©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thermal Stability and Domain-Domain Interactions in Natural and Recombinant Protein C (*)

Leonid V. Medved (§) , Carolyn L. Orthner , Henryk Lubon , Timothy K. Lee , William N. Drohan , Kenneth C. Ingham

From the (1) J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Protein C (PC)() 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.

Protein C is a glycoprotein of M 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.


MATERIALS AND METHODS

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.

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, 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) .

The Gla-(EGF) 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.


RESULTS

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.




DISCUSSION

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 (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) .

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. 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.

In addition to the Gla module at least one of the EGF modules of protein C possesses a Ca-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.

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 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



FOOTNOTES

*
This work was supported by a grant from the Mathers Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0731; Fax: 301-738-0794.

The abbreviations used are: PC, protein C; APC, activated protein C; DSC, differential scanning calorimetry; dG-PC, Gla-domainless protein C; dG-rPC, Gla-domainless recombinant protein C; EGF, epidermal growth factor-like module (domain); Gla, -carboxyglutamic acid-containing module (domain); PPACK, D-Phe-Pro-Arg chloromethyl ketone; rPC, recombinant human protein C; SP, serine protease module.

T. K. Lee, W. N. Drohan, and H. Lubon, submitted for publication.

C. Orthner, manuscript in preparation.

This peak was also possible to describe by two two-state transitions using the dependent scheme, but the fit was inferior.


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