From the
Scanning microcalorimetry and spectrofluorimetry were applied to
a study of the thermal stability and interaction of the modules within
natural human protein C (PC) and recombinant protein C (rPC), a
potential therapeutic anticoagulant expressed in transgenic pigs. Upon
heating in the presence of 2 mM EDTA, pH 8.5, each protein
exhibited a similar heat absorption peak with a
T
Protein C (PC)
Protein C is a glycoprotein of
M
Recombinant Gla-domainless
protein C (dG-rPC) and recombinant Gla fragment were prepared from rPC
in the same manner as their natural counterparts. Sequence analysis of
dG-rPC revealed three major sequences in proportion 1:0.6:0.4, starting
at Ser
The
Gla-(EGF)
The major goal of this study was to characterize the
stability of and interactions between individual modules in human
protein C. Another goal was to determine whether proper folding,
stability, and interaction between domains are preserved in the
recombinant protein. This was accomplished through a detailed study of
the denaturation process, first with the recombinant protein, to
determine optimum experimental conditions for physical-chemical
characterization, and then with both recombinant and natural proteins
as well as their proteolytic fragments.
We found that the two EGF
modules in both recombinant and natural PC are folded independently and
interact with each other. They are also extremely thermostable,
although the EGF domains of rPC denatured at lower temperature than
those of PC. The extreme thermal stability of the EGF domains does not
necessarily mean that they are unusually stable toward other forms of
stress under physiological conditions. Previous analysis of the
homologous EGF domains of factor IX
(25) as well as small type I
finger domains of fibronectin
(36) revealed that in spite of
their high thermostability, their free energy of stabilization
(
Previous scanning calorimetry experiments
with factor IX and some other proteases revealed the presence in the SP
module of two independently folded domains, which merge together into
one cooperative unit at neutral pH but melt independently at low
pH
(23, 24, 25, 34) . Numerous
crystallographic data also indicate that different serine proteases
comprise two interacting lobes (domains) separated by a cleft in which
the catalytic triad resides. It seems that these enzymes utilize a
two-domain structure to bring catalytic residues together for optimal
function. The interaction between two SP domains may play an important
role in the maintenance of the proper geometry of the catalytic triad
and may be responsible for the regulation of enzyme
activity
(34, 39) . The results presented here clearly
indicate that the SP module of both recombinant and natural protein C
also consists of two domains, which, in contrast to other studied
cases, melt independently even at neutral pH. At the same time, when
PPACK was bound covalently to catalytic His and Ser which are situated
on different SP domains, the two domains melted together in a sharp
highly cooperative transition. Thus, in the absence of the inhibitor,
the interaction between the two SP domains of protein C is too weak to
influence their melting behavior at the temperature of denaturation.
It is well established that the conformation of the Gla domain of
protein C is altered when it binds calcium
ions
(27, 40) . Previous results obtained with
prothrombin fragment 1
(35) and Gla-containing fragments of
human factor IX
(25) showed that their Gla domains formed a
compact structure only in the presence of Ca
In addition to the Gla module at least one
of the EGF modules of protein C possesses a
Ca
In summary, our
results indicate that the Gla and two EGF modules in protein C each
form an independently folded domain while the serine protease module
consists of two domains. The two EGF domains are extremely thermostable
and interact with each other. The Gla module is folded into a compact
domain only in the presence of Ca
of
62 °C corresponding to
the melting of the serine protease (SP) module. Deconvolution of this
peak indicated that the SP module consists of two domains that unfold
independently. At pH below 3.8, a second peak appeared at extremely
high temperature corresponding to the unfolding of the two interacting
epidermal growth factor-like (EGF) domains. This second peak occurred
at a temperature about 20 °C lower in rPC than in PC indicating
that the EGF domains in the recombinant protein are less stable. The
isolated 6-kDa
-carboxyglutamic acid-rich (Gla) fragment as well
as a 25-kDa Gla-(EGF)
fragment both exhibited a sigmoidal
fluorescence-detected denaturation transition in the same temperature
region as the SP domains, but only in the presence of
Ca
. In 2 mM Ca
, the first
heat absorption peak in both intact proteins became biphasic,
indicating Ca
-induced structural changes. By
contrast, Ca
had very little effect on the melting of
Gla-domainless protein C. This indicates that not Ca
itself but the Ca
-loaded Gla domain is
responsible for conformational changes in the SP domain of the parent
protein. Detailed analysis of the shape of the endotherms obtained in
Ca
and EDTA suggests that Ca
induces compact structure in the Gla domain which appears to
interact strongly with the SP domain(s) of protein C.
(
)
is a major component of
the anticoagulant pathway. It is converted to activated protein C (APC)
at the endothelial cell surface by the thrombin-thrombomodulin complex.
APC regulates the coagulation cascade by proteolytic inactivation of
procoagulant cofactors Va and VIIIa. This terminates activation of
factor X and prothrombin and thus limits thrombin
production
(1) . Congenital or acquired deficiency of PC is
associated with thrombotic disease, which has been successfully treated
by replacement therapy with purified PC
concentrate
(2, 3, 4, 5) . Biologically
active recombinant protein C (rPC) has been expressed in different cell
lines
(6, 7, 8) and in the milk of transgenic
pigs
(9, 10) . High level production of the recombinant
protein in the latter compared to that in cell cultures makes it
feasible to consider its therapeutic use. Comparison of this protein
with the natural one revealed some differences
(10, 11) including lower anticoagulant activity, which may be
related to the number and complexity of post-translational
modifications
(10) and/or altered conformation. Further analysis
is required to better understand the structure-function relationships
of this protein and to critically assess the differences between the
natural and recombinant forms.
62,000 containing 25% carbohydrate
(12) .
It is synthesized in the liver as a single polypeptide that undergoes
extensive processing including removal of prepro- and propeptides,
vitamin K-dependent
-carboxylation of nine glutamic acid residues
in the NH
-terminal region,
-hydroxylation of
Asp
, glycosylation of four Asn residues, and
endoproteolytic removal of the Lys
-Arg
dipeptide
(13) . Mature PC is a two-chain disulfide-linked
heterodimer consisting of a 155 amino acid 21-kDa light chain and a 262
amino acid 41-kDa heavy chain. PC is a typical mosaic protein
consisting of four modules whose homologous copies are found in many
different proteins. Among these modules, the NH
-terminal
-carboxyglutamate-rich (Gla) module and the following two
epidermal growth factor-like (EGF) modules comprise the light chain
while the COOH-terminal serine protease (SP) module comprises the heavy
chain containing the catalytic triad residues. Although the SP module
of PC is primarily responsible for proteolytic inactivation of factors
Va and VIIIa, the Gla and/or EGF modules are required for
Ca
-dependent protein-protein and protein-membrane
interactions that increase the efficiency of PC activation as well as
the effectiveness of APC as an anticoagulant (14-20). In
addition, the Gla module of PC/APC is required for its
Ca
-dependent binding to an endothelial cell
receptor
(21, 22) . Based on previous studies with other
modular proteins
(23, 24, 25) , one can expect
that protein C modules are folded into autonomous structural and
functional domains. However, the expression of some functions may
require the presence of neighboring domains. For example, it was shown
that a fragment of protein C containing the Gla and EGF modules was
able to inhibit the anticoagulant effect of APC in a clotting assay or
inhibit PC activation by the thrombin-thrombomodulin complex whereas
the individual modules could not
(17, 26) . It was
suggested that the Gla and EGF domains in protein C
(26, 27) and related proteins
(25, 28, 29) interact with each other. This interaction may play an
important role in the expression of their functions. This paper
addresses the issue of intramolecular domain-domain interactions in
protein C through detailed studies of the thermal stability and folding
properties of its modules in both the natural and recombinant proteins.
Proteins
Natural protein C (PC) was prepared
from human cryoprecipitate-poor plasma by ion exchange and
immunoaffinity chromatography
(30) . Recombinant protein C (rPC)
was expressed in transgenic pig milk
(9) and purified to
homogeneity by the procedure described earlier
(11) . rPC was 95%
pure according to SDS-PAGE and sequence analysis and had about
70-75% of both amidolytic and anticoagulant activity of the
natural protein. Detailed biochemical characteristics of both natural
and recombinant proteins used in this study have been
presented
(11) . Activated protein C was prepared by incubation
of PC with human -thrombin, followed by its removal by ion
exchange chromatography
(30) . Alternatively, PC was activated by
protein C activator
(31) . PPACK-inhibited activated protein C
(PPACK-APC) was prepared by incubating APC at 1 mg/ml with 500
µM PPACK for 60 min at room temperature.
Proteolytic Fragments
6-kDa and 56-kDa fragments
were prepared from a 10-min chymotrypsin digest of PC by ion exchange
chromatography on a Q-Sepharose column according to the procedure
described in Ref. 14. NH-terminal sequence analysis of the
56-kDa fragment, performed with a Hewlett-Packard G1000S sequencer,
revealed two major sequences starting at Ser
and
Asp
, indicating that it corresponds to Gla-domainless
protein C (dG-PC) in which the 1-41 NH
-terminal
region of the light chain was entirely removed while disulfide-linked
heavy chain starting at Asp
was preserved. In addition,
two minor sequences (total not exceeding 15%) starting at Gly
and Val
were detected, indicating the presence of
cleavages in the heavy chain. The 6-kDa fragment exhibited only one
sequence starting at Ala
. Taking into account the
determined NH
terminus of the light chain of dG-PC, this
fragment represented the Gla module consisting of
Ala
-Trp
.
, Arg
, and Asp
. The
first sequence indicated that the Gla module was entirely removed. The
two other sequences reflected the presence of two different forms of
the heavy chain of rPC occurring due to alternative cleavage of the
one-chain protein upon processing.(
)
The amount
of other sequences in each cycle did not exceed 5%, indicating that in
contrast to dG-PC derived from the natural protein, dG-rPC contained no
detectable amount of cleavages in the heavy chain. The 6-kDa Gla
fragment from rPC was sequenced for 30 cycles. Only one sequence
corresponding to the NH
terminus of the light chain
starting at Ala
was revealed; all glutamic acids were
-carboxylated as was reported previously
(11) .
25-kDa fragment was prepared by proteinase K
digestion of PC followed by size exclusion and immunoaffinity
chromatography by a method described elsewhere.
(
)
The 25-kDa fragment comigrated with the light chain of PC
upon SDS-PAGE analysis and was of only slightly smaller molecular mass
upon reduction. Sequence analysis revealed three major sequences in
proportion 1:0.5:0.5, starting at Ala
, Thr
,
and Ser
, respectively, indicating that it consists of the
light chain and disulfide-linked small remnants of the heavy chain.
Activity Measurements
To determine the effect of
heating on catalytic activity, 100 µg/ml APC in 50 mM
Tris, 20 mM NaCl, pH 7.5, with 2 mM CaCl
or EDTA, was equilibrated to 25 °C. Aliquots were removed and
assayed for amidolytic activity on the chromogenic substrate S-2366 by
measuring the rate of change of absorbance at 410 nm with a kinetic
microtiter plate reader. The temperature was then ramped up and
aliquots removed at various temperatures and assayed immediately as
described above and expressed as a percentage of base-line activity.
The activity of a control sample of APC maintained at 25 °C for the
duration of the experiment was unchanged.
Fluorescence Study
Fluorescence measurements of
thermal unfolding were performed by monitoring the intrinsic
fluorescence intensity at 350 nm or the ratio of the intensity at 350
nm to that at 320 nm with excitation at 280 nm in an SLM 8000-C
fluorometer. Right-angle light scattering at 350 nm was measured
simultaneously in the same fluorometer. Protein concentrations were
0.04 mg/ml.
Calorimetric Study
Differential scanning
calorimetry (DSC) measurements were made with a DASM-4M or with an
updated DASM-1M calorimeter at a scan rate of 1°/min. The latter
instrument allows measurements up to 115 °C, while the former
extends to 130 °C (32). Protein concentrations varied from 0.8 to
2.5 mg/ml. Melting temperatures (T ),
calorimetric (
H
) and van't Hoff
(
H
) enthalpies were determined from the DSC
curves using software provided by Dr. Filimonov (Institute of Protein
Research, Pouschino, Russia). Deconvolution analysis was performed
according to Refs. 32 and 33, using the same software. The software
allows one to perform the analysis by either independent or dependent
schemes. The former is based on the assumption that each domain unfolds
independently, regardless of the state of the neighboring domains. The
latter assumes an ordered process in which constituent domains unfold
sequentially, implying the occurrence of interactions between domains
such that the unfolding of any given domain depends on the status of
its neighbor. The relative error of the experimental enthalpy values is
estimated at ±5% and that of the T
values at ±0.2 °C. The corresponding errors in the
parameters of individual transitions obtained by deconvolution of
complex endotherms are estimated at ±10% and ±1.0 °C.
Fluorescence-detected Thermal Denaturation of PC and
rPC
Fluorescence spectroscopy, which requires a relatively small
amount of protein per experiment, was used to monitor the denaturation
process of both recombinant and natural protein C in different
conditions to find those in which the proteins unfold reversibly and/or
without significant aggregation. At pH > 9.5 denaturation of both
proteins was irreversible and, in some cases, was accompanied by strong
aggregation. The changes of the fluorescence signal at 3.0 < pH <
5.0 upon heating were small and difficult to interpret. The most useful
results were obtained at pH 8.5, where both proteins exhibited a
sigmoidal transition that was reversible in the presence of EDTA, but
irreversible in Ca (Fig. 1). The
irreversibility in Ca
may be due in part to
aggregation of the denatured molecules since simultaneous light
scattering measurements indicated an increase in turbidity following
denaturation in the presence of Ca
, but not EDTA.
This effect was more pronounced with the natural protein (Fig. 1,
dottedcurves). Addition of 2 M urea
partially restored the reversibility in Ca
(Fig. 1, curve4). This screening allowed
us to select the appropriate conditions for studying the denaturation
of the proteins with DSC, which requires much higher protein
concentrations but gives more structural information.
Figure 1:
Fluorescence-detected thermal
denaturation of recombinant and natural protein C in different
conditions. Fluorescence-detected melting curves were obtained upon
heating (solidlines) and cooling (dashedlines) of rPC (curves1, 3,
and 4) or PC (curve2) in 100 mM
glycine buffer, pH 8.5, containing 2 mM EDTA (curve1), 2 mM Ca (curves2 and 3), or 150 mM NaCl, 2 mM
Ca
, and 2 M urea. Curve4 was arbitrarily shifted along the vertical axis to improve
visibility. Twodottedcurves marked at the
right as PC-turb and rPC-turb represent
turbidity changes upon heating in 100 mM glycine buffer, pH
8.5, with 2 mM Ca
, and were registered
simultaneously with curves2 and 3 by light
scattering.
Calorimetric Study
When heated in the calorimeter
at pH 8.5, rPC exhibited a heat absorption peak in the same temperature
range where the reversible sigmoidal transition was observed by
fluorescence (Fig. 2). Following this endothermic unfolding
transition, the heat capacity decreased above 95 °C, suggesting the
beginning of some exothermic process, probably aggregation. At pH 3.8
and 3.4 the peak was still present, although substantially
destabilized, beginning near room temperature. Under these conditions,
the transition was irreversible since the peaks were not reproduced
when protein was heated first up to 70 °C (dottedcurves) and then cooled and heated again (solidcurves). At pH 3.0, this low temperature transition was
not observed at all when the sample was exposed to room temperature
prior to loading into the calorimeter (lowercurve).
At the same time, the high temperature exothermic process seemed to be
abolished at low pH, allowing an additional high temperature heat
absorption peak to be clearly observed. At pH 3.8, an incomplete
endothermic transition with T of 112
°C was revealed. This transition became more obvious at lower pH
where it was shifted to lower temperature, allowing its completion to
be registered by the instrument. These results indicate that rPC
contains two structures with vastly different stabilities.
Figure 2:
DSC curves of recombinant protein C in
different conditions. Experiments were performed in 100 mM
glycine buffer with 2 mM EDTA (pH 8.5) or without EDTA (other
pH) on the DASM-4 calorimeter. The samples at pH 3.4 and 3.8 were kept
at 4 °C during preparation and loading; they were first heated up
to 70 °C (first run, dottedcurves), then cooled
and heated again up to 130 °C (second run, solidcurves); the sample at pH 3.0 was kept at room
temperature prior to loading and heating. Dashedlines indicate the manner in which the excess heat capacitieswere determined. All but the lower curve were arbitrarily shifted
along the vertical axis to improve
visibility.
Characterization of the Low Temperature Transition in
Different Species of PC
The uppertwocurves in Fig. 3A compare the melting of
recombinant and natural PC at pH 8.5 in the presence of EDTA. The heat
absorption peaks are very similar, suggesting that the structures which
undergo denaturation in this temperature range have similar stability
and are presumably folded in the same manner in both proteins.
Gla-domainless natural and recombinant protein C also exhibited
endotherms, which were similar to each other and to those of the
full-length proteins. This similarity excludes the possibility that the
Gla module is involved in this transition. It also indicates that the
heterogeneity revealed by sequence analysis of dG-PC and dG-rPC (see
``Materials and Methods'') does not influence substantially
the stability of these proteins. Activation of PC increased slightly
the T and changed the shape of the
endotherm suggesting involvement of the SP module in the denaturation
process. The enthalpies of this transition for all species
() are close to those reported for the SP module in other
serine proteases (24, 25), supporting this suggestion. Since
chloromethylketone inhibitors, which bind covalently to the active site
Ser and His residues, are known to affect the cooperativity and
stability of the SP module in different serine proteases
(34) ,
we treated APC with PPACK. The treated protein exhibited a heat
absorption peak whose shape differed dramatically from that of APC,
further implicating SP in this transition. Finally, measurements of the
amidolytic activity of APC at different temperatures revealed
inactivation of the enzyme in the same temperature region where the
first transition occurred (Fig. 3B), clearly indicating
that this transition is connected with the melting of the serine
protease module.
Figure 3:
DSC curves of different species of protein
C obtained in the presence of EDTA. Panel A represents melting
curves of recombinant and natural protein C (rPC and PC, respectively),
Gla-domainless recombinant and natural protein C (dG-rPC and dG-PC,
respectively), activated protein C (APC), and PPACK-inhibited activated
protein C (PPACK-APC). All experiments were performed in 100
mM glycine buffer, pH 8.5, with 2 mM EDTA, on the
DASM-1M calorimeter. Dashedlines indicate the manner
in which the excess heat capacities were determined. All but APC curve
were arbitrarily shifted along the vertical axis to improve visibility.
Panel B represents heat-induced changes in amidolytic activity
of APC in the presence of Ca (filledrectangles) or EDTA (openrectangles).
Thermodynamic parameters of the heat absorption
peaks of different species of protein C are summarized in .
Both recombinant and natural protein C as well as Gla-domainless and
activated protein C exhibited similar calorimetric enthalpies
(H
) equal to 109-120 kcal/mol. The
van't Hoff enthalpies (
H
), calculated
from the shape of the peaks, were more than twice as low, suggesting
the melting of at least two independently folded domains. This
suggestion was reinforced by the fact that the peak in all cases was
well described by two two-state transitions using the independent
scheme of deconvolution (Fig. 4, A-C, and
). At the same time, the sharp heat absorption peak for
PPACK-APC had a
H
/
H
ratio of 1.1 and was well described by a single two-state
transition
(
)
(Fig. 4D), suggesting
the merging of two domains into one cooperative unit. These results
indicate that the SP module of protein C in the presence of EDTA
consists of two rather independent domains, which seem to interact
strongly with each other when the PPACK-inhibitor is covalently bound.
Figure 4:
Deconvolution of the excess heat capacity
functions of different species of protein C. The peaks represent excess
heat capacities of the corresponding curves presented in Fig. 3. The
solidunevencurvesrepresent
experimental endotherms, while the dottedsmoothcurves represent the component two-state transitions
obtained by deconvolution; the sums of the transitions that are close
to the experimental curves are also presented by smoothdottedcurves. Thermodynamic parameters obtained
by this analysis are summarized in Table II.
Characterization of the High Temperature Transition in
rPC and PC
Since the SP module melts in the low temperature
transition and the Gla module is expected to form compact structure
only in the presence of Ca(25, 35) ,
the high temperature peak observed at acidic pH must be connected with
the melting of the remaining structures, namely one or both EGF
modules. Thermodynamic analysis of the high temperature peaks of rPC
presented in Fig. 2and 5A revealed that in all cases
the
H
to
H
ratio
was higher than unity but lower than 2 (), suggesting that
two interacting domains contribute to this peak. Furthermore, the peak
in all cases was described by two two-state transitions using only the
dependent scheme (an example is presented in Fig. 5A,
and results of the deconvolution are in ). Thus the
analysis indicates that in rPC, both EGF modules are folded
independently and seem to interact with each other.
Figure 5:
Deconvolution analysis of the excess
heat capacity function of the high temperature peaks of protein C. The
molar heat capacity curves for the recombinant (panel A) and
natural (panel B) protein were obtained in 100 mM
glycine, pH 3.2, on the DASM-4 calorimeter. The samples were
preincubated at 40 °C to eliminate the low temperature transition.
The original melting curves are presented by the solidlines with dashedlines indicating the
manner in which the excess heat capacity curves, (also solidlines) were determined. The results of the deconvolution
of the excess heat capacity curves are represented by dotted
lines. The original curves were arbitrarily shifted along the
vertical axis to improve visibility.
The natural
protein, when heated at acidic pH, exhibited a downward turn in the
heat capacity curve, which distorted the high temperature heat
absorption peak (Fig. 5B). This turn is connected, most
probably, with aggregation of the denatured protein since the turbidity
measurements revealed an increased tendency for aggregation upon
denaturation of natural PC versus rPC (Fig. 1). Results
obtained at pH 3.2 are shown in Fig. 5B, where the onset
of the heat-absorption peak can be seen to occur approximately 20
°C higher than for the recombinant protein. Similar results were
obtained at pH 3.0 (Tables I and II). Meanwhile, when correction for
aggregation was made in the arbitrary manner presented by the
brokenline in Fig. 5B, a reasonable
heat absorption peak could be obtained (lowercurve).
It was described by two two-state transitions using the dependent
scheme. Although this deconvolution cannot be regarded as unambiguous,
the conclusion that the high temperature transition in PC occurs about
20 °C higher than in rPC is obvious even from the raw data.
Denaturation of the Gla Module
To clarify the
folding status of the remaining Gla module, we prepared 6-kDa Gla and
25-kDa Gla-(EGF) fragments from PC and 6-kDa Gla fragment
from rPC in amounts sufficient for spectral study. When heated in the
fluorometer while registering the change in fluorescence intensity, the
Ca
-loaded Gla-fragment from PC exhibited a downward
sigmoidal transition with a T
at about 55
°C (Fig. 6, dashedcurve1). The
Gla fragment from rPC exhibited similar behavior (not shown). The
change in fluorescence intensity seems to be connected with aggregation
of the denatured fragment, which starts at about 45 °C as revealed
by simultaneous measurements of turbidity (solidcurve1). The Gla-(EGF)
fragment exhibited
a transition in the same temperature region according to the light
scattering data (solidcurve2), but this
transition was not clearly expressed by fluorescence (dashedcurve2), perhaps because of additional Trpresidues in the EGF region. No transitions were observed in any of
the fragments in the presence of EDTA. Thus the data indicate that the
Gla module forms a compact structure only in the presence of
Ca
and that this structure melts in the same
temperature region as the SP module.
Figure 6:
Fluorescence and turbidity changes upon
heating of the 6-kDa Gla fragment (curves 1) and 25-kDa
Gla-(EGF) fragment (curves 2) of PC. Dashedcurves represent changes in relative fluorescence
(F/F), while solidcurves represent
changes in turbidity, which were monitored simultaneously by light
scattering. All experiments were performed in 50 mM glycine
buffer, pH 8.5, with 0.15 M NaCl and 2 mM
Ca
.
Denaturation of PC and rPC in the Presence of
Ca
rPC in 2 mM Ca exhibited a biphasic low temperature peak whose shape differed
dramatically from that in EDTA (compare Fig. 3A,
uppercurves, and 7A). Essentially the same
curve was observed in 1 mM Ca
(Fig. 7B), indicating that
Ca
-induced changes are completed at this ion
concentration. The natural protein exhibited a similar melting curve
(Fig. 7A, dotted curve), except that its first
heat absorption peak was shifted to higher temperature by about 2
°C. The determination of the excess heat capacity for these curves
is complicated by the broadness of the transitions and the downward
changes in the heat capacity function (C
) above 95
°C. The curve obtained upon second heating (Fig. 7B,
curve 2) reflects C
of the denatured
state since denaturation of both PC and rPC in the presence of
Ca
was found to be irreversible (see Fig. 1).
It coincides perfectly after 85 °C with the C
function obtained upon first heating (Fig. 7B,
curve1), allowing the end of the transition and the
excess heat capacity function to be determined with greater confidence
(see dashedlines). The total enthalpies of
denaturation of both PC and rPC are presented in . The
results indicate that in the presence of Ca
, the
total enthalpy of the process increased by about 40 kcal/mol in
comparison with that in the presence of EDTA. This strongly suggests
the appearance of an additional Ca
-induced compact
structure, most probably the Gla module, which was shown above to melt
in this temperature region only in the presence of
Ca
. This concept was essentially proven by
measurements with both natural and recombinant Gla-domainless protein
C, which exhibited curves that, even with Ca
, are
similar to that of the full-length protein in EDTA (Fig. 7, C and D, and ).
Figure 7:
DSC curves of different species of protein
C obtained in the presence of Ca. All experiments
were performed in 100 mM glycine buffer, pH 8.5, on the
DASM-1M calorimeter. Two uppercurves (A)
represent superimposed melting curves of rPC (solidline) and PC (dottedline) in 2
mM Ca
. CurvesB represent
melting of rPC in 1 mM Ca
, first
(1) and second (2) heating. CurvesC represent superimposed melting curves of Gla-domainless PC
(solidline) and Gla-domainless rPC (dotted line) in
1 mM Ca
. CurvesD represent Gla-domainless rPC in 2 mM
Ca
, first (1) and second (2)
heating. Brokenlines in all cases indicate the
manner in which the excess heat capacities were determined (see text).
All curves were arbitrarily shifted along the vertical axis to improve
visibility.
Deconvolution results
obtained with rPC in the presence of EDTA and Ca are
presented in Fig. 8(A and C, respectively). In
the former case, two transitions of similar enthalpy were obtained,
corresponding to the melting of two domains in the SP module. Addition
of Ca
would be expected to introduce a third
transition if the Gla domain melted independently of the other two.
However, only two were obtained when the endotherm in Ca
was deconvoluted by the independent scheme. One was shifted to
higher temperature relative to EDTA, with little change in enthalpy.
The other was shifted downward with substantial gain in enthalpy. What
are the possible interpretations? First of all, since EGF domains are
extremely thermostable even at acidic pH, the thermogram must reflect
melting of compact structure formed only by the remaining Gla and SP
modules. One possibility is that the second smaller transition
corresponds to the Gla module and the first larger one to the SP module
whose two domains are somehow destabilized, one by
5 °C and
the other by
10 °C, and merged into a highly cooperative
structure that melts in a single two-state transition. This seems
improbable since it would require the small Gla domain to have an
unusually high specific enthalpy. Furthermore, deconvolution of the
endotherm for the Gla-domainless protein in Ca
gave a
pattern (Fig. 8B) that was very similar to that of the
full-length protein in EDTA. An alternative explanation is that the
large transition represents one of the SP domains whose melting is
sufficiently intertwined with that of the Gla module that only a single
transition is resolved by the independent scheme. In fact, when the
dependent scheme was used (Fig. 8D), a third small
transition could be extracted consistent with the notion that Gla
interacts with at least one of the SP domains. Thus it is not the mere
presence of Ca
but the presence of a
Ca
-loaded Gla domain that is responsible for the
effects on the melting of the SP domains.
Figure 8:
Deconvolution of the excess heat capacity
functions of different species of protein C in the presence of
Ca. Panel A, excess heat capacity and
deconvolution results of rPC in 2 mM EDTA, presented for
comparison. Panel B, excess heat capacity and deconvolution
results of Gla-domainless rPC in 1 mM Ca
.
Panels C and D, excess heat capacity and
deconvolution results of rPC in 1 mM Ca
using independent and dependent schemes, respectively (see text).
The solidunevencurves in all panels
represent experimental endotherms, while the dottedsmoothcurves represent the component two-state transitions
obtained by deconvolution; the sum of the transitions that are close to
the experimental curves are also presented by smoothdottedcurves. Thermodynamic parameters obtained
by this analysis are summarized in Table II.
G) at physiological temperature is relatively low.
Although we did not determine
G for the EGF domains of
PC, one can expect that it is similar to that of factor IX considering
the similarity in their T
and melting
enthalpies. Since the
Gversus temperature
profile has a shallow nature for small
domains
(25, 36, 37) , even small changes in free
energy of stabilization may cause substantial changes in
T
. In this case, the difference in
T
for the EGF domains in natural and
recombinant protein C could simply reflect a slightly lower
G in the latter. This may be caused by altered folding or by
differences in post-translational modifications if they are important
for stability. Protein C undergoes numerous post-translational
modifications, including
-hydroxylation of Asp
in the
first EGF and glycosylation at Asn
in the second. Evidence
for altered glycosylation of the light chain of the recombinant protein
used in this study has already been reported
(11) . It is
relevant that a strong stabilizing effect of carbohydrate was found
recently for the eighth finger domain in the gelatin binding region of
fibronectin
(38) .
.
Likewise, with protein C, only in Ca
-containing
buffer did the 6- and 25-kDa fragments exhibit an unfolding transition.
This transition occurred in the same temperature range for both
fragments, suggesting that the Gla domain was not stabilized by the
neighboring EGF domain(s) as in factor IX
(25) . An interaction
between Gla and EGF domains in protein C was suggested earlier based on
fluorescence measurements or study with a monoclonal
antibody
(26, 27) . Our results indicate that if this
interaction exists, it is either non-stabilizing or too weak to be
detected by our approach.
-binding site of higher affinity (16, 41). The SP
module of PC was suggested to also contain a
Ca
-binding site analogous to that in
trypsin
(42) . Indeed, the present study showed that
Ca
has a profound effect on the melting of the SP
domains, stabilizing one and destabilizing another. However, this
effect was attributed not to the binding of Ca
directly to the SP domains, since the effect was not seen with
Gla-domainless protein C, but to the presence of a
Ca
-loaded Gla domain, which appeared to interact with
one or both SP domains to influence their melting behavior. This
implies a folding back of the molecule to allow direct contact between
the NH
-terminal Gla domain and COOH-terminal SP domain(s).
Such interaction can be expected to impose constraint on the
supertertiary structure of the molecule, as shown schematically in
Fig. 9
. A similar situation was found with tissue plasminogen
activator in which the NH
-terminal finger-like and/or EGF
modules interact with the COOH-terminal SP module rendering the
molecule more compact
(23) . There is also immunochemical data
suggesting that Ca
binding to the Gla module alters
the conformation of the SP module in both factor IXa
(43) and factor Xa (44). In addition, molecular modeling of the
serine protease domain of protein C revealed a hydrophobic surface,
which was proposed to be involved in an interaction with its EGF and/or
Gla domains
(42) .
Figure 9:
Schematic representation of
Ca-free (upper figure) and
Ca
-loaded (lower figure) protein C,
illustrating the effect of domain-domain interaction on its
supertertiary structure. The interactions between domains are denoted
by cross-hatches.
The effects of Ca on rPC
and Gla-domainless rPC were very similar to those of their natural
counterparts aside from a slight destabilization of the first peak of
rPC in Ca
(Fig. 7, curvesA).
This indicates that both proteins undergo similar
Ca
-induced conformational changes, i.e. that
the ability of the Gla and SP modules that melt in this temperature
region to bind Ca
and to interact with each other is
preserved in the recombinant protein. The repertoire of fragments was
insufficient for us to detect any interaction between the second EGF
and the SP module analogous to that which was clearly demonstrated for
factor X by crystallography
(45) and predicted for PC from
molecular modeling
(42) . At the same time, if this interaction
exists, one might expect the SP domain(s) to be stabilized by the
neighboring thermostable EGF domain(s). In this case, the small
destabilization of the first peak on the thermogram of rPC in
Ca
in comparison with that of natural PC
(Fig. 7, curvesA) could be a consequence of
the lower stability of its EGF domains.
and seems to
interact strongly with one of the distant serine protease domains. All
domains in recombinant protein C fold into compact structures similarly
to those in the natural protein and undergo similar
Ca
-induced conformational changes. At the same time
both EGF domains of rPC are substantially destabilized in comparison
with the EGF domains of the natural protein.
Table:
Thermodynamic parameters of melting of protein C
in different conditions
Table:
Deconvolution results of the heat absorption
peaks in protein C
-carboxyglutamic acid-containing
module (domain); PPACK, D-Phe-Pro-Arg chloromethyl ketone;
rPC, recombinant human protein C; SP, serine protease module.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.