(Received for publication, November 29, 1995, and in revised form, October 21, 1996)
From the Instituto de Química-Física
Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain and
§ Chimie-Physique des Macromolécules aux Interfaces,
CP206/2, Universite Libre de Bruxelles,
B-1050 Bruxelles, Belgium
Differential scanning calorimetry has been used to
characterized the thermal denaturation of gastric
(H+,K+)-ATPase. The excess heat capacity
function of (H+,K+)-ATPase in highly oriented
gastric vesicles displays two peaks at 53.9 °C
(Tm1) and 61.8 °C
(Tm2). Its thermal denaturation is an
irreversible process that does not exhibit kinetic control and can be
resolved in two independent two-state processes. They can be assigned
to two cooperative domains located in the cytoplasmic loops of the
-subunit, according to the disappearance of the endothermic signal
upon removal of these regions by proteinase K digestion. Analysis of
the thermal-induced unfolding of the enzyme trapped in different
catalytic cycle intermediates has allowed us to get insight into the
E1-E2 conformational
change. In the E1 forms both transitions are
always observed. As Tm1 is shifted to
Tm2 by vanadate and ATP interaction,
the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions.
Stabilization of the E2 forms results in the
disappearance of the second transition at saturation by K+,
Mg2+-ATP, and Mg2+-vanadate as well as in
significant changes in Tm2 and
H1. The catalytic domain melts following a
process in which intermolecular interactions either in the native or in
the unfolded state might be involved. Interestingly, the
E2-vanadate-K+ form displays
intermediate properties between the E1 and
E2 conformational families.
The (H+,K+)-ATPase (EC 3.6.1.36) is the
electroneutral ion pump responsible for acid secretion in the gastric
mucosa. In this process cytosolic H+ is exchanged 1:1 for
extracellular K+. From a structural point of view this
enzyme is a protomer composed of a catalytic -subunit (95 kDa) and a
glycosylated
-subunit (52 kDa). The
-subunit, a membrane
multispanning polypeptide, is responsible for the coupling of ATP
hydrolysis with ion transport across the membrane, whereas the
-subunit, a single membrane spanning polypeptide, is required for
the proper assembly and targeting of the entire protein. According to
the current models of (H+,K+)-ATPase (1), the
-subunit has 795 amino acid residues exposed on the cytoplasmic side
(i.e. extravesicular side), 172 amino acids embedded, and 66 amino acid residues located in the vesicle interior (intravesicular
side). The
-subunit is mainly intravesicular, with 225 amino acid
residues inside the vesicle, 40 amino acid residues on the cytoplasmic
side, and a single transmembrane segment of 27 residues.
The gastric (H+,K+)-ATPase is a member of the
P-ATPase family and shares many features, including sequence homology
and enzymatic mechanism, with other members such as the
Ca2+-ATPase (2) and especially the
(Na+,K+)-ATPase (3). Two major conformations,
called E11 and
E2, have been identified. In the
E1 conformation the proton binding site is
cytosolic, and the phosphorylated enzyme reacts with ADP to form ATP
(4, 5). The E1-phosphorylated enzyme converts
spontaneously to the E2-phosphorylated form of
the enzyme, which is no longer sensitive to ADP but is rapidly
dephosphorylated in the presence of K+ (6). While definite
conformational changes take place during the catalytic cycle as
demonstrated from fluorescence of fluorescein-labeled -subunit (7,
8) and from limited trypsin digestion pattern (5), little is known on
the nature of these conformational changes. Amide hydrogen/deuterium
exchange kinetics for Neurospora plasma membrane
H+-ATPase have shown that at least 175 amino acid residues
are shielded from the solvent in an E2
conformation (9). Membrane disposition of the H5-H6 hairpin of the
homologous (Na+,K+)-ATPase
-subunit has
shown to be ligand-dependent (10).
Differential scanning calorimetry can provide information on the structural organization in cooperative domains and on domain-domain interactions in proteins (11-14). However, as discussed by others (11, 12, 14), the number of cooperative units does not always exhibit a straightforward relation with the number of structural domains identified by x-ray crystallography. While this approach has been widely used for water-soluble proteins, there are still only a few studies on membrane proteins (15). We have used this approach in the present study to investigate the structural organization into cooperative domains of the (H+,K+)-ATPase and its modulation by ligand binding. Proteinase K digestion of highly oriented gastric vesicles was used to locate the melting cooperative domains in the different topological regions of the protein. Analysis of the variations induced by ligand binding on the thermal denaturation of the enzyme has allowed us a tentative assignment of these cooperative domains in terms of their specific binding activities and has revealed the existence of structural rearrangements during the catalytic cycle.
The materials used throughout the whole experimentation were of the highest purity grade available. Proteinase K, vanadate, ATP, phenylmethanesulfonyl fluoride, nigericin, ouabain, and oligomycin were obtained from Sigma. SDS-PAGE reagents were of electrophoresis grade from Bio-Rad.
Gastric Vesicles Isolation and PurificationGastric
vesicles were isolated from hog gastric fundus by differential
centrifugation and discontinuous sucrose density gradient ultracentrifugation as described previously (16, 17). The material
collected at the 8-30% sucrose interface is referred to as gastric
vesicles in this paper. SDS-PAGE reveals essentially a major band of 95 kDa, corresponding to the -subunit, and a smear of 60-90 kDa, due
to the
-subunit. This smear is only resolved as a single band after
deglycosylation of the
-subunit (see below).
ATPase activity was measured in 40 mM Hepes-Tris, pH 7.2, containing 2 mM ATP and 2 mM MgCl2 in presence or in absence of 20 mM KCl. Sucrose (8%) was present or absent as indicated to create isotonic or osmotic shock conditions, respectively. Reaction was performed at 37 °C for 15 min and stopped by addition of SDS (final concentration 1.75%). Inorganic phosphate released from ATP was quantified according to Stanton (18), except that coloration was developed with ascorbate.
Proteinase K DigestionVesicles containing the
(H+,K+)-ATPase (2.5 mg ml1
protein concentration) in 50 mM Hepes-Tris, pH 7.2, containing 8% sucrose (conservation buffer) were incubated at 37 °C
with proteinase K in a protease/protein ratio of 1:4 (w/w). Proteolysis
was halted by the addition of phenylmethanesulfonyl fluoride (6 mM final concentration). The digested vesicles were
adjusted to 0.5 M NaCl and shaken on ice for a few minutes.
Then the NaCl concentration was set at 0.25 M by adding 1 mM Hepes buffer, and the samples were centrifuged at
45,000 × g for 45 min. The resulting pellets were
resuspended in the conservation buffer, pelleted again in the same
conditions, and stored at
20 °C. Aliquots were removed after 5 min, 30 min, 1, 4, 7, 24, 48, and 72 h from the digestion mixture.
Since ATPase digestion reaches a plateau after 1 h, as measured
from the lipid/protein ratio in the reisolated vesicles, 1-h incubation
periods were routinely used for the experiments described in this
paper.
Calorimetric measurements
were performed in a Microcal MC-2 differential scanning calorimeter
(Microcal Inc., Northampton, MA) at a heating rate of 0.5 K
min1, unless otherwise stated and under an extra constant
pressure of 2 atm. The standard DynaCp, CpCalc, DA-2, and Microcal
Origin softwares were used for data acquisition and analysis. The
excess heat capacity functions were obtained after base-line
subtraction and correction for the instrument time response. In all
cases thermal denaturation was found to be irreversible. Since
buffer-buffer base lines had the same shape as rescans, the latter
could be used as instrument base lines because the offset in Cp due to aggregation or other causes was unimportant (19). Gastric vesicle samples were prepared at a 0.5-2.0 mg/ml protein concentration in 50 mM Hepes-Tris, pH 7.2, containing 0.25 M
sucrose, 1 mM EGTA, and the required ligands. The presence
of 0.25 M sucrose in the buffer is essential to maintain
the integrity of gastric vesicles. Samples requiring 2 mM
MgCl2 or CaCl2 were prepared by centrifugation at 75,000 × g for 15 min after a 4-fold dilution in 50 mM Hepes, pH 7.2, and further resuspension of vesicles in
an EGTA-depleted buffer containing 2 mM divalent cation
chloride. Variations in the buffer pH with temperature were negligible
in the 20-80 °C range (7.20 ± 0.15) and followed by a sharp
drop to pH 6.65 at 95 °C. The addition of ionizable ligands at the
concentration required in the present experiments did not cause
additional modifications of pH. The
(H+,K+)-ATPase Mr
considered for calculations was 147,000 assuming a 90% of total
protein abundance.
Purity of gastric vesicle
(H+,K+)-ATPase as well as proteinase K
digestion products was analyzed by SDS-PAGE on 7.5% acrylamide gels
(20). Protein concentration was determined using the BCA kit (Pierce)
or a modified Lowry assay (21), using bovine serum albumin as standard
in both cases. Lipids were assayed with the MPR2 enzymatic test
specific for the choline derivatives (Boehringer Mannheim), assuming
that lipids carrying a choline group represent 50% of the total of the
lipids (22). The -subunit of the
(H+,K+)-ATPase was deglycosylated using
N-glycosidase F (Boehringer Mannheim) under denaturing
conditions. Gastric vesicles (20 ml of 1 mg/ml protein concentration
solutions) were boiled for 3 min in SDS 1% (w/v) and
-mercaptoethanol 1% (v/v). Then, 25 ml of
n-octyl-
-D-glucopyranoside 10% (w/v), 150 ml
of phosphate-buffered saline/EDTA buffer (137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4,
8.1 mM Na2HPO4, and 20 mM EDTA), and 30 ml of N-glycosidase F (250 units/ml) were added before incubation at 37 °C during 24 h.
Gastric vesicle
thermal denaturation has been studied by differential scanning
calorimetry. The result of a typical scan of gastric vesicles in 50 mM Hepes-Tris, pH 7.05, containing 0.25 M
sucrose and 1 mM EGTA at 0.5 K min1 scan rate
is shown in Fig. 1 (trace b). Such a result is
representative of those obtained with at least three different enzyme
preparations and varying the protein concentration in the 0.5-2.0 mg
ml
1 range. The standard deviation of the mean in five
independent experiments amounted to ± 25 kcal mol
1
for the overall
H (281 kcal mol
1) and
±0.2 °C in peak positions. The thermogram, in the 15-95 °C temperature range, is characterized by the presence of two overlapping peaks, at 53.9 Tm1 and 61.8 °C
(Tm2), in agreement with previous
studies (23). Since about 90% of gastric vesicle total protein is
(H+,K+)-ATPase and the thermal-induced
transitions occur within the temperature range described for most
proteins, including (Ca2+,Mg2+)-ATPase from
sarcoplasmic reticulum (24, 25), it seems likely that both peaks
correspond to the (H+,K+)-ATPase denaturation.
This assignment is further supported by the sensitivity of both peaks
to (H+,K+)-ATPase-specific ligands (see
below).
Thermal denaturation of (H+,K+)-ATPase is an
irreversible process under all conditions employed, as indicated by the
fact that no endotherm is seen on rescanning the samples under any of
the experimental conditions employed. Partial heating experiments show
that both transitions are irreversible and proceed independently (data
not shown). The effect of scanning rate on the heat capacity profile
and thermodynamic parameters was studied (Fig. 1, traces A-C) in order to inspect a possible kinetic control in the
denaturation process. Tm1 values are
scanning rate independent, within the experimental uncertainty.
Tm2 is initially shifted toward lower
values as the heating rate decreases and then becomes constant at the
lowest scanning rate tested (0.5 and 0.33 K min1). This
type of dynamic effect is consistent with the existence of a slow
relaxation process between native and reversibly unfolded forms and
excludes a kinetic control by the irreversible steps. Thus, application
of equilibrium thermodynamics to the experimental traces would not lead
to significant errors (26). In fact, irreversibility appears to be a
general feature of membrane protein thermal denaturation arising from
the aggregation of the unfolded state in the membrane plane (15,
27).
(H+,K+)-ATPase thermal-induced denaturation can
be resolved into two independent two-state processes
(HvH/
H = 1) with enthalpy changes of 165 and 116 kcal mol
1 for the first and second
transitions, respectively (Fig. 1, Table I). Identical
values for the calorimetric and the van't Hoff enthalpies are
found.
|
To get insight into the protein regions involved in thermal
transitions, (H+,K+)-ATPase was digested with
proteinase K to differentiate the contributions of different
topological regions (cytoplasmic, membrane-embedded, or vesicle
internal parts). Accounting for asymmetric distribution of the - and
-subunits of (H+,K+)-ATPase across the
membrane, treatment with proteases will also allow separation of the
-subunit contribution (
-subunit is the main cytoplasmic
protruding fraction) from the
-subunit, provided that gastric
vesicles remain sealed during the isolation process. Activity
measurements were carried out to check the integrity and orientation of
the (H+,K+)-ATPase in the isolated vesicles.
Enzymatic activities are 30 (expressed as µmol
Pi/(mg·h)) in the absence of KCl (basal unspecific activity) and 122 in the presence of KCl after vesicle opening by
osmotic shock by dilution in a sucrose-free assay medium. Therefore, the full access of ATP and K+ to both side of the membrane
results in a supplementary activity of 92 µmol
Pi/(mg·h). In the absence of osmotic shock, the
supplementary activity was 6 µmol of Pi/(mg·h),
accounting for the open vesicles present in the preparation (6.5%) and
increases to 81 in the presence of the H+/K+
exchanger nigericin. These results indicate that at least 88% of the
vesicles are sealed and exhibit the ATP binding site on the outside of
the vesicle, which agrees with the data reported elsewhere (22, 27).
Since the ATP binding site is located on the largest cytoplasmic loop
of the
-subunit (loop C3 limited by H4 and H5 hydrophobic regions),
the above results demonstrate that at least 88% of the isolated
gastric vesicles exhibit that region directed outside the vesicle.
Before DSC experiments, gastric vesicles were first treated with
proteinase K in iso-osmotic conditions. Separation from digested peptide fragments and protease followed, as described under
"Experimental Procedures." According to the
(H+,K+)-ATPase current model, a large fraction
of the -subunit should be removed by this treatment, whereas most of
the
-subunit should remain intact. SDS-PAGE analysis of the
deglycosylated
-subunit give molecular masses of 31 kDa for the
untreated
-subunit and 30.5 kDa (starting at Gly-31 (28)), without
intensity loss, after proteinase K digestion. The
-subunit band
completely disappears when the digestion is performed under osmotic
shock conditions (data not shown). Therefore, the protease protection
observed in iso-osmotic conditions is due to the intravesicular
disposition of the largest portion of the
-subunit, which agrees
with the above described model. Regarding the
-subunit, SDS-PAGE
analysis reveals that its 95-kDa polypeptide chain practically
disappears (more than 95%) upon proteinase K digestion, even in the
absence of osmotic shock (data not shown). The largely cytoplasmic
localization of this subunit is confirmed because no fragments with a
mass greater than 20 kDa were detected.
No thermal transition was observed in the thermograms of proteinase
K-digested gastric vesicles (Fig. 1, trace D), even at protein concentrations of 2.0 mg ml1. This finding
excludes an independent contribution of the largest part of the
-subunit to the heat capacity profile of
(H+,K+)-ATPase obtained with intact gastric
vesicles. The contribution of the transmembrane stalk to the heat
capacity function is also excluded, since the structure of this region
is preserved in the protease treatment (28-30). Therefore, the thermal
transitions observed in intact gastric vesicles should be assigned to
the melting of two cooperative domains located in the cytoplasmic protruding segments of the
-subunit. This assignment is also supported by the finding of two different transitions in the
sarcoplasmic reticulum (Ca2++Mg2+)-ATPase (24),
homologous protein that does not contain
-subunit.
As demonstrated for the
(Na+,K+)-ATPase and other P-ATPases, the
catalytic cycle of the (H+,K+)-ATPase is
characterized by the presence of two families of conformations, E1 and E2, both in
phosphorylated and dephosphorylated forms, with a conformational
transition linked to cation transport. The E1
conformation presents a cytoplasmic-facing H+ site and
exhibits a high affinity for ATP. After phosphorylation of the
-subunit by ATP, an E2 conformation appears,
characterized by its high affinity for K+ ions and the low
affinity for ATP and H+ (for a more complete description
see Ref. 31). Among the different ligands potentially affecting the
(H+,K+)-ATPase conformational equilibrium,
K+ is known to promote the E2
conformation. Fluorescence of fluorescein-labeled
-subunit (7) and
limited trypsin digestion (5) revealed qualitative differences in the
tertiary structure of the protein when bound by different ligands.
Secondary structure probed by Fourier transform infrared spectroscopy
(32) did not show significant modification. However, it must be noted
that some variation in the
-helical content had been observed before
by circular dichroism (23). Since very little is known about the nature
of these conformational changes, we have investigated here the effect
of the E1-E2
ligand-induced transitions on the structural organization of the
enzyme. In addition, the analysis of the effect of ligand binding on
(H+,K+)-ATPase thermal denaturation can provide
further insight into the functional role and, thus, the location of the
unfolding cooperative units.
Thermal denaturation of ligand-bound (H+,K+)-ATPase is irreversible under all conditions tested and exhibits the same scanning rate dependence as the unligated protein. It should be mentioned that instrumental artifacts, resulting in exothermic peaks at high temperatures, may be observed at fast scanning rates in irreversible denaturations (33).
Thermal Denaturation of E1 Enzyme FormsVanadate
binding drives the enzyme into the E1-van form.
DSC scans of (H+,K+)-ATPase in the presence of
increasing vanadate concentration are reported in Fig. 2,
and the thermodynamic parameters are listed in Table I. Vanadate
interaction produces a progressive increase in
Tm1 that results in merging of both
transitions above 0.14 mM concentration (Fig. 2).
Therefore, deconvolution analysis was performed assuming either a
random model for two independent elementary transitions (domains
independently undergo a transition between the folded and denatured
forms) or a sequential model for two sequential two-state transitions
(domain stability is dependent on whether other domains are folded or
unfolded) (13, 34, 35). At saturating vanadate concentrations,
deconvolution assuming the sequential model gives best fit of the
experimental data (Table I) revealing interdomain communication.
However, when Tm1 and
Tm2 are separated and the overlap is
minimal (vanadate concentrations below 1 mM) consideration of one non-two-state transition is required for best fit. Taking into
account the value of the Kd of vanadate (30 µM) (36), these deviations observed under non-saturating
conditions probably arise from the reequilibration of the binding
equilibrium coupled to protein denaturation, as the temperature is
increased (37). Reequilibration of the binding and the unfolding
processes with temperature causes a deviation of the first peak from
the ideal two-state transition, and such distortion is also manifested in the apparent thermodynamic parameters derived, under these conditions, for the second peak. The observed effects upon vanadate interaction with the (H+,K+)-ATPase (increment
of Tm1 and H1,
and invariability of Tm2 and
H2) is consistent with a direct interaction of this ligand with the cooperative domain involved in the lower temperature transition. Since vanadate is thought to bind at the phosphate site in the C3 loop of the
-subunit, near Asp-385 (23), it
is reasonable to assign the first transition to a cooperative domain
containing this region.
Binding of ATP in the presence of Ca2+ results in ATP
hydrolysis and the appearance of the phosphorylated
E1-P form of the enzyme, insensitive to
K+ ions (7). This form exists in equilibrium with the
E2-P conformation and can react with free ADP to
dephosphorylate the initial E1 state. Addition
of 2 mM Ca2+ modifies the thermal denaturation
pattern of (H+,K+)-ATPase (Fig. 3,
Table I). Again, the sequential model fits slightly better the
experimental curve. The first transition is upward shifted, and its
enthalpy change is increased up to 196 kcal mol1. The
second transition is downward shifted to 60.5 °C, with minor modifications in its enthalpy change. A more accurate analysis of
Ca2+ effect was prevented by the appearance of a drop in
the Cp function, immediately after the end of the second transition,
upon increasing either cation or protein concentrations. With these
limitations, the experimental results indicate a direct cation-enzyme
interaction that affects both transitions. ATP, in the presence of 2 mM Ca2+, further stabilizes the first
transition and counteracts the apparent Ca2+-induced
destabilization of the second transition (Fig. 3, Table I). As in the
previous cases, the experimental curves are better described in terms
of a sequential model rather than a random model. Changes produced by
Ca2+ and Ca2+-ATP on the first transition
resemble those observed with vanadate and, therefore, could reflect the
interaction of both ligands as cosubstrates with the catalytic domain
contained in the
-subunit C3 loop. On the other hand, the antagonist
action of both cosubstrates on the second transition could arise from a
decrease in the free Ca2+ concentration due to cation
chelation by ATP. A direct effect of ATP on the second transition
cannot be discarded either because the main ATP binding is located on
the second domain or because of the existence of multiple ATP binding
sites (38).
Inclusion of EDTA in the medium permits uncoupling of ATP binding from
enzyme phosphorylation and linked events. Addition of EDTA prevents ATP
hydrolysis, by cosubstrate chelation, protecting the enzyme from total
proteolysis (5). EDTA (1 mM) itself does not modify the
thermal denaturation profile of the enzyme (data not shown). Addition
of 1 mM ATP in the presence of 1 mM EDTA results in the appearance of an exotherm at low temperatures followed by the double peak endotherm characteristic of
(H+,K+)-ATPase thermal unfolding (Fig. 3, Table
I). The area of the exotherm, centered near 25 °C, is about 170
kcal mol
1, and its molecular basis is under
investigation. ATP binding modifies the first transition in a manner
similar to vanadate but to a lesser extent. The second transition is
slightly stabilized.
Therefore, E1 forms of
(H+,K+)-ATPase are characterized by the
organization of the -subunit cytoplasmic loops into at least two
cooperative unfolding domains. Interdomain communication is evidenced
by the analysis of their calorimetric transitions.
Binding
of K+ drives the enzyme into the
E2-K+ conformation. Fig.
4 and Table II show the effects of increasing
concentrations of KCl on the heat capacity function of the protein and
in the derived thermodynamic parameters. It should be noted that the observed effects are K+-specific since equal concentrations
of NaCl do not produce significant modifications (Fig. 4, Table II).
The effect of K+ on the first transition is saturable and
shifts Tm1 to 57.4 ± 0.2 °C
without modifications in the calorimetric enthalpy change,
H1 = 164 kcal mol
1. However,
its
HvH increases, probably due to
aggregation of the denatured form of this cooperative domain. The
second peak undergoes major changes.
H2
continuously decreases with increasing K+ down to a
negligible value. Tm2 remains nearly
constant. The van't Hoff enthalpy change increases to 170 kcal
mol
1, probably due to the aggregation of the denatured
form. The effect of 10 mM KCl is reversed upon addition of
CaCl2.
|
Addition of ATP in the presence of Mg2+ blocks the
E2-P form of the enzyme. As demonstrated above
for Ca2+, Mg2+ alone binds to the enzyme,
inducing significant protein conformational changes (5, 39). Fig.
5 shows the effect of 2 mM Mg2+ in
the absence and in the presence of ATP. It must be mentioned that, in
the presence of Mg2+, the experimental traces obtained at a
protein concentration of 1 mg ml1, in the absence or
presence of additional ligands (see below), exhibited a constant abrupt
drop in the post-transitional base line that is minimized at lower
protein concentration. Therefore, protein concentrations of 0.5 mg
ml
1 were employed in these cases. Mg2+
binding results in a notable destabilization of the second transition, whereas the first transition remains unaltered (Fig. 5 and Table II).
Addition of Mg2+-ATP progressively increases
Tm1 and
H1.
The second transition is further downward shifted and its enthalpy
change decreases without varying its van't Hoff enthalpy change.
In the presence of Mg2+ and vanadate, the conformational
equilibrium is shifted to the E2-van
conformation. The variation with temperature of the excess heat
capacity curve is characterized by a single peak centered at 60.7 °C
with a total enthalpy change of 176 kcal mol1 (Fig. 5,
Table II). The
HvH/
H ratio
increases to 1.7 due to the high increase of the van't Hoff enthalpy,
suggesting either intermolecular interactions between native catalytic
domains or their association upon thermal denaturation. Differentiation
between both possibilities is prevented by the appearance of an abrupt decay of the base line around 75 °C at high protein concentrations. Tetrameric arrangements of the native enzyme in the presence of Mg2+-vanadate have been reported (40).
Thermal denaturation of the
E2-van-K+ form, stabilized by (2 mM) Mg2+ + (1 mM) vanadate + (10 mM) K+, is depicted in Fig. 5 (trace
e). In this case the denaturation process is characterized by two
peaks with Tm values of 63.0 and 56.4 °C (Table
II). For the peak at 63 °C, the
HvH/
Hcal ratio is
1.9, suggesting again that the cooperative unit of the catalytic domain
could be a dimer, under the above conditions. The
E2-van-K+ form of the enzyme can be
described as an intermediate conformation in which hallmarks of
E2 (increased cooperativity of the catalytic domain melting) and of E1 (two calorimetric
transitions) are seen.
In conclusion, the analysis of the thermal denaturation of the enzyme in E2 forms reveals major changes in both transitions, whereas ligands maintaining the ATPase in the E1 conformation mainly affect the first transition.
The excess heat capacity curves of gastric vesicles reveal the
presence of at least two different peaks. The abundance of the
(H+,K+)-ATPase in these membranes (more than
90% of protein content) and the sensitivity of these transitions to
enzyme-specific ligands indicate that both endotherms arise from
denaturation of the (H+,K+)-ATPase molecule.
The total value of 281 kcal mol1 measured for the
enthalpy change, i.e. 1.9 cal/g (Mr
147,000), is much lower than the enthalpy change associated with the
unfolding of water-soluble proteins (the average value 7.8-7 cal/g has
been compiled in 41 from 42). It is even lower than the values of 2.9, 2.7, and 2.4 cal/g reported for the cytochrome oxidase from
Paracoccus denitrificans, beef heart, and yeast,
respectively (41, 43, 44). In the case of the oxidase, a protein
fraction corresponding to the membrane-embedded segments (45% of the
amino acid residues) was not denatured (41). The present results
suggest that an even larger proportion of the
(H+,K+)-ATPase could remain structured below
80 °C. In fact, Fourier transform infrared spectroscopy data
obtained in our laboratory (28) indicate that up to 55% of the protein
could be located in the membrane. While thermal denaturation of
membrane proteins affects primarily the extramembrane regions (15),
resulting in a reduced specific overall enthalpy change, the
contribution of a partial unfolding of the regions responsible for the
observed transitions cannot be discarded.
In the absence of ligands, (H+,K+)-ATPase
thermal denaturation reveals the presence of two independent elementary
transitions at 53.9 and 61.8 °C with enthalpy changes of 165 and 116 kcal mol1, respectively. The origin of both transitions
in terms of structural domains is difficult to assess a
priori considering the complexity of the system that involves two
different polypeptide chains and a variety of topological regions. The
use of proteases to specifically cleave the protein fraction protruding
from the membrane is legitimate only if the initial system is
homogeneously oriented with respect to the membrane and if the
structure of the so-isolated membrane region is maintained. The fact
that rupture of the vesicles by hypotonic conditions reveals about the
same ATPase activity as collapsing the electrochemical gradient by
nigericin indicates that the ATP binding site of the ATPase is highly
oriented toward the outside of the vesicles. Other works on
membrane-embedded proteins support the idea that interactions between
membrane-embedded helices stabilize their own assembly and that
external connecting loops are not essential. Retinal binding and loop
connections in bacteriorhodopsin were found to make a small
contribution to stability by a variety of techniques (45). Rhodopsin
can be proteolytically cleaved into three fragments which remain
associated (46). Functional Escherichia coli lactose
permease is obtained from genes which yield complementary fragments of
the protein (47, 48). Dimerization of glycophorin A only depends on the presence of some specific amino acids (49) in its single
-helix transmembrane sequence (50). The fact that no thermal transition is
observed in the proteolytically isolated membrane region of the protein
is consistent with a high stability of the tertiary and secondary
structures embedded in the membrane. This is due in part to the absence
of competitor hydrogen bond donor or acceptor groups as already
discussed elsewhere (41, 51-55). The total disappearance of
calorimetric signal in (H+,K+)-ATPase after
proteinase K treatment could then be attributed to the loss of those
segments responsible for the excess heat capacity function observed in
intact vesicles. Therefore, the two transitions can be assigned to two
different cooperative domains located in the cytoplasmic loops of the
-subunit.
The experimental data also bring evidence for two major families of
conformations based on their thermal denaturation mechanism. Under
conditions in which the E1 form of the enzyme is
promoted, as in the case of unligated E1,
E1-van and E1-P, the
thermal denaturation process can be described in terms of two two-state
transitions. The first transition is sensitive to vanadate and ATP and
is therefore suggested to contain a protein domain that encompasses the
ATP and vanadate binding sites on the -subunit (Table I). The strong stabilization of the first transition upon Mg2+-ATP and
Mg2+-vanadate interaction could reflect the substrate or
analog binding to this domain. The second transition is hardly
affected. Yet some degree of interdomain communication can be
concluded. As described below, there are also clues that ATP in the
presence of Mg2+ could have a direct effect on the second
domain.
Addition of those ligands promoting the E2
conformational family such as K+, Mg2+,
Mg2+-ATP, Mg2+-vanadate, and
Mg2+-vanadate-K+ dramatically modifies the
previous picture. Importantly, the absence of effect of Na+
on the thermogram underlines the specificity for K+ and
rules out any unspecific ionic strength effect on the protein conformation. Both transitions are affected by K+ binding.
The second transition displays a progressive disappearance upon
K+ addition until it completely vanishes from the scan at
100 mM KCl. It must be mentioned here that KCl was shown to
protect the (H+,K+)-ATPase against trypsin
digestion, whereas the enzyme is completely degraded into small
fragments in its absence (5). The K+ binding site has been
suggested to be on the H6 segment (31), a part of the H5-H6 hairpin,
accessible to trypsin cleavage in the absence of ligands but not in the
presence of K+ (30). This region can be found either
membrane-embedded, when occluding cations are present, or in the
aqueous phase in their absence in the
(Na+,K+)-ATPase (56). Therefore, it is a
possibility, although not demonstrated by the present experiments, that
the second cooperative domain contains the H5-H6 hairpin. Whether or
not the disappearance of the second calorimetric transition is due to
membrane insertion of this domain remains to be demonstrated. Indeed,
base-line problems in this region might distort the DSC scan profile.
The saturable effect of K+ on the first transition
parameters (Table I) might be the result of either a domain-domain
interaction (if K+ binds the other domain) or an enzyme
conformational change on going from E1 to
E2. Alternatively, it could be the consequence of a direct interaction of K+ with this domain. In good
agreement with fluorescence emission of the fluorescein-labeled enzyme
(7) is the partial reversal of the E2 to
E1 conformation observed here upon
Ca2+ addition which results in the recovery of the
calorimetric signal corresponding to thermal denaturation of the second
transition. DSC data thereby confirm the direct competition of
Ca2+ for Rb+ at the entrance of the cation
occlusion site and its failure to replace Rb+ in inducing a
conformation with a 19-kDa peptide protected against trypsin hydrolysis
in the (Na+,K+)-ATPase (57). The reversibility
of the first transition to the E1 state upon
Ca2+ addition is not completely achieved. On the other
hand, the thermal signal corresponding to the second transitions is
95% recovered (H2 = 105 kcal
mol
1) (Table II).
Induction of the E2 conformation by
Mg2+, Mg2+-ATP, and Mg2+-vanadate
also results in a destabilization of the second transition observed in
free (H+,K+)-ATPase, which is accompanied by a
progressive disappearance of the thermal transition and characterized
by a reduction in the calorimetric enthalpy
(H2<
HvH2).
Mg2+-ATP and Mg2+-vanadate produce essentially
the same effect as K+ on the second domain but also modify
the first domain, as expected from the observation that free ATP or
vanadate mainly affect the first transition (Table I). The specific
effect of ATP on the second transition in the presence of
Mg2+ (but not in its absence) raises the possibility that
ATP could interact with the second domain in the presence of
Mg2+. Alternatively, Mg2+ could interact with
bound ATP (58) and induce a protein structure rearrangement. In the
E2-van-K+ the first domain is
further stabilized probably as a result of the increased affinity of
the enzyme for vanadate in the presence of K+ (36).
In conclusion, the study reported herein offers new points of view on
the conformational multiplicity of P-ATPases, establishing the
differences among forms in terms of the structural organization into
cooperative domains of the -subunit.