 |
INTRODUCTION |
The detailed structure of membrane proteins is only known in a few
cases. Among P-type ATPases, which are responsible for active cation
transport, the best structure to date is only known to about 14 Å resolution, from cryo-electron microscopy; this is for
Ca2+-ATPase, which catalyzes uptake of Ca2+
from the cytosol to the endoplasmic or sarcoplasmic
Ca2+-storing compartment (1-3). However, large
three-dimensional crystals of the protein are not available as yet, and
the ATPase structure has been mainly deduced from electron microscopy
studies of two-dimensional crystals (3-6), combined with studies by
other techniques like chemical labeling, Förster-type resonance
energy transfer, immunoreactivity of antibodies with various epitopes, site-directed mutagenesis, and proteolysis studies (reviewed in Refs.
7-9). By comparing structures of Ca2+-ATPase with and
without bound nucleotide, suggestions have been made concerning the
likely location of the ATP-binding site in the cytoplasmic portion of
this pump (10), but the actual structure of the ATP-binding site and
the mechanism of ATP hydrolysis are still largely unknown.
Nevertheless, it has been hypothesized that this region of the
Ca2+-ATPase might be organized like water-soluble
phosphokinases, with a hinge transiently bringing an ATP-binding and a
phosphate-accepting region in close apposition during the ATPase
enzymatic cycle (Ref. 11 and reviewed in Refs. 8 and 12).
As a first step toward determining the structure of the entire ATPase,
it would be useful to determine the structure of smaller fragments,
provided that such fragments reflect the structure of the corresponding
region in the intact protein. A few studies have paved the way in this
direction, by trying to express in bacterial systems soluble fragments
of ATPase with anticipated autonomous functional properties (13, 14).
Proteolysis experiments can contribute to the success of such
studies, since protease-resistant fragments probably constitute domains
whose structural properties can be investigated with minimal risk of
instability. Following our previous analysis of Ca2+-ATPase
proteolysis by proteinase K (15), we now report on the properties of
closely related proteolytic fragments with molecular masses of 29/30
kDa, derived from the large cytosolic loop, which we show retain many
of the properties of the corresponding region in intact
Ca2+-ATPase. In view of the stability and functional
properties of these fragments, we suggest that such a soluble 30-kDa
fragment of Ca2+-ATPase could be a reasonable candidate for
future three-dimensional structure determination. From our results we
also infer possible implications for the organization of the cytosolic
portion of P-type ATPases.
 |
EXPERIMENTAL PROCEDURES |
Most of the methods used in this report have been described in
detail previously, including the isolation of
SR1 vesicles (16) and their
FITC labeling (17), preparation of purified ATPase by deoxycholate
extraction (18), proteolysis and analysis of the resultant peptides
(15, 19, 20), handling of lanthanide ions (21), and general
fluorescence methods, including the use of TNP-nucleotides (22).
Fluorescence recordings were obtained with a SPEX fluorolog instrument;
SDS-PAGE gels were digitized with a Bio-Rad camera and Molecular
Analyst software. Total amino acid analysis after acid hydrolysis of
the peptides was performed by F. Baleux, Unité de Chimie
Organique, Département de Biochimie et Génétique
Moléculaire at Institut Pasteur, Paris. CD Spectra were run on a
Jobin-Yvon CD6 spectrodichrograph and were analyzed with the Dichroprot
software.2
Mass Spectrometry--
In this work, mass spectrometry was not
performed after ElectroSpray Ionization (as in our previous work, see
Ref. 15), but we used the matrix-assisted laser desorption
ionization-time of flight technique (23). MALDI-TOF spectra of
proteolytic peptides were obtained with a Voyager-Elite Biospectrometry
Workstation mass spectrometer (PerSeptive Biosystems Inc., Framingham,
MA). Each sample was first desalted and then loaded on the target by the dried droplet method; the matrix was
3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, Aldrich). Spectra
were calibrated externally with a two-point calibration using the [M + H+] and the [M + 2H+] ions from a protein
standard, horse apomyoglobin (m/z = 16952.5 and 8476.8, respectively). The analysis was performed in the positive and linear
modes, with an accelerating voltage of 25,000 V. Different extraction
delays were used (150, 275, or 400 ns), and around 120 scans were
averaged. Typically, the amount of peptide analyzed was 5-10 pmol.
MALDI-TOF spectra were run to determine the C termini of the soluble
fragments produced by proteolysis (see Fig. 1) with various proteolytic
enzymes. After proteolysis, samples were processed in one of two ways.
In the case of Asp-N, the supernatant of Asp-N-treated SR was simply
desalted by passing through a Sephadex G25M column equilibrated with a
10 mM ammonium bicarbonate buffer at pH 8.1 and examined
directly by mass spectrometry. Since the N terminus of the 30-kDa
proteolytic peptide was already known from N-terminal sequencing,
determination of its total mass by MALDI-TOF (to within 10 Da) allowed
us to determine its C-terminal residue without ambiguity. In the case
of elastase and proteinase K, which are proteolytic enzymes with
relatively poor specificity (and also after use of Asp-N, for control),
the peptides found in the supernatants of treated SR were further
cleaved with trypsin (as shown for proteinase K in Fig. 5), and the
smaller peptides generated in this way were then desalted by binding to
a C18 reversed phase resin (Sep-Pak, Waters Corp., Milford, MA) and
elution with 80% acetonitrile and 0.1% trifluoroacetic acid (v/v) in
deionized water (24). In this case, mass spectrometry revealed two
groups of peaks (see Fig. 6) in the 16-17-kDa range and in the
11-12-kDa range, respectively, corresponding to the N-terminal and
C-terminal parts of the original peptides, respectively, and confirmed
that these peptides had been cut by trypsin at a single site,
Arg505-Ala506 (as shown for proteinase K in
detail, see "Results"). The masses measured for the 16-17-kDa
peptides confirmed the results of the previous N-terminal sequencing
experiments. The masses measured for the 11-12-kDa peptides, which
corresponded to peptides starting at the trypsin cleavage site
(Arg505-Ala506) and ending at the C termini of
the original peptides, allowed to identify the latter unambiguously in
all cases (again to within 10 Da).
Binding of [
-32P]ATP to Soluble Fragments, as
Deduced from Ultrafiltration Experiments--
To measure directly
equilibrium binding of ATP to the soluble p29/30 fragments resulting
from Ca2+-ATPase proteolysis, we took advantage of the
ability of appropriate ultrafiltration membranes (Centricon 10 devices,
Amicon) to concentrate these soluble peptides while allowing free
passage of ATP. Samples for ultrafiltration (2 ml) were transferred to
Centricon 10 tubes and centrifuged for 150 min at 5000 rpm in a Beckman
JA-12 rotor. After centrifugation, the various samples were
concentrated to about the same extent (about 30-fold), as indicated by
measurements of protein concentrations. Protein-bound ATP
versus free ATP was determined from the excess ATP in the
concentrated sample over that present in the ultrafiltrate, after
correction for the small "blank" value (a few %) found in
experiments performed with protein-free samples. Control measurements
with intact SR vesicles were included. Experiments were performed on
samples containing various concentrations of ATP, to which a constant
concentration of [
-32P]ATP tracer was added, to
measure competition between nonlabeled and labeled ligand
(e.g. Ref. 65). [3H]Glucose was also added as
an inert tracer, and the concentrations of ATP were deduced from the
ratios of 32P and 3H counts. The data were
analyzed in terms of Michaelian binding, according to Equation 1.
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(Eq. 1)
|
where [EL] and [L] are the concentrations of bound and free
ligand (ATP), respectively; [Etot] and
Kd are the concentration of binding sites and the
binding affinity, and
corresponds to a small fraction of
nonspecific binding. Plotting the fraction of ATP bound
(i.e. [EL]/[L]) as a function of free ATP ([L]), as in
Fig. 4, thus permits us to directly estimate Kd from
the concentration of free ATP for which this fraction is reduced to
half of its maximal value, obtained at low ligand concentration; in
turn, this maximal value, which is equal to
{[Etot]/Kd} +
, permits estimation of the
concentration of the ATP-binding sites in the sample
(Etot). A full description of the method will be given
elsewhere.3
In the case of p29/30 peptides, a special problem arises because these
peptides are prepared as the high speed supernatant of protease-treated
SR membranes; in such a protocol, a small fraction (possibly a few
percent) of the original membranes may escape pelleting, and these
non-pelleted membranes may contain a small fraction of either residual
intact ATPase or only partially cleaved ATPase (e.g. the
membranous p83C peptide, which is able to bind ATP and be
phosphorylated, see Ref. 15). Unfortunately, for estimation of a
binding affinity, contamination of a sample (with an anticipated
relatively poor affinity for its ligand) by a component of higher
affinity is a real complication (e.g. see Ref. 66). To
eliminate such contamination, we submitted the p29/30-containing
supernatant to a preliminary step of centrifugation on an
ultrafiltration membrane with a 100,000-dalton cut-off (2-ml samples
were loaded onto Centricon 100 devices and centrifuged for 135 min at
2400 rpm in a Beckman JA-12 rotor); SDS-PAGE showed that contaminating
SR membranes were retained by the Centricon membrane but that p29/30
peptides were recovered in the filtrate. These peptides were
subsequently used for the ultrafiltration experiments with Centricon
10.
Metal-binding Properties of Bis-Tris Buffer--
In some cases,
for treatment of SR vesicles with proteinase K at pH 6.5, we replaced
the Bis-Tris buffer of our original standard medium (15) by a Mops/NaOH
buffer at the same pH. This is because we found that Bis-Tris buffer
(but not Mops/NaOH buffer) binds Ca2+ with an apparent
dissociation constant around 25 mM at this pH, so that in
the presence of 100 mM Bis-Tris and a certain amount of
total Ca2+, the free [Ca2+] is about 5-fold
lower than the total [Ca2+]. Although this has no
implication for the present results, it explains why, in our previous
work (15), unexpectedly high concentrations of Ca2+ were
required to protect the ATPase transmembrane bundle from denaturation
during proteinase K treatment.
 |
RESULTS |
Protease-resistant Soluble Fragments of
Ca2+-ATPase--
The starting point for the
present work was the observation that treatment of sarcoplasmic
reticulum Ca2+-ATPase with a variety of proteases generally
leads to accumulation of soluble fragments of about 30 kDa, which are
relatively resistant to further proteolysis. Fig.
1 shows an experiment in which SR vesicles were treated with elastase (El, left), proteinase K
(prK, central lanes), or Asp-N (AspN,
right). Aliquots of the treated samples were centrifuged,
and peptides found in either the total sample (Tot), the
pellet (Pel), or the supernatant (Sup) were separated by SDS-PAGE. In all three cases, irrespective of the proteolytic enzyme, the supernatant (lanes 4, 8, and
12) contains, in addition to the protease itself
(asterisk), one or two major proteolytic fragment(s) of
about 30 kDa (see arrows). In the case of Asp-N, a faint
band at the position of intact ATPase also shows up in the supernatant,
but this is presumably due to contamination of the protease with
phospholipase. Treatment of SR vesicles with endoproteinase Glu-C
protease also results in appearance of a 31-kDa fragment (not
shown).

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Fig. 1.
Accumulation of soluble fragments of
Ca2+-ATPase in the 29-30-kDa range after proteolysis by
various proteases. SDS-PAGE (using a 12.5% acrylamide Laemmli gel
prepared in the absence of Ca2+) was used to separate the
peptides present in various protein samples. Lane 14,
molecular mass standards (low molecular weight standards)
(LMW). Lane 1, control intact SR vesicles
(SR); the ATPase is indicated, as well as calsequestrin
(Cals) and M55, two extrinsic proteins. Lanes
2-4, SR vesicles (at a final concentration of 1 mg/ml) were
incubated for 30 min with 30 µg/ml pancreatic elastase
(El), in a medium containing 100 mM Bis-Tris and
1 mM EGTA at pH 6.5 and 22 °C, and the reaction was
stopped with 0.5 mM diisopropyl fluorophosphate; 100-µl
samples were then centrifuged (Beckman rotor TLA 100, 75,000 rpm, about
200,000 gav, for 60 min), and aliquots of total
sample (Tot), resuspended pellet (Pel), and
supernatant (Sup) were loaded onto the gel. Lane
5, elastase alone (El, asterisk). Lanes
6-8, same experiment, except that SR vesicles were now incubated
for 30 min with 30 µg/ml proteinase K (prK), in a medium
containing 100 mM Bis-Tris and 0.3 mM
Ca2+ at pH 6.5 and 20 °C, and the reaction was stopped
with 0.5 mM PMSF. Lane 9, proteinase K alone
(prK, asterisk). Lanes 10-12, same
experiment, except that SR vesicles were incubated with Asp-N (AspN), instead of proteinase K, and stopped with 1 mM EDTA; in this case, proteolysis was performed with only
3 µg/ml Asp-N, allowing for limited proteolysis but also limited
ATPase solubilization by the contaminating phospholipase. Lane
13, Asp-N alone; Asp-N is hardly visible but can be detected at
the location indicated by the asterisk. The various
arrows point to the soluble proteolytic fragments.
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The fragments found in the supernatants were identified as peptides
originating from the large cytosolic domain of Ca2+-ATPase
by immunoblotting with sequence-specific antibodies and N-terminal
sequencing (15); mass spectrometry experiments (some of which will be
described below in Fig. 6; see "Experimental Procedures") were also
performed for identification of C termini. The conclusion from all
these experiments regarding the identification of the soluble peptide
fragments is shown in Table I. It appears that the region surrounding the Arg505-Ala506
peptidic bond, known to be highly reactive to trypsin, is not a major
target for the other proteases. Cleavage by Asp-N is found to occur at
locations just N-terminal of an aspartic residue
(Ser350-Asp351 and
Arg615-Asp616 bonds), and cleavage by
endoproteinase Glu-C occurs after glutamic acid residues
(e.g. at Glu340-Thr341), as
expected. Cleavage by proteinase K does not appear to be particularly
specific of certain amino acid residues (see also Ref. 15) but occurs
at a few sites only, presumably because of the local peptide
conformation. This also seems to apply to elastase. A remarkable
finding is that all fragments more or less coincide, as they all start
close to the ATPase phosphorylation site, Asp351, and end
close to Ser610, in the
Lys605-Arg615 region. This suggests the
existence, between Asp351 and Ser610, of a
region of relatively compact structure resistant to proteolysis, characteristic of a structural domain in the cytoplasmic portion of the
ATPase. In the following sections, we mainly used peptides derived from
proteinase K-treated SR vesicles (a mixture of "p29" and "p30"
peptides, starting at Thr357 and Ser350,
respectively, and ending after either Met608 or
Ser610), to ask to what extent the isolated peptides retain
properties resembling those of the polypeptide chain in the native
structure. To do this, we studied nucleotide- and metal-binding
properties of p29/30 fragments as well as their sensitivity to trypsin
and reactivity toward FITC, and we characterized the secondary
structure of these fragments as well as their hydrodynamic and
spectroscopic properties.
Retention of Nucleotide- and TNP-nucleotide-binding Properties for
p29/30 Fragments, as Deduced from TNP-nucleotide Fluorescence--
To
study the nucleotide-binding properties of these soluble
Ca2+-ATPase fragments, we took advantage of the fact that
the fluorescence of a TNP-nucleotide is 3- or 4-fold higher when it is
bound to Ca2+-ATPase than when it is free in solution (14,
25, 26). This is shown in panel A of Fig.
2, which shows control experiments with
TNP-ATP and intact SR. Using excitation and emission wavelengths optimal for detection of TNP-ATP fluorescence (410 and 540 nm, respectively), addition of TNP-ATP to an SR-containing cuvette results
in an increase in the intensity of the signal which is almost
completely reversed when TNP-ATP is chased off the nucleotide-binding sites by subsequent addition of a high concentration of ATP. The difference between the residual signal intensity in the presence of
both ATP and TNP-ATP and the signal intensity in the absence of both
nucleotides corresponds to the fluorescence of free TNP-ATP. This can
be seen in experiments in which TNP-ATP is added to an SR-free sample;
in this case (see panel B experiment, in which the
supernatant of a centrifuged SR suspension provides such a sample), the
base-line signal before TNP-ATP addition is very low (because this
signal is directly related to the amount of light scattered by the
sample) and the small TNP-ATP-dependent signal is not
altered upon ATP addition. Thus, the large
TNP-ATP-dependent signal observed in the presence of SR
vesicles, partially reversed by ATP, reveals TNP-ATP binding to the
ATPase nucleotide site.

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Fig. 2.
Nucleotide binding to p29/30 fragments, as
deduced from TNP-ATP fluorescence measurements. To detect TNP-ATP
fluorescence (panels A-D), the excitation wavelength was
set at 410 nm (bw = 5 nm), and the emission wavelength was set at
540 nm (bw = 20 nm). Experiments were performed with control SR
vesicles (panels A and B) or proteinase K-treated
vesicles (panels C and D). SR vesicles at 1 mg/ml
were incubated for 30 min at 20 °C in the absence (A and
B) or presence (C and D) of 30 µg/ml
proteinase K, in a medium containing 100 mM Mops-NaOH and
0.3 mM Ca2+ at pH 6.5; the reaction was stopped
by addition of 0.5 mM PMSF; 1.5-ml aliquots of the samples
were centrifuged (Beckman 50 Ti rotor at 40,000 rpm, about 125,000 gav, for 80 min), and supernatants (Sup, B and D) as well as total
non-centrifuged samples (Tot, A and C)
were examined. In the fluorescence cuvette, 0.5-ml samples were mixed
with 0.5 ml of 200 mM KCl, 2 mM EDTA, and 30 mM Tris-HCl at pH 9; the final suspension pH was 7. Then, 5 µM TNP-ATP was added (17 µl of a 300 µM
stock solution), followed by several additions of 6 mM ATP
(60 µl of 100 mM ATP each time). Finally, sample aliquots
were analyzed by SDS-PAGE (panel E on the
right).
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Panels C and D in Fig. 2 show the outcome of
corresponding experiments performed on proteinase K-treated SR
vesicles. With both total sample (panel C) and supernatant
(panel D), ATP is still effective in reducing the signal
observed in the presence of TNP-ATP, suggesting ATP-displaceable
TNP-ATP binding to isolated p29/30 fragments. SDS-PAGE analysis of the
various samples used in this experiment is shown on the
right of Fig. 2, confirming degradation of
Ca2+-ATPase and formation of appreciable amounts of p29/30
after proteinase K treatment, as in Fig. 1. In complementary
experiments, we found that both the TNP-ATP-dependent
signal in the supernatant of proteinase K-treated samples and the
magnitude of the ATP-induced drop in this signal (as in panel
D) depend on the duration of the proteolysis period; they rise to
a maximal value after about 30 min of proteolysis at pH 6.5 and
subsequently decline, in close agreement with the amount of p29/30
fragments which can be estimated from SDS-PAGE gels (data not shown).
In Fig. 2, the fact that changes in TNP-ATP fluorescence are smaller
upon binding to non-centrifuged but proteinase K-treated ATPase
(panel C) than upon binding to intact
Ca2+-ATPase (panel A) is probably due to a
combination of factors, such as proteolysis-induced modification of the
environment of TNP-ATP in its site (resulting in reduced fluorescence
enhancement upon binding), lower affinity of TNP-ATP for p29/30 than
for Ca2+-ATPase (see below), and slow conversion of p29/30
to smaller fragments (which no longer bind TNP-ATP). On the other hand,
the fact that changes in TNP-ATP fluorescence are smaller upon binding to the supernatant of treated ATPase (panel D) than upon
binding to non-centrifuged samples (panel C) is probably due
in part to the fact that the membranous fraction still contains a few
ATPase chains either completely intact or only partially cleaved by
proteinase K, such as the membranous 83-kDa peptide which retains full
reactivity toward ATP (15). In addition, p29/30 fragments may weakly
bind to portions of the ATPase still attached to the membrane and be partially pelleted together with the membrane fraction (see lane 7 in Fig. 1).
Retention of Nucleotide and TNP-nucleotide-binding Properties for
p29/30 Fragments, as Deduced from Intrinsic Fluorescence
Measurements--
In p29/30 fragments, one Trp residue,
Trp552 (out of a total of 13 Trp residues in intact
Ca2+-ATPase) is present. Thus, it is also possible to study
nucleotide binding to p29/30 by monitoring changes in intrinsic (Trp)
fluorescence. TNP-ATP is known to quench Ca2+-ATPase
intrinsic fluorescence, both by a saturable mechanism due to
Förster transfer from Trp552 to the TNP moiety of
bound TNP-ATP (Ref. 27; R0 = 24 Å, see Ref. 22)
and by a non-saturable quenching due to light absorption by the added
nucleotide (an "inner filter" effect). These effects can be seen in
panel A of Fig. 3, which
illustrates a control experiment with intact Ca2+-ATPase
and TNP-ATP; in this case, there is a well defined break in the
quenching curve (27), indicating high affinity binding of the
TNP-nucleotide. When the experiment is repeated with p29/30 peptides
(panel B of Fig. 3), there is also evidence for a saturable component in the quenching curve but with lower affinity (the apparent
dissociation constant is a few µM for p29/30 peptides, compared with much less than 1 µM for intact SR, see also
Refs. 14 and 25). The same result is obtained when TNP-ADP is added, instead of TNP-ATP (see below). Interpretation of saturable quenching in terms of binding to the nucleotide site is strengthened by the
demonstration that this quenching is reversed when the TNP-nucleotide is chased off its binding site by addition of nucleotide. This is shown
in panels C and D of Fig. 3, in which ADP was
added to samples pretreated with TNP-ADP; both for control ATPase
(panel C) and for p29/30 fragments (panel D), a
high concentration of ADP does reverse the quenching resulting from
previous addition of TNP-ADP. This reversal is partial, because
although Förster quenching is reversed, the reduction in signal
intensity due to inner filter and dilution effects is of course not
reversed, and added ADP further contributes to the latter effects. Note
that this experiment started with addition of EDTA (ED),
which, by chelating contaminating Ca2+, had a clear effect
on the fluorescence of intact Ca2+-ATPase, as expected
(28), but did not affect the fluorescence of p29/30 fragments, since
these fragments are no longer connected to the ATPase transmembrane
domain responsible for Ca2+ sensitivity (29).

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Fig. 3.
Binding of nucleotides to p29/30 fragments,
as deduced from Trp fluorescence measurements. Experiments were
performed either (i) with the supernatant of proteinase K-treated
vesicles (prK Sup, panels B and D; here, SR
vesicles at 2 mg of protein/ml were treated for 30 min with 30 µg/ml
proteinase K in a medium containing 300 mM sucrose, 1 mM Mg2+, 0.01 mM Ca2+,
and 10 mM Tricine-Tris, at pH 8 and 20 °C, and 3-ml
aliquots were centrifuged with a Beckman 45Ti rotor at 40,000 rpm,
about 185,000 gav, for 90 min), or (ii) with
control SR, incubated under the same conditions but in the absence of
proteinase K (SR, panels A and C).
SDS-PAGE analysis of the resulting peptides is shown in panel
E on the right. Intrinsic fluorescence was detected
with ex = 300 nm (bw = 2 nm), to minimize
absorption by nucleotides, and with em = 330 nm (bw = 10 nm). The fluorescence level of p29/30 fragments was more than
10-fold lower than that of intact Ca2+-ATPase; the data
were normalized, 100% corresponding to the fluorescence of each sample
in the absence of Ca2+ and nucleotide. In all cases,
100-µl protein aliquots were added to 1.9 ml of a medium containing
100 mM KCl and 50 mM Mops-Tris at pH 7 and
20 °C. Panels A and B, concentration
dependence of fluorescence quenching by TNP-ATP, in the additional
presence of 0.5 mM EDTA. The ordinate scale is logarithmic;
dashed lines represent the inner filter component of
quenching. Panels C and D, 2 µl of EDTA 0.5 M was first added to the sample (ED) to chelate contaminating Ca2+ (and Mg2+ contributed by the
proteolysis medium); then, four additions of TNP-ADP were made,
followed by several additions of ADP itself; panel C,
additions of 0.4 µl of 10 mM TNP-ADP followed by
additions of 20 µl of 200 mM ADP; panel D,
additions of 1 µl of 10 mM TNP-ADP followed by additions
of 50 µl of 200 mM ADP. The dotted lines show
the "true" fluorescence levels in the presence of nucleotide, taking dilution and inner filter effects into account.
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These results were obtained with a supernatant of SR vesicles that had
been treated with proteinase K in a pH 8 sucrose medium instead of the
previous pH 6.5 medium (see legend to Fig. 3). In this case, the ratio
of p29 and p30 peptides is more in favor of p29 than after proteolysis
under the previous conditions (compare SDS-PAGE results in panel
E of Fig. 3 with those in panel E of Fig. 2), but the
outcome of the above Trp fluorescence experiments was the same when
proteolysis was performed in the pH 6.5 medium instead of the pH 8 sucrose medium (not shown). Note also that similar results were
obtained with supernatants of SR vesicles that had been treated with
elastase (as in Fig. 1) instead of proteinase K (data not shown). All
these results provide unambiguous evidence for binding of both
TNP-nucleotides and nucleotides themselves to 29/30 peptides, with a
reduced but significant affinity.
Binding of [
-32P]ATP to p29/30 Fragments, as
Deduced from Ultrafiltration Experiments--
For intact SR, binding
of ADP or ATP in the absence of TNP-nucleotide can also be directly
detected as a rise in ATPase intrinsic fluorescence (e.g.
Ref. 30). However, when, in similar experiments, 1 mM ATP
aliquots were sequentially added to p29/30 fragments, the resulting
rise in fluorescence level was small; despite our efforts to reduce
inner filter artifacts due to nucleotide absorbance as much as possible
by excitation of Trp residues at 300 nm, this rise could only, after
the first addition of ATP, compensate for the dilution and inner
filter-induced artifacts expected and indeed observed after subsequent
ATP additions (data not shown). Evaluation with this method of the
affinity of p29/30 fragments for ATP was therefore not possible.
Thus, to measure directly the equilibrium binding of
[
-32P]ATP to the soluble p29/30 peptides, we designed
a protocol based on the opportunity provided by ultrafiltration
membranes (Centricon 10 devices, Amicon) to concentrate these soluble
peptides but let free [
-32P]ATP flow through them;
measurement of the amount of [
-32P]ATP in the
concentrate then corresponds to the sum of free ligand (as can be
measured in the filtrate) and bound ligand (see "Experimental Procedures"), and including various amounts of nonradioactive ATP
together with a constant amount of radioactive ATP tracer makes it
possible to evidence competition between nonradioactive and radioactive
ligands (e.g. Ref. 65). These experiments were conducted in
the absence of Mg2+, and for comparative reasons
included measurements with intact SR vesicles. The results of the
experiments with intact SR (circles in Fig.
4), in which the membranes were loaded
onto the Centricon 10 device at an initial concentration of 0.16 mg of
protein/ml and were concentrated to 4.8 mg/ml (on average), are
consistent with a Kd for ATP binding of 34 µM and a concentration of ATP-binding sites of about 15.3 µM, hence 3.2 nmol/mg, values which are similar to those
previously described after filtration experiments with intact SR under
the same conditions (e.g. Ref. 30). Triangles in
Fig. 4 show similar experiments performed with p29/30 peptides at a
molar concentration about 9-fold higher than that of ATPase in the SR
samples of the previous experiments (see legend to Fig. 4). In this
case, the amount of [
-32P]ATP bound, at low total
concentrations of ATP, was about twice lower and was displaced by
higher concentrations of nonradioactive ATP, consistent with binding of
[
-32P]ATP to p29/30 peptides with an affinity 20-fold
lower than in intact SR ATPase (Kd = 680 µM, total number of sites 138 µM).

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Fig. 4.
Comparison of binding of
[ -32P]ATP to p29/30 fragments and to SR ATPase, as
measured in the absence of Mg2+. Ultrafiltration
experiments were performed with either intact SR (initially at a
concentration of 0.2 mg protein/ml; these experiments are illustrated
by circles) or p29/30 fragments (obtained after 30 min
proteolysis of SR vesicles at 2 mg protein/ml and centrifugation, essentially as described in the legend to Fig. 3 except that in addition, the supernatant was loaded onto a Centricon 100 device and
the ultrafiltrate was collected, to eliminate contamination of soluble
peptides by non-pelleted membranes; these experiments are illustrated
by triangles). To 1.6-ml aliquots of these protein samples
(in 300 mM sucrose, 1 mM Mg2+, 0.01 mM Ca2+, and 10 mM Tricine-Tris at
pH 8 and 20 °C), we added 0.2 ml of a concentrated solution
containing radioactive ATP and glucose, KCl, Mops-Tris, and EDTA, as
well as 0.2 ml of another solution of nonradioactive ATP at various
concentrations; final concentrations were 100 mM KCl, 50 mM Mops-Tris (final pH was 7.0, at 20 °C), 240 mM sucrose, 0.8 mM Mg2+, and 1.5 mM EDTA; in the various samples, [3H]glucose
was present at an activity of 2 µCi/ml and a total concentration of 1 mM, whereas [ -32P]ATP was present at a
constant activity of 0.4 µCi/ml but at various total concentrations,
ranging from 10 µM to 3 mM. These 2-ml
samples were loaded onto Centricon 10 devices, and after centrifugation
10-µl aliquots were taken out of the concentrate for double labeling
counting (i.e. estimation of total
[ -32P]ATP concentrations) and measurement of the
protein concentrations. After taking into account blank experiments
performed in the absence of any added protein, the
protein-dependent increase in [ -32P]ATP
concentration in the concentrate, for a given total ATP concentrations,
was corrected for slight variations in protein concentration. On
average, the concentration of protein rose from 0.16 to about 4.8 mg/ml
in SR samples. In the experiments with proteinase K-treated SR, we
estimated a similar 30-fold increase in peptide concentration. Since
90% of the 29/30 domains was recovered after the proteolysis and
Centricon 100 recentrifugation steps, and since the initial SR protein
concentration was 10-fold higher in the experiment with proteinase
K-treated SR than in that with intact SR (2 mg/ml instead of 0.2 mg/ml), this resulted in a molar concentration of ATP-binding sites
9-fold higher for p29/30 fragments (triangles) than for
intact SR ATPase (circles). The data were fitted to
Michaelian binding (see "Experimental Procedures") with Kd = 34 µM, Etot = 15.3 µM (i.e. 3.2 nmol/mg protein), and = 0.02 for intact SR ATPase, and K'd = 680 µM, E'tot = 138 µM, and ' = 0.06 for p29/30 fragments.
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Retention by p29/30 Fragments of High Sensitivity to Trypsin and
Reactivity with FITC--
After having demonstrated the ability of
p29/30 fragments to bind nucleotides, we asked whether these fragments
also retain two other properties characteristic of intact ATPase,
i.e. strong susceptibility to trypsin of the
Arg505-Ala506 peptidic bond, and high
reactivity toward FITC of Lys515. The results of
experiments designed to explore these aspects are shown in Fig.
5.

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Fig. 5.
Sensitivity to trypsin of the
Arg505-Ala506 bond in p29/30 fragments, and
reactivity of Lys515 toward FITC in these fragments.
In this experiment, the same medium was used both for proteolysis with
proteinase K and/or trypsin and for labeling with FITC; it contained
0.3 M sucrose, 1 mM Mg2+, 10 µM Ca2+, and 10 mM Tricine-Tris
at pH 8 and 20 °C. After samples were run on a SDS-PAGE/Tricine gel,
the gel was examined immediately for fluorescence (panel B)
and subsequently stained with Coomassie Blue (Coom. blue)
(panel A). Lane 7, molecular mass markers (see panel A); the large spot in lane 7 of panel
B corresponds to the tracking dye in the migration front.
Lane 3, FITC-labeled SR vesicles. Labeling was partial and
corresponded to 1 nmol FITC/mg of protein. Lanes 4-6, the
same labeled vesicles (2 mg/ml) were submitted to proteolysis, either
(i) for 2 min with 125 µg/ml trypsin (lane 4; in
panel A, fragments A and B are apparent, as well as A1, A2
superimposed with trypsin (Tr), and the trypsin inhibitor
(Tr Inh)), or (ii) for 30 min with 30 µg/ml proteinase K
(lane 5, p29/30 peptides are indicated), or again (iii) with
proteinase K first, followed by trypsin (lane 6).
Lanes 1 and 2, a sample similar to the one for
lane 5, but prepared from unlabeled SR vesicles, was
centrifuged. The supernatant was then incubated with 2 µM
FITC for 30 min and subsequently either (i) loaded onto lane
1, or (ii) submitted to trypsin treatment and then loaded onto
lane 2, in the latter case, trypsin (Tr), trypsin
inhibitor (Tr Inh), and three smaller peptides (designated
as p17/18 and p12) are apparent (panel A). For fluorescence
examination, the gel was deposited in a transparent box before acid
fixation and Coomassie Blue staining and illuminated with UV light (302 nm). Fluorescence (panel B) was detected with a Kodak
Wratten 55 green filter; the white dots are due to
fluorescent defects in the plastic box. Panel C, the N
termini of p12 peptides derived from trypsin cleavage of either
nonlabeled or heavily FITC-labeled p29/30 fragments (see text) were
sequenced. PTH-amino acids found after each cycle of Edman degradation
were compared for the labeled "p12-FITC" peptide and the unlabeled
"p12" peptide (the ratio is plotted). The second alanine residue
was not determined quantitatively, hence the ×.
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Panel A of this figure shows the result of Coomassie Blue
staining of an SDS-PAGE/Tricine gel after separation of the fragments resulting from proteolysis of various samples with proteinase K,
trypsin, or both. As a reference, intact Ca2+-ATPase and
the associated Ca2+-binding proteins of SR vesicles are
shown in lane 3. The well known bands A and B, as well as A1
and A2 (e.g. Ref. 31), resulting from SR treatment with
trypsin are shown in lane 4, whereas treatment with
proteinase K (lane 5) results in formation of a number of membranous fragments as well as of the soluble p29/30 fragments (see
Ref. 15 and Fig. 1). Lane 6 in panel A shows that
treatment with proteinase K, followed by treatment with trypsin,
results in additional fragments. To permit identification of the
fragments derived from p29/30, a separate proteinase K-treated sample
was centrifuged, and although an aliquot of its supernatant, mainly containing p29/30 peptides, was deposited in lane 1, the
rest of the supernatant was treated with trypsin and deposited in
lane 2. Five bands are observed in lane 2, of
which the two upper ones correspond to trypsin and the trypsin
inhibitor; p29/30 fragments are absent: thus, these fragments are
cleaved down to three fragments only, two of which (designated as p17
and p18) migrate close to each other, whereas the third one (designated
as p12) migrates at a faster rate.
It should be noted that in the experiment illustrated by lanes
3-6, the SR vesicles had been labeled with FITC at
Lys515 prior to proteolysis. Before Coomassie Blue
staining, we were therefore in a position to examine which of the
various peptides formed were fluorescent. Panel B in Fig. 5
shows that, as expected, fluorescence shows up in
Ca2+-ATPase, the tryptic B fragment, and proteinase K
fragments p81/83 and p29/30 (15); in addition, fluorescence shows up in
the smallest (p12) of the three peptides resulting from p29/30 tryptic
cleavage. This p12 peptide was blotted onto polyvinylidene difluoride
(15) and subjected to N-terminal sequencing; it was found that its N-terminal sequence corresponds to cleavage of the
Arg505-Ala506 peptidic bond (see also below).
We conclude that, like in intact Ca2+-ATPase, the
Arg505-Ala506 bond in p29/30 fragments
resulting from proteinase K treatment is highly sensitive to
trypsin.
Mass spectrometry (MALDI-TOF) experiments were fully consistent with
this conclusion and extended its validity to the fragments resulting
from Asp-N and elastase treatment of Ca2+-ATPase. Fig.
6 shows these results. A preliminary
experiment was performed with a control protein, apomyoglobin; this
allowed us both to calibrate the spectrometer response and to observe the presence of unexpected minor peaks (see asterisk in
panel A) presumably due to formation of adducts between the
polypeptide chain and the matrix with which it is initially
co-crystallized. In the experiment illustrated in panel B,
the peptides found in the supernatant of Asp-N-treated SR (Fig. 1) were
further treated with trypsin (as in the case of proteinase K fragments,
Fig. 5), and the smaller peptides generated were examined (see
"Experimental Procedures"). Besides the minor adducts
(asterisks), mass spectrometry reveals two major peaks
(panel B), corresponding to the N-terminal part and the
C-terminal part of the initial fragment. Because of its larger size,
the peptide corresponding to the peak on the right is less efficiently
desorbed by the laser beam than that corresponding to the peak on the
left, but both masses can be estimated unambiguously. The measured
m/z value of the larger peptide (17,227)
corresponds closely to the theoretical value (17,225) expected for a
peptide starting at Asp351 (the N-terminal residue of Asp-N
fragments, as deduced from the sequencing experiments) and ending after
Arg505. The Arg505-Ala506 bond is
therefore highly reactive to trypsin in fragments resulting from Asp-N
treatment of Ca2+-ATPase. The mass of the smaller peptide
then allows us to determine the C-terminal residue of the fragment
without ambiguity, in this case Arg615 (as indicated in
Table I). The same experiment was repeated with fragments resulting
from Ca2+-ATPase treatment with elastase (panel
C); again two groups of peaks showed up, and the measured masses
were consistent with the previously determined N-terminal residues and
the existence of a unique tryptic cleavage site at the
Arg505-Ala506 bond (see figure legend). The
same conclusion was also true for proteinase K fragments (panel
D). Thus, all fragments retain an exquisite sensitivity to trypsin
of their Arg505-Ala506 peptidic bond.

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Fig. 6.
MALDI-TOF mass spectrometry of proteolytic
fragments, after further cleavage by trypsin. Panel A shows
a control experiment with apomyoglobin, with adducts between the
polypeptide and the matrix (sinapinic acid) showing up as minor peaks
indicated by asterisks, both in this panel and in
the other ones. For the experiments illustrated in panels
B-D, the supernatant of SR treated with Asp-N (panel
B), elastase (panel C), or proteinase K (panel
D) (as for Fig. 1) was further treated with trypsin (4 µg/ml, 30 min at 20 °C). The resulting peptides were desalted (see
"Experimental Procedures") and examined by MALDI-TOF mass
spectrometry. The peptides corresponding to peaks in panel B
are indicated in parentheses in the figure, together with
their theoretical [M + H+] values. For the
peaks in panels C and D, these
peptides and the theoretical [M + H+] values are as
follows: panel C, C-terminal half-peptides: 12,086 (Ala506-Cys614, 12,078 theoretical value),
11,542 (Ala506-Gly609, 11,533 theoretical
value), 11,629 (Ala506-Ser610, 11,621 theoretical value), 11,126 (Ala506-Lys605,
11,117 theoretical value), panel C, N-terminal
half-peptides: 17,321 (Ser350-Arg505, 17,312 theoretical value), 16,886 (Gly354-Arg505,
16,880 theoretical value); panel D, C-terminal
half-peptides: 11,623 (Ala506-Ser610, 11,621 theoretical value), 11,480 (Ala506-Met608,
11,476 theoretical value), 11,117 (Ala506-Lys605, 11,117 theoretical value);
panel D, N-terminal half-peptides: 16,619 (Thr357-Arg505, 16,609 theoretical value),
17,318 (Ser350-Arg505, 17,312 theoretical
value), 16,300 (Gln360-Arg505, 16,293 theoretical value).
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FITC is a covalent specific reagent for Lys515 in the
Ca2+-ATPase cytoplasmic domain (32). To study the
reactivity toward FITC of Lys515 in p29/30, we performed
two kinds of experiments. The results of the first approach can be seen
in lanes 1 and 2 in Fig. 5B. In the
experiment illustrated by these lanes, the supernatant of proteinase
K-treated Ca2+-ATPase was prepared from unlabeled SR
vesicles, and p29/30 fragments were subsequently labeled by adding FITC
directly to this supernatant, under less than stoichiometric conditions
(to reduce unreacted FITC to a minimum); subsequently, as explained
above, these fragments were treated with trypsin and processed for
SDS-PAGE. Comparison of lanes 2 and 6 in Fig.
5B shows that FITC added to p29/30 only labels the p12
peptide and neither the p17 nor the p18 peptides, although the p12
peptide contains fewer lysine residues than the two other peptides (7 lysine residues compared with 12 or 13). Selectivity of labeling is
thus demonstrated. In the second approach, the same protocol was used,
but the unlabeled p29/30-containing supernatant was now either (i)
reacted with an excess of FITC (16 µM) first, followed by
elimination of unreacted FITC (by passing through a Pharmacia PD10
column) and subsequent treatment with trypsin, or (ii) treated with
trypsin in the absence of any FITC labeling. In both cases, this was
followed by SDS-PAGE separation of the resulting peptides,
electrotransfer onto polyvinylidene difluoride membranes, and
N-terminal sequencing of p12. In the peptide derived from the
FITC-treated sample, we expected that sequencing would not be able to
identify the FITC-modified residue. Indeed, the yield of the 10th
residue (corresponding to Lys515) was much lower in the
FITC-treated sample (designated as p12-FITC) than in the non-reacted
one (designated as p12) (see panel C in Fig. 5). This
confirms that selective labeling of Lys515 has occurred in
the p29/30 fragment, as in intact Ca2+-ATPase, resulting in
disappearance of at least close to 50% of Lys515 residues.
The reason why the other half of the Lys515 residues
appears not to have been modified was not investigated further. It is
possible that labeling is less selective for p29/30 fragments than for
ATPase; it is possible also that the labeling period in our experiments
(30 min at pH 8 only) is simply not sufficient for 100% labeling of
the proteolytic fragments, although it is sufficient for intact
ATPase.
Hydrodynamic Properties of p29/30 Fragments, Secondary Structure,
and Spectroscopic Characterization of p29/30 Fragments--
The
hydrodynamic properties of p29/30 fragments were examined by gel
filtration on a TSK G3000 SWXL column (33). As shown in Fig.
7, a sharp major peak showed up in the
eluant, together with minor smaller peptides. On the basis of column
calibration with water-soluble proteins, the position of the major peak
corresponds to a Stokes radius of 24 Å. Since
Rmin for a 29.5-kDa protein can be estimated to
be 20.6 Å (using a partial specific volume of 0.745 cm3/g), this Stokes radius corresponds to a frictional
ratio of 1.17. This frictional ratio is compatible with an almost
perfectly globular shape of the p29/30 fragments, i.e. a
compact structure, consistent with the resistance of the fragments to
most proteolytic enzymes.

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Fig. 7.
Gel filtration of p29/30 fragments.
Here, p29/30 fragments were prepared by proteolysis with agarose-bonded
proteinase K (Sigma P9290), as follows: 5 mg of washed agarose beads
were suspended in 500 µl of buffer (100 mM Bis-Tris and
2.5 mM EGTA at pH 6), and SR was added to a final
concentration of 1 mg/ml; after 2 h at 18 °C under gentle
stirring, the reaction was stopped (0.5 mM PMSF), and low
speed centrifugation on a table-top centrifuge was performed to remove
the beads, followed by high speed centrifugation (Beckman rotor
TLA-100, 90,000 rpm, 1 h at 4 °C); the supernatant was then
concentrated on Centricon 10 (Amicon), and 40 µl at 2 mg/ml protein
were finally loaded onto the column. These fragments were
chromatographed (33) at 20 °C on a TSK G3000 SWXL column (0.78 × 30 cm, with a precolumn) equilibrated with 100 mM KCl, 0.1 mM Ca2+, and 10 mM Tes (pH
7.5), and eluted with the same buffer at a flow rate of 0.5 ml/min;
p29/30 is seen eluting as a sharp peak between the void volume
(V0) and the total volume
(Vt) of the column, together with minor amounts of
smaller peptides. The inset shows a calibration of the
column, i.e. Stokes radius (Rs) as a
function of the distribution coefficient KD (KD = (Ve V0)/(Vt V0)) for the following proteins (with Stokes
radius in parentheses): ferritin (Ferr, 6.3 nm), bovine
serum albumin (BSA, 3.5 nm), ovalbumin (Ova, 2.8 nm), -lactoglobulin ( Lgb, 2.75 nm), chymotrypsinogen (CTG, 2.25 nm), and pancreatic ribonuclease
(RNase, 1.75 nm).
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The fact that p29/30 fragments have a well organized structure was also
checked by circular dichroism (CD) measurements. As shown in
panel A of Fig. 8, the
supernatants of SR vesicles treated with proteinase K for 15 and 30 min
(dashed and solid lines, respectively) have CD
spectra whose shapes are closely similar and whose amplitudes only
slightly differ, consistent with almost complete conversion of ATPase
to p29/30 fragments after 30 min. After 120 min proteolysis (dashed-dotted line), the slightly more pronounced trough at
205-207 nm is indicative of further hydrolysis of p29/30 to small
disorganized peptides (random coils exhibit strongly negative
ellipticities below 200 nm); however, this trend is much more apparent
after an additional 10 h proteolysis at 37 °C (dotted
line). After analysis of the total amino acid contents of the
supernatant resulting from 30-min proteolysis, the measured ellipticity
was converted to molar ellipticity: for this 30-min supernatant, the
result, expressed in degrees·cm2·dmol
1,
is shown by the solid line in panel B of Fig. 8.
Since such a supernatant sample in addition to p29/30 peptides contains
the very small peptides derived from the 608/610-734/747 region (15), we also attempted to obtain purer p29/30 peptides by passing this supernatant over a Sephadex G25M column before measuring ellipticity and amino acid contents of the fraction eluted in the void volume; the
result is shown by the dashed line in panel B,
which, as expected, reveals a larger (negative) molar ellipticity at
222 nm (characteristic of
helices) and a smaller relative
contribution of the trough at 205-207 nm in the Sephadex eluate,
compared with the initial supernatant. For comparison, the molar
ellipticity of C12E8-solubilized deoxycholate-extracted ATPase is also
shown in panel B as a dashed-dotted line.

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Fig. 8.
CD spectra of soluble proteolytic
fragments. For these experiments, proteolytic cleavage of
Ca2+-ATPase was performed in a medium containing 4 mg/ml SR
protein, 30 µg/ml proteinase K, 10 mM NaCl, and 10 mM
Na2HPO4/NaH2PO4 at pH
7.5. For panel A experiments, proteolysis was stopped with PMSF after 15, 30, or 120 min at 20 °C (dashed line, solid
line, and dashed-dotted line, respectively), samples
were centrifuged, and supernatants, after 4-fold dilution in the same
medium, were examined for their CD spectra in a cell of 0.2-mm optical
path length. Problems due to too high absorbance were restricted to the
180-184-nm region (hatched box), and the base line was
subtracted. We also prepared a sample which, after 120 min proteolysis
at 20 °C, was centrifuged to remove membranous fragments but to
which PMSF the proteinase K inhibitor was not added; this supernatant was incubated for an additional 10 h at 37 °C (dotted
line), to produce extensive hydrolysis of p29/30 fragments (as
tested by SDS-PAGE; data not shown). For panel B
experiments, 0.9 ml of the supernatant after 30 min proteolysis was
passed through a Sephadex G25M column (Pharmacia PD10) to separate
larger peptides from the smaller peptides, and the larger peptides
(p29/30) eluting in the void volume were pooled and diluted 2-fold for
CD measurements (dashed line). The measured ellipticity was
compared with that of two other samples, total supernatant, diluted
four times (same sample as in panel A, solid
line) and intact deoxycholate-extracted ATPase, solubilized in the
presence of 4 mg/ml C12E8, the CD of which was measured at 0.2 mg of
protein/ml. For all three samples illustrated in panel B,
the total amino acid contents were also determined to make it possible
to plot molar ellipticities.
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All these spectra were analyzed to estimate, according to various
methods, the percentage of the various secondary structures present in
p29/30 peptides. Depending on the method, as made possible by the
"Dichroprot" software, 28 (±5)% of amino acids were estimated to
be involved in
-helices in the p29/30-containing Sephadex eluate
(compared with 43 (±5)% in C12E8-solubilized ATPase). According to
some of these methods, e.g. the "variable selection
method" (34, 35), the proportion of amino acids involved in
-structures (either parallel or anti-parallel) was higher for p29/30
fragments than for the entire ATPase, 20 (±3)% compared with 14 (±3)%, and
-turns represented 19 and 15%, respectively (again
±3%). Thus, an unambiguous outcome of these measurements is the
demonstration that a significant fraction of amino acids in p29/30
fragments are organized in defined secondary structures.
We finally performed a few experiments to characterize from a
spectroscopic point of view the soluble p29/30 subdomain. We first
focused on intrinsic fluorescence. After proteinase K treatment, the
fluorescence spectrum of non-centrifuged samples is similar to that of
control ATPase, although slightly less intense (not shown). As shown in
panel A of Fig. 9, normalized
excitation spectra reveal that the Trp shoulder at 290 nm is less
prominent for the p29/30-containing supernatant of such samples
(dotted line) than for the total samples (solid
lines); in addition, the emission spectrum of the
p29/30-containing supernatant is red-shifted by about 6 nm compared
with the fluorescence of the total sample, which probably indicates
that the environment for Trp552 in p29/30 peptides is more
polar than, on average, the environment of the other 12 Trp residues of
Ca2+-ATPase; the latter residues in native ATPase are
thought to reside in or close to the ATPase transmembrane sector
(2).

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Fig. 9.
Spectroscopic characterization, in p29/30
fragments, of Trp fluorescence, FITC fluorescence, and energy transfer
from Trp to FITC. Panel A, excitation (left) and
emission (right) intrinsic fluorescence spectra. SR vesicles
were treated for 45 min as for Fig. 2, and spectra for non-centrifuged
samples (Tot) and supernatants (Sup)
(solid and dotted lines, respectively) were
examined after 10-fold dilution (to 100 µg/ml protein for
non-centrifuged samples) into a medium containing 100 mM
KCl, 5 mM Mg2+, and 50 mM Mops-NaOH
at pH 7 and 20 °C. ex was 290 nm for emission spectra, and em was 330 nm for excitation spectra (hence
the Rayleigh peaks). Spectra were normalized to the same maximal level. Panel B, excitation spectra for FITC ( em = 520 nm, hence the Rayleigh peaks), after 50-fold dilution into the same
medium as for panel A of samples in which FITC was either
(i) bound to intact FITC-labeled Ca2+-ATPase (solid
line), or (ii) bound to p29/30 fragments (dashed line)
in the supernatant of FITC-labeled vesicles treated for 30 min like in
panel A experiment, or (iii) bound to the small peptides
resulting from extensive proteolysis of FITC-labeled SR vesicles after
an additional 6 h at 37 °C in the presence of 0.6 mg/ml
proteinase K (dashed-dotted line), or (iv) free in buffer, at a similar concentration (dotted line; the excitation
spectrum of this sample is close to that of the previous one, but its
Rayleigh peak is smaller, as expected). Inset, Stern-Volmer
plot of FITC quenching by KI, with FITC either bound to
Ca2+-ATPase (circles), bound to the supernatant
peptides after 15 or 45 min proteolysis (inverted triangles
and triangles, respectively), or free in solution
(squares). Panel C, emission spectra revealing fluorescence transfer from Trp to bound FITC in p29/30 fragments. Unlabeled SR vesicles (2 mg/ml) were treated for 30 min with proteinase K as for Figs. 3 and 4 and centrifuged, and the supernatant was incubated for 30 min with or without 16 µM FITC. Samples
were diluted 20-fold in a pH 7 medium as for panels A and
B. Emission spectra (with ex = 290 nm) were
recorded for (i) unlabeled supernatant (solid line), (ii)
free FITC (dashed line), and (iii) FITC-labeled supernatant
(dotted line). All spectra are corrected for Raman scattering.
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We also examined the characteristics of the fluorescence of FITC,
either free or bound to p29/30 fragments or intact
Ca2+-ATPase. Panel B in Fig. 9 shows that the
excitation spectrum of FITC bound to intact Ca2+-ATPase
(solid line) is red-shifted by almost 10 nm with respect to
that of free FITC (dotted line). This fact, together with
the lower pKa observed for ATPase-bound FITC
compared with free FITC (pKa of 5.8 instead of 6.4, see Ref. 36), indicates that bound FITC experiences an environment
different from that for free FITC. Panel B also shows that
the excitation spectrum of bound FITC after proteinase K
treatment is an intermediate one, in both the non-centrifuged samples
(data not shown) and the p29/30-containing supernatants (in Fig.
9B, the dashed line corresponds to a supernatant
obtained after 30 min proteolysis). Complete disappearance of p29/30
fragments after extensive proteolysis results in an excitation spectrum
similar to that of free FITC (dashed-dotted line). These
results suggest the environment of bound FITC in p29/30 fragments
produced by proteinase K treatment is somewhat different from its
environment in intact Ca2+-ATPase but is not changed to the
extent that it would become completely exposed to the aqueous medium.
This conclusion is strengthened by experiments in which solvent
accessibility of FITC is examined with the quenching agent iodide (see
inset to Fig. 9B); as previously found (36), FITC
accessibility to iodide is higher for free FITC than for FITC bound to
Ca2+-ATPase (slopes for Stern-Volmer plots are 6.5 M
1 and 2.7 M
1,
respectively), suggesting restricted accessibility to FITC, a
suggestion supported by immunoreactivity data (37). In p29/30 fragments, this accessibility has an intermediate value, close to that
of FITC in intact Ca2+-ATPase (slopes for Stern-Volmer
plots are 3.1 or 3.9 M
1 for supernatants
after 15 or 45 min proteolysis, respectively; in the latter case,
degradation of p29/30 fragments to much smaller peptides contributes to
the larger slope). Thus, the environment of FITC in p29/30 fragments is
still significantly protected from the aqueous solvent.
Finally, panel C in Fig. 9 shows that Trp552 and
bound FITC are close enough in p29/30 fragments to allow fluorescence
transfer from the indole moiety to the fluorescein moiety to occur.
Since nothing is known about the relative orientations of these two fluorophores, we cannot, however, derive from the data a distance between these two moieties. For random relative orientation of the
indole and fluorescein moieties, the modest transfer observed in Fig.
9C would imply a distance between the two fluorescent moieties larger than 25 or 30 Å, the Förster radius
R0. Alternatively, the distance could be smaller
if the two fluorescent moieties are positioned relative to each other
in a relatively rigid way unfavorable for energy transfer.
Demonstration of Metal-binding Sites in p29/30--
When the
measurements of either TNP-ATP fluorescence or Trp fluorescence
illustrated in Figs. 2 and 3 were repeated with p29/30 fragments in the
presence of Mg2+ (5 mM), we found that the
fluorescence responses due to nucleotide binding to the fragments were
reduced, presumably because of a decreased binding affinity (data not
shown). The same counterproductive effect of Mg2+ on
TNP-ATP binding was previously noted by Moutin et al. (14) with the heterologously expressed isolated large cytoplasmic loop. Nevertheless, in our intrinsic fluorescence measurements, as those in
Ref. 14, there was no direct evidence for binding of
Mg2+ per se to the soluble p29/30 fragments; two
additions of Mg2+, 5 mM each time, performed in
the absence of nucleotide, had no effect on the intrinsic fluorescence
level of p29/30 fragments (data not shown).
In contrast, by using lanthanide ions, we obtained direct evidence for
metal binding to p29/30 in the absence of nucleotides. This was
investigated by using Nd3+ ions and SR vesicles previously
labeled with FITC at Lys515. Nd3+ has
previously been shown to quench the fluorescence of bound FITC by up to
40%, even at high ionic strength (39, 40), probably because of
efficient Förster transfer from the fluorescein moiety of bound
FITC to an Nd3+ ion bound very close to it on the ATPase,
at a distance of about 1 nm. The specificity of this interaction is
suggested by the fact that Mg2+ competes with
Nd3+, and it has been argued that the Nd3+ ion
responsible for FITC quenching may be bound to a metal-binding subsite
normally occupied by Mg2+ and located (because of the short
FITC-Nd3+ distance) in the ATPase active cleft (39). Thus,
in the experiment illustrated in Fig.
10, we examined quenching by
Nd3+ of FITC fluorescence in FITC-labeled p29/30 fragments.
These fragments were prepared by first labeling SR vesicles with FITC (17), followed by treatment with proteinase K and centrifugation. Increasing concentrations of Nd3+ were added either to
non-centrifuged samples (closed symbols in Fig. 10) or to
their supernatants (open symbols in Fig. 10).

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Fig. 10.
Binding of Nd3+ ions to
FITC-labeled Ca2+-ATPase. FITC-labeled SR vesicles
were treated with proteinase K for various periods under conditions
identical to those described in Fig. 2; aliquots were centrifuged, and
lanthanide-induced quenching of FITC fluorescence in the various
samples was measured. Closed symbols represent total samples
and open symbols represent supernatants. Circles refer to control experiments with intact SR-FITC; for the other experiments, proteolysis duration was 30 (open diamonds) or
45 min (closed and open triangles). Aliquots of
the treated samples were diluted 50-fold in a medium containing 5 mM Mg2+, 0.1 mM Ca2+,
500 mM KCl, and 40 mM Mops-NaOH at pH 7 and
20 °C (this high ionic strength is designed to reduce nonspecific
interactions with the trivalent Nd3+ ion, see Ref. 39).
FITC fluorescence was examined with ex = 495 nm (bw = 2 nm) and em = 520 nm (bw = 5 nm). The
fluorescence data are normalized and plotted as percent of the initial
fluorescence level in the absence of lanthanide. Additional experiments
include quenching by Nd3+ of FITC free in solution
(open squares) and quenching by Nd3+ of
covalently bound FITC after an additional 6 h of proteolysis in
the presence of 0.6 mg/ml proteinase K at 37 °C (closed
squares); the dotted line corresponds to an experiment
performed with La3+ instead of Nd3+. For
millimolar Nd3+ concentrations, inner filter effects caused
by Nd3+ absorbance begin to be detectable (not
shown).
|
|
When examined at excitation and emission wavelengths commonly used for
free FITC (495 and 520 nm, respectively), total FITC fluorescence
slightly varies during proteolysis (data not shown), presumably in part
because of the small spectral change of bound FITC after proteolysis
documented in Fig. 9B. When treated samples are centrifuged,
the proportion of FITC fluorescence recovered in the supernatant
increases with the proteolysis period, up to almost 100% (data not
shown), in agreement with almost complete degradation of the region
around Lys515 to soluble fragments. As proteolysis
proceeds, the well known sensitivity of covalently bound FITC to high
affinity Ca2+ binding to Ca2+-ATPase (41)
progressively vanishes, again as expected (data not shown).
Nevertheless, after 30 or 45 min proteolysis, quenching by
Nd3+ is retained to a significant extent for proteolyzed
samples as well as for their supernatants. Analysis of such quenching
curves is best performed when the data are normalized, and residual
fluorescence is expressed as percent of the initial fluorescence in the
absence of Nd3+, as shown in Fig. 10. For 30-300
µM Nd3+, quenching by Nd3+ of
FITC fluorescence in p29/30 fragments (triangles and
diamonds) is definitely observed, although to a lesser
extent than in Ca2+-ATPase (closed circles).
Open and closed squares are two control experiments designed to confirm the insensitivity to Nd3+
of (i) FITC in solution (open squares) and (ii) FITC bound
to the ATPase peptides resulting from extensive proteolysis
(closed squares; in this case, proteolysis was performed as
in the experiment corresponding to the dashed-dotted line in
Fig. 9B). The dotted line illustrates a different
control experiment showing that, as expected because of the lack of
absorbance properties, La3+ does not quench fluorescence of
FITC bound to intact Ca2+-ATPase. These data demonstrate
that soluble p29/30 fragments retain some residual ability (although
with reduced affinity, as in the case of nucleotide binding) to bind
lanthanide ions relatively close to FITC bound at
Lys515.
 |
DISCUSSION |
In this report, we show that by using various proteolytic enzymes
(Fig. 1), it is possible to prepare fragments of
Ca2+-ATPase closely related to each other and all
originating from the cytosolic portion of this membranous enzyme (Table
I). According to hydrodynamic and circular dichroism analysis, these
"p29/30" fragments have a compact globular shape with a relatively
high content of secondary (
/
) structure (Figs. 7 and 8). They
have a significant affinity toward nucleotides (Figs. 2-4) and also
retain reactivity toward trypsin and FITC, like intact
Ca2+-ATPase, and at the same sites (Figs. 5 and 6). All
this suggests that these proteolytic fragments, which contain a major
fraction of the ATPase cytosolic head, are released without loss of
basic structural features from a region which, in the native tertiary structure, constitutes an autonomous structural domain.
The reasons why the properties of p29/30 fragments have not been
recognized earlier are probably related to the fact that most previous
proteolytic investigations on Ca2+-ATPase have been
performed with trypsin. Studies with this enzyme have never resulted in
production of fragments comparable to those described in the present
work because trypsin has an extraordinary potency for cleavage of
Ca2+-ATPase in the middle of the molecule, at
Arg505-Ala506. We found no evidence for a
particular sensitivity of the Arg505-Ala506
peptidic bond to proteases other than trypsin; thus, the high sensitivity to trypsin of this bond is probably due to a particular conformation of the Arg505-Ala506 peptidic
bond, favorable for trypsinolysis, rather than to a strategic location
of this bond at the boundary between two distinct domains of
Ca2+-ATPase. It is remarkable that proteolysis fragments
similar to our p29/30 fragments have in fact been already described for
Na+,K+-ATPase and
H+,K+-ATPase, after treatment with either
trypsin (42) or other proteases (43-46).
Our extensive peptide sequencing and mass spectrometry data allow us to
unambiguously conclude that p29/30 fragments start close to the
phosphorylation domain, around residue Asp351, and end in
the 605-615 region, well in advance of the conserved region starting
around residue 670 (the so-called "hinge" region). N-terminal
cleavage sites are consistent with a rather superficial localization of
the phosphorylatable Asp351 residue, to which
phosphorylating substrates must of course have access, in the region
delimited by the CSD351KTGTLT motif. However, p29/30
fragments are probably not primary cleavage products, and the
possibility therefore remains that accessibility to proteolytic attack
is somewhat modified by previous cuts in the N-terminal and C-terminal
regions of the ATPase (15). Concerning the C-terminal end in the
605-615 region, a region that has not been given much attention up to
now, it is of interest that it is flanked by two conserved motifs,
present in most eucaryotic P-type ATPases, D601PPR and
MIT625GD. According to the Rost and Sander algorithm (47), the 608-616 region is surprisingly predicted to be
-helical and thus should be resistant to proteolytic enzymes; in contrast, the
vulnerability of this region to proteolytic cleavage could indicate
that this region forms a loop connecting p29/30 to the rest of the
ATPase. It is worth noting also that according to the prediction in
Ref. 2, 118-120 residues in the sequence corresponding to the isolated
p29/30 fragments should be
-helical, i.e. more than 45%,
whereas our CD measurements suggested a significantly smaller (about
28%) fraction of
-helical residues in these fragments.
Irrespective of the detailed conformation and location of the
N-terminal and C-terminal boundaries of p29/30 fragments, it is clear
from the present results that these fragments form a rather compact
domain and that this domain is able to bind nucleotides with an
affinity which is quite respectable but reduced compared with that of
intact ATPase (Figs. 2-4). It is certainly not a surprise that a
fragment lacking several highly conserved and functionally critical
regions of the ATPase (including residues that obviously interact with
the ATP-binding pocket like Arg678 and Lys684,
see Ref. 12 for review) has a reduced affinity for its substrates. The
important fact is the ability of p29/30 fragments to bind these ligands
at all. Moutin et al. (14) previously concluded that the
nucleotide-binding site was at least partially preserved in the ATPase
peptide Lys329-Phe740 (prepared by heterologous
expression in Escherichia coli), which corresponds to the
entire "large cytosolic loop" of Ca2+-ATPase; our
results show that the same conclusion is valid for our smaller p29/30
fragments, which only comprise
Thr357/Ser350-Ser610/Met608
peptides. The observation that the presence of Mg2+ does
not favor ATP or ADP binding to p29/30 fragments is of interest. It is
known that in intact Ca2+-ATPase, an enhancing effect of
Mg2+ on the affinity of nucleotide binding is only observed
for ATP but not for ADP (30); thus, such an enhancing effect of
Mg2+ is not characteristic of the portion of the site that
recognizes the adenine moiety. In Na+,K+-ATPase
also, high affinity binding of ATP is not dependent on Mg2+. In addition, there is in
Na+,K+-ATPase evidence for electrostatic
interaction between the
-phosphate of ATP and the phosphorylatable
residue (48, 49); corresponding interactions will obviously be affected
by proteolysis in our p30 or p29 peptides (starting at
Ser350 and Thr357, respectively) since the
region around the phosphorylation site (Asp351) is either
absent (p29) or presumably poorly structured (p30). Thus, the fact that
MgATP does not appear to bind to p29/30 fragments (nor to bind to the
entire large cytosolic loop, see Ref. 14) with an affinity higher than
that of Mg2+-free ATP should not be taken as an objection
against the nucleotide binding ability of p29/30 fragments, which we
think is demonstrated here unambiguously. It is also worth pointing out
that the nucleotide binding abilities of the proteolysis-derived p29/30
fragments (this work) and of the heterologously expressed large
cytosolic loop (14) are of the same order of magnitude, despite the
fact that the former fragments are 35-40% shorter than the latter
(residues 350/357-608/610 compared with residues 329-740).
Although P-type ATPases lack the classical glycine-rich Walker sequence
motifs found in water-soluble kinases (50), it has previously been
suggested that like these kinases, the large cytosolic loop of
Ca2+-ATPase comprises two distinct domains, a
phosphorylation domain and an ATP-binding domain (2). Mainly based on
the existence of a tryptic split at
Arg505-Ala506, between the phosphorylation site
(Asp351) and the ATP-protected FITC-binding site
(Lys515), the Arg505-Ala506 bond
was originally thought to be involved in the separation between
phosphorylation and ATP-binding subdomains. Subsequent work, however,
indicated that residues located N-terminally of Arg505 also
play an important role in nucleotide binding (e.g. Ref. 51).
Based on the present data, we speculate that in Ca2+-ATPase
and probably also in other P-type ATPases, the ATP-binding region
mainly consists of the p29/30 domain, with the phosphorylation site
located at the N-terminal border of this domain. In this view,
interaction with the rest of the molecule of the rather compact p29/30
domain would be made possible by flexibility of the regions connecting
this domain to the rest of the molecule; on the one hand, the strategic
location of the phosphorylation site at the N-terminal boundary of the
domain would be consistent with the demand, from an energy-transducing
enzyme, of conformational flexibility at the active site; on the other
hand, the 605-615 region at the C-terminal boundary of the domain
might constitute the real hinge with conformational flexibility. In
this view, the conserved region starting around residue 670 (and whose
previous description as a hinge was probably
misleading)4 would be part of
a second lobe, perhaps not strictly necessary for nucleotide binding
(see Ref. 52) but whose precise positioning could set the stage for a
nucleophilic attack on the
-phosphate of ATP and its transfer to the
phosphorylatable Asp351 residue; note that binding of
Ca2+ to the ATPase transport sites changes the precise
positioning, with respect to the nucleotide-binding site, of
Lys684 in this putative second lobe (Refs. 53 and 54; see
Ref. 12 for review).
With respect to the above considerations it should be pointed out that
it is not possible to completely exclude the possibility (as
considered, for instance, in Ref. 45) that in intact ATPase the
ATP-binding site could consist of a groove formed between the p29/30 subdomain and the rest of the molecule (the latter would
then again presumably include the conserved region starting around
residue 670, see review in Ref. 12). In this case, however, the
affinity for ATP binding on one side of an isolated p29/30 domain would
probably exhibit a reduction of much more than 1 order of magnitude
compared with intact ATPase, which was not the case (see Fig. 4). In
addition, assuming that the FITC planar chromophore is located at the
same place as the nucleotide base in intact ATPase, it would be
difficult to understand why FITC bound to p29/30 fragments remains
significantly shielded from the aqueous environment (Fig.
9B). Thus, a more likely hypothesis might be that a cavity
in the p29/30 domain provides most of the ligands for binding the
adenine moiety of nucleotides. The answer to the question of the exact
nature of the ATP-binding site will hopefully be provided by x-ray
crystallography analysis of this domain with or without ATP bound to
it.
In addition to nucleotides, we have shown here that p29/30 fragments
are able to bind Nd3+ ions (Fig. 10); this observation
deserves special comment. Although lanthanide binding to the ATPase
Ca2+ transport sites has sometimes been advocated in the
past, it has been demonstrated that these trivalent ions have an even
higher affinity for other sites in the protein (21, 55-57), and the present study shows that some of these sites are located in the region
of the ATPase cytosolic domain corresponding to p29/30. Whether these
sites have any functional relevance is the next question. Based on the
idea that the fluorescein moiety of FITC bound to Lys515 in
intact ATPase occupies the nucleotide-binding cleft (32, 58), Highsmith
(39) suggested that the large quenching effect exerted by
Nd3+ (in competition with Mg2+) on FITC
fluorescence was due to Nd3+ binding at a metal subsite in
the catalytic active cleft of Ca2+-ATPase normally occupied
by Mg2+. From other functional studies we know that
replacement of Mg2+ by other metal ions (presumably in the
catalytic cleft) can indeed occur, with the result that in the presence
of lanthanide ions the ATPase is severely inhibited (e.g.
Refs. 59-61). Along this line, the observed quenching by
Nd3+ of FITC fluorescence in p29/30 fragments might suggest
that p29/30 fragments provide some of the liganding groups for binding
of catalytically active Mg2+. In
Na+,K+-ATPase, a role of Asp586 (in
the DPPR motif of the homologous domain) in coordinating the
Mg2+ ion required for ATPase activity was also recently
proposed (62-63). Note, however, that residues much further away
toward the C-terminal end of the Ca2+-ATPase large
cytosolic loop have also been proposed as Mg2+-binding
sites (e.g. Refs. 52 and 64), and further studies are needed
to clarify this issue. As a completely different and somewhat less
attractive possibility, lanthanide ions might bind to p29/30 fragments
at sites that are not relevant for ATPase activity. Nevertheless, even
if this were the case, the present demonstration of lanthanide binding
to p29/30 fragments could be useful for future phasing of x-ray
diffraction diagrams. Before crystallization of p29/30-like domains,
however, we have to prepare samples with a high purity. Our
hydrodynamic and spectroscopic characterization of freshly prepared
proteolysis-derived fragments (Figs. 7-9) will then help check that
purification procedures have not subsequently led to irreversible
changes in conformation for the purified peptide(s).
We thank Françoise Baleux (Institut
Pasteur, Paris) for total amino acid analysis; Bernard Lagoutte (CEA,
Saclay) for kindly performing a few N-terminal sequencing experiments;
Béatrice de Foresta for critically reading our manuscript;
Jean-Pierre Le Caer (ESPCI and CNRS, Paris) for permanent assistance
with the mass spectrometry experiments; and François Penin and
Gilbert Deléage (IBCP, Lyon) for their expert help with
deconvolution of CD spectra and the Dichroprot software they made
accessible to us.