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
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
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ABSTRACT |
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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.
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.
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
p 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 p
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 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 [
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 [
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).
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 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.
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.
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).
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
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 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.
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).
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
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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
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
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.
E1sp1A or
pAd-lox, were conducted by the DEAE-dextran method (16).
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.
-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.
-32P]ATP and 1 mM CaCl2, or 1 µM [
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
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).
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+
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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."
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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.
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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 [ -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.
Effects of mutations on Ca2+ binding in the presence of 3 µM free Ca2+ and no ATP
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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.
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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 [ -32P]ATP and 1 mM
CaCl2, or 1 µM [
-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 [ -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.
Ala and Pro812
Ala mutants, and
moderate inhibition in the Pro821
Ala,
Pro824
Ala, and Pro827
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).
Ala and Gln920
Ala mutants sustained ATPase activity at rates equal to the WT
enzyme, while the Glu918
Ala and Asn919
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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,
, as in Fig. 8 but k1f = 4 × 106 M
1
s
1, and k8r = 5 × 104 M
1
s
1;
, 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
does not, but
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;
, as in Fig. 8 but
k5f = 10 s
1 and
k5r = 3 s
1;
,
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.
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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 -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.
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).
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
-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.
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ACKNOWLEDGEMENT |
---|
Manuscript and figures were edited by Lisa Schuetz.
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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.
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
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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.
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