisk (*) refers to enzyme acquisition of the low affinity state for Ca2+. The bidirectional rate constants (25 °C) for the partial reactions were obtained by kinetic and equilibrium experimentation and related analysis as described previously (20).


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Fig. 9.   Simulated steady state velocities of WT and mutant ATPase at various Ca2+ concentrations. A and B, , rate constants as shown in Fig. 8. A, black-square, as in Fig. 8 but k1f = 4 × 106 M-1 s-1, and k8r = 5 × 104 M-1 s-1; black-diamond , as in Fig. 8 but k2f = 24 s-1, k2r = 5 s-1, k5f = 6 s-1, and k5r = 10.5 s-1. Note that black-square does not, but black-diamond  does, correspond to the experimental data obtained with the aspartate cluster mutant (Fig. 2A). The analysis is consistent with mutational perturbation of a rate-limiting step in the Ca2+ binding and activation mechanism. B, , rate constants as in Fig. 8; black-square, as in Fig. 8 but k5f = 10 s-1 and k5r = 3 s-1; black-diamond , k5f = 5 s-1 and k5r = 1.5 s-1. Note the correspondence to the experimental patterns obtained with the Lys and Arg mutants (Fig. 2C). The analysis is consistent with mutational perturbation of the phosphorylation domain, affecting enzyme phosphorylation with ATP in the presence of Ca2+, and with Pi in the absence of Ca2+. Note that any manipulation of rate constants was balanced in the forward and reverse directions so as to maintain an identical overall equilibrium constant for the entire reaction cycle.

To simulate the triple mutant (Asp cluster) behavior we considered that the mechanism of Ca2+ binding and ATPase activation includes three steps: fast binding of the first Ca2+, followed by a relatively slow isomeric transition, and cooperative binding of the second Ca2+ (21). Enzyme activation occurs only after binding of the second Ca2+, when involvement of transmembrane helices M4, M5, M6 and M8 in Ca2+ complexation is complete (12). We then found in our simulation that a simple inhibition of the enzyme affinity for Ca2+ through reduction of fast steps (reactions 1 forward and 8 reverse in Fig. 8) yields a displacement of the Ca2+ curve, with full activation at higher Ca2+ (Fig. 9A, squares). This type of curve was not observed experimentally (Fig. 2A). Combined inhibition of the slow Ca2+ induced transition and of enzyme phosphorylation with ATP (reactions 2 and 5 in Fig. 8) is required to generate a pattern (Fig. 9A, diamonds) matching precisely the pattern observed experimentally with the cluster Asp813, Asp815, and Asp818 to Asn mutant (Fig. 2A, diamonds). Thus the simulation suggests that the aspartate negative charges are not involved in direct coordination of Ca2+, but play an important role in maintaining the structural integrity of the L67 loop, thereby permitting Ca2+ binding and transmission of the activation signal to the phosphorylation domain.

Simulating the behavior of the Lys and Arg mutant is more straightforward, and is obtained just by lowering the rate constants of ATP phosphorylation by ATP and Pi (reactions 5 forward and 11 reverse in Fig. 8). The simulations reproduce satisfactorily the curves observed experimentally, including the low steady state velocity and an apparent saturation by Ca2+ at lower concentrations than observed with the WT enzyme (Compare Figs. 2B and 9B). This indicates that the observed reduction of overall steady state ATPase velocity at 25 °C can be attributed to mutational perturbation of the phosphorylation domain and consequent inhibition of phosphoenzyme formation (Fig. 6).

Structural Considerations-- L67 is an extended loop, consisting of ~25 residues. Its pathway seems to be determined by a hydrogen bond network with other protein segments; that is, with the L89 loop near its N-terminal end, with helices in the P domain and M5 in the middle part, and with M3 and M5 near its C-terminal end. Two pairs of Pro may also play an important part in determining the L67 path, by restricting the main chain torsion angles (Fig. 10).


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Fig. 10.   Three-dimensional structure of the Ca2+-ATPase around the loop (L67) connecting M6 and M7 transmembrane helices. The side chains of all the mutated residues (and others involved in hydrogen bonds) are shown and marked. Potential hydrogen bonds are indicated by red dotted lines. The residues the mutations of which disturb the proper folding of the protein are marked by yellow boxes. To conform with chicken sequence, Arg819 in the original coordinates (1EUL; Ref. 8) is replaced with Lys. Asp351, the phosphorylation residue, is located at the top of the beta -strand 1 in the P domain. One of the two bound Ca2+ appears as a purple sphere at the bottom of the figure. Color changes gradually from the N terminus (blue) to the C terminus (red). Prepared with MOLSCRIPT (24). Single-letter amino acid code is used on figure.

The effect of L67 aspartate mutations on Ca2+ binding are likely to be indirect, since their distance from the transmembrane domain (8) precludes their direct participation in the binding of the two activating calcium ions. As illustrated in Fig. 10, Asp813, Asp815, and Asp818 are involved in a hydrogen bonding network with Arg751 and Asn755 (cytosolic extension of M5), Ser917 and Gln920 (L89), and Ala617 (P2). Our experiments suggest that involvement of Asp813 in these bonds is most important. Since aspartate mutations impair Ca2+ binding, the hydrogen bonding network is likely to maintain M6 (with its three Ca2+ binding residues) and neighboring segments in optimal position for Ca2+ binding and Ca2+ dependent enzyme activation.

It is of interest that the L67 apex sustains a crucial role in protein folding, as the Pro820 right-arrow Ala mutant fails to yield significant levels of expressed protein even though transcription occurs normally. Mutation of the neighboring Arg751 (in the cytosolic extension of the long M5 transmembrane helix) also interferes with expression and functional competence of recombinant ATPase (22). It is shown in Fig. 10 that Pro820 is very close to Arg751, and hydrogen-bonded at the carbonyl oxygen atom of the preceding residue (Lys819). Thus this hydrogen bond appears critical for proper folding of the protein and Pro820 is likely to be important for the correct positioning of this carbonyl group (presumably by restricting the main chain torsion angle).

Mutations of Lys819 and Arg822, also within the L67 apex, produce strong functional inhibition and specific interference with formation of the phosphorylated enzyme intermediate. Lys819 and Arg822 are in close proximity and hydrogen-bonded to residues of the P2 (Asp616) and P1 (Glu340) helices of the phosphorylation domain (Fig. 10). Mutation of Ser346, which is located in the loop connecting beta 1 and P1, and is hydrogen-bonded to Glu696 (Fig. 10), also produces catalytic interference (23). It is likely that, in the process of enzyme activation, M4 and M5 undergo large conformational changes (8), and M6 is repositioned by engagement of three of its residues (Asn796, Thr799, and Asp800) in Ca2+ complexation (12). Thereby, the L67 loop and the P1 and P2 helices are affected as well. This would in turn affect the neighboring beta -strand (strand 1 in Fig. 10) that includes the residue undergoing phosphorylation (Asp351). This strand is connected to transmembrane segment M4, which is also involved in Ca2+ complexation through the intervention of Glu309. The entire region appears to be stabilized by the M5 helix that extends from the lumenal surface of the membrane up to the end of the P domain (8). Thus, precise conservation of structural interactions in this region is required for competence of the phosphorylation and the Ca2+ binding domains, and their long range functional linkage.

    ACKNOWLEDGEMENT

Manuscript and figures were edited by Lisa Schuetz.

    FOOTNOTES

* This work was supported by National Institutes of Health Program Project HL27867 and by grants-in-aid for scientific research and for international scientific research from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 410-706-3220; Fax: 410-706-8297; E-mail: ginesi@umaryland.edu.

Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010813200

    ABBREVIATIONS

The abbreviations used are: SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; WT, wild type; MOPS, 3-(N-morpholino)propanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; PCA, perchloric acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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