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J. Biol. Chem., Vol. 277, Issue 32, 28987-28995, August 9, 2002

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Thermodynamic Linkage between the S1 Site, the Na⁺ Site, and the Ca²⁺ Site in the Protease Domain of Human Activated Protein C (APC)

SODIUM ION IN THE APC CRYSTAL STRUCTURE IS COORDINATED TO FOUR CARBONYL GROUPS FROM TWO SEPARATE LOOPS^*

Amy E. Schmidt^§, Kaillathe Padmanabhan^¶, Matthew C. Underwood, Wolfram Bode, Timothy Mather^**, and S. Paul Bajaj

From the  Departments of Pharmacological & Physiological Sciences and Internal Medicine, Saint Louis University Health Sciences Center, St. Louis, Missouri 63110, the ^¶ Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824,  Abteilung Strukturforschung, Max-Planck-Institut fur Biochemie, D82152 Martinsried, Germany, and the ^** Department of Cardiovascular Biology Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Received for publication, February 26, 2002, and in revised form, May 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

The serine protease domain of activated protein C (APC) contains a Na⁺ and a Ca²⁺ site. However, the number and identity of the APC residues that coordinate to Na⁺ is not precisely known. Further, the functional link between the Na⁺ and the Ca²⁺ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na⁺ binds to the substrate-occupied APC with K_d values of ~24 mM in the absence and ~6 mM in the presence of Ca²⁺. Sodium-occupied APC has ~100-fold increased catalytic efficiency (~4-fold decrease in K_m and ~25-fold increase in k_cat) in hydrolyzing S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) and Ca²⁺ further increases this k_cat slightly (~1.2-fold). Ca²⁺ binds to the protease domain of APC with K_d values of ~438 µM in the absence and ~105 µM in the presence of Na⁺. Ca²⁺ binding to the protease domain of APC does not affect K_m but increases the k_cat ~10-fold, and Na⁺ further increases this k_cat ~3-fold and decreases the K_m value ~3.7-fold. In agreement with the K_m data, sodium-occupied APC has ~4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca²⁺ site in APC is similar to that in trypsin, and the Na⁺ site is similar to that in factor Xa but not thrombin. Collectively, the Na⁺ site is thermodynamically linked to the S1 site as well as to the protease domain Ca²⁺ site, whereas the Ca²⁺ site is only linked to the Na⁺ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na²⁺-APC-Ca²⁺ form, which has 110-fold increased proteolytic activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

Activated protein C (APC)¹ circulates in blood as an inactive zymogen, protein C (PC), which is a disulfide-linked two-chain vitamin K-dependent glycoprotein with a M_r value of ~62,000 (1, 2). The N-terminal light chain of PC contains a -carboxyglutamic acid (Gla) domain and two epidermal growth factor domains (EGF1 and EGF2), whereas the heavy chain contains the latent serine protease domain (3-5). Although -thrombin alone can activate PC to APC, the rate is inappropriately slow for a physiologically important reaction (1). The physiologic activator for conversion of PC to APC is the thrombin-thrombomodulin complex at the endothelial surface that may also involve the endothelial PC receptor (6-8). During conversion of PC to APC, a short peptide consisting of 12 residues in human (2) and 14 residues in bovine (9) is removed from the N terminus of the heavy chain. The resultant enzyme is a serine protease that inactivates two key cofactors of coagulation, namely, factors Va and VIIIa (8). This represents a major anticoagulant mechanism involved in the regulation of hemostasis (8).

The Gla domain of the light chain of APC contains several Gla residues, which are required for calcium binding to this domain (10). In addition, the EGF1 domain contains a high affinity Ca²⁺-binding site in the native PC (or APC) molecule (8). Further, Gla domainless PC also contains a single high affinity Ca²⁺-binding site (11), and by mutagenesis experiments this site is implicated to be located in the protease domain (12). Thus, it appears that removal of the Gla domain may impair binding of calcium to the EGF1 domain. The protease domain of APC also contains a Na⁺-binding site, and residue c225² has been implicated in determining the Na⁺-induced allosteric regulation of catalytic activity of this as well as of other serine proteases (13).

The proteolytic activity of APC, including the hydrolysis of amide and ester substrates, is regulated by Na⁺ and Ca²⁺ (13-19). Initially, Castellino and co-workers (14-18) published a series of elegant kinetic and biophysical studies describing the potentiation of amidolytic and esterolytic activities of bovine APC. In these studies, they observed a sigmoidal dependence of the reaction velocity at a subsaturating concentration of substrate as a function of Na⁺. In the absence of structural information available at that time, these authors proposed that APC contained two sites or classes of Na⁺ sites that may be allosterically linked. In this report, we present data that support a heterotropic allosteric linkage of the single Na⁺-binding site in the protease domain to the S1 site of the substrate. Further, in a recent study, He and Rezaie (19) attempted to establish linkage between the protease domain Ca²⁺ site and the Na⁺ site. Here, we present data which strongly indicate that occupancy of the S1 site in APC increases the affinity of Na⁺ ~4-fold. He and Rezaie (19) did not address this point and only measured the affinity of Na⁺ at a single subsaturating concentration of substrate. Such data led to obtaining a high K_d app value for Na⁺ binding and therefore precluded determination of an accurate linkage between the Na⁺ site and the Ca²⁺ site (19). Moreover, although it is recognized that the c221-c225 loop in APC is most likely involved in binding to Na⁺ (13, 19), the site is not structurally defined. Here, we present crystallographic evidence that the Na⁺ site in APC involves not only the c221-c225 loop but also the c183-c189 loop. Based upon our findings, we predict that the Na⁺ site in factors VIIa and IXa will be similar to that in APC but not thrombin.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

Reagents-- H-D-Ile-Pro-Arg-p-nitroanilide (S-2288) was obtained from Diapharma Inc. Polyethylene glycol 8000 (PEG) and p-aminobenzamidine (PABA) were purchased from Sigma. D-Phe-Pro-Arg-chloromethylketone (FPRck) and diisopropyl fluorophosphate were obtained from Calbiochem. Human PC was purified as a by-product of the purification of factor VII (20, 21). A total of ~25 mg of PC obtained from six isolations, each starting with 8 liters of plasma, was used for these studies. It showed a single band (~62,000 Da) on nonreduced and two bands (~41,000 and ~21,000 Da) on reduced SDS-PAGE (22). For Ca²⁺ binding studies, FPRck-APC and Gla domainless FPRck-APC were prepared and purified using Mono Q fast flow anion exchange resin as outlined by Esmon et al. (23, 24) and Mather et al. (25). Decarboxylated FPRck-APC was obtained by thermal decarboxylation of Gla residues as described earlier for prothrombin (26) and factor X (27). For kinetic studies, human APC was obtained either from Enzyme Research Laboratories Inc. or from Hematologic Technologies, Inc. Both proteins gave comparable results. Gla domainless APC was prepared by chymotrypsin treatment of APC as outlined by Hill and Castellino (18). It was also purified according to their method with the exception that we used a Mono Q fast protein liquid chromatography column instead of the Q Sepharose column (18). Each protein sample was freed of Na⁺ by dialysis and/or by a desalting column exactly as described by Wells and Di Cera (28). Although this step resulted in some loss of APC activity, it was necessary for preparing APC free of Na⁺. The final concentration of Na⁺ after these steps was <1 mM as measured by a conductivity meter as outlined by Wells and Di Cera (28). The proteins were frozen at 80 °C in 20-µl aliquots, thawed, and used immediately. This freezing and thawing step did not result in a measurable loss of activity. All of the final kinetic data presented in this paper were obtained using APC obtained from Enzyme Research Laboratories or Hematologic Technologies, Inc.

Amino Acid Sequencing and Gla Analysis-- Automated Edman degradation of each protein component was performed using an Applied Biosystems model 477A gas phase sequencer. Approximately 0.5 nmol of protein was loaded on the filter cartridge. The proteins from SDS gels were transferred to polyvinylidene difluoride membranes as described by Rosenberg (29). The N-terminal sequence analysis of Gla domainless APC or Gla domainless FPRck-APC revealed two sequences of approximately equimolar amounts, one corresponding to the heavy chain (Leu-Ile-Asp-Gly-Lys) and the other corresponding to the modified light chain (Ser-Lys-His-Val-Asp). This indicates that Gla domainless APC does not contain the Gla domain (N-terminal 41 residues of the light chain) of APC. Decarboxylated APC contained <0.5 Gla/mol as compared with APC, which contained 7.9 Gla/mol as measured by the specific ³H incorporation (30). APC concentrations were determined from the absorbance at 280 nm using an extinction coefficient of 14.5 for 1% solution (2).

SDS-PAGE Analysis-- SDS gel electrophoresis was performed using the Laemmli buffer system (22). The acrylamide concentration was 15%, and the gels were stained with Coomassie Brilliant Blue dye. All of the proteins used in the present study were ~98% pure.

Measurement of S-2288 Amidolytic Activity of APC Proteins-- The concentration of APC or Gla domainless APC used was between 4 and 40 nM. The S-2288 concentration ranged from 10 µM to 3 mM. The buffer used was 50 mM Tris-HCl, pH 7.4, containing 0.1% PEG and various salt combinations given in the legends to the appropriate figures. Chloride salt of choline (Ch⁺), a bulky monovalent cation, was used to keep the ionic strength constant to 0.2 M. p-Nitroaniline (pNA) release was measured continuously (A_405 nm/min) for up to 30 min using a Beckman DU65 spectrophotometer equipped with a Soft-Pac kinetics module. An extinction coefficient of 9.9 mM¹ cm¹ at 405 nM was used in calculating the amount of pNA released (31). All of the reactions were performed in triplicate. The K_m and V_max values were obtained using the Enzyme Kinetics program from Erithacus Software. The K_d app of binding of Na⁺ or Ca²⁺ to the substrate-bound APC was calculated from the kinetic data using the following equation.

(Eq. 1)

where V is the apparent maximum velocity under a specified cation concentration represented by x, V is the V_max at saturating concentration of the cation, b is the maximum velocity at 0 concentration of the cation, and K_d app is the K_d of the specified cation for the APC(S) (substrate bound APC). The values for the correlation coefficient (r), a measure of the strength of the goodness of fit, were obtained using the program SPSS, version 10.0.

Ca²⁺ Binding to Decarboxlated FPRck-APC and Gla Domainless FPRck-APC-- Calcium ion activity was determined by using a Ca²⁺-specific electrode and a model 601A digital Ionlyzer (Orion Research). Titrations of the protein at 29 µM in 4 ml of Tris/NaCl, pH 7.4 (50 mM Tris-HCl, 200 mM NaCl), were performed by adding 1-2-µl increments of 40 mM CaCl₂ at room temperature. In these titrations, bound Ca²⁺ was taken as the difference between the measured free Ca²⁺ concentration and the total added. The data were fitted to the following equation using the program GraFit from Erithacus Software.

(Eq. 2)

where K_d app is the dissociation constant for binding of Ca²⁺ to the protein, Ca_B is the maximum Ca²⁺ binding capacity, and Ca_f and Ca_b represent the free and bound concentrations of Ca²⁺, respectively.

Determination of K_d PABA of Binding of PABA to APC-- The K_d app of binding of PABA to APC was determined by its ability to competitively inhibit S-2288 hydrolysis in the absence and presence of Na⁺ and/or Ca²⁺. The details are provided in the legend to Fig. 6. The IC₅₀ (concentration of PABA required for 50% inhibition) was determined by fitting the data to the following IC₅₀ four-parameter logistic equation from Halfman (32) given below.

(Eq. 3)

where y is the rate of pNA release in the presence of a given concentration of PABA represented by x, a is the maximum rate of pNA release in the absence of PABA, and s is the slope factor. Each point was weighted equally, and the data were fitted to Equation 3 using the nonlinear regression analysis program GraFit from Erithcus Software. To obtain K_d PABA values for the interaction of PABA with APC, we used the following equation as described by Cheng and Prusoff (33) and further elaborated by Craig (34).

(Eq. 4)

where [S] is the S-2288 concentration. The K_m values obtained under different conditions (listed in Table II) were used to obtain the K_d PABA value.

Further Refinement of the Gla Domainless FPRck-APC Structure-- The x-ray intensity data and the atomic coordinates (Protein Data Bank code 1aut) used for refinement were provided earlier by Mather et al. (25). The crystallization drop contained 500 µM total calcium concentration, which could provide sufficient free Ca²⁺ for it to bind to the c70-c80 loop in the protease domain. Examination of the (2F_obs  F_cal) and (F_obs  F_cal) electron density maps revealed that Ca²⁺ and appropriate solvent water molecules could be added to the structure. Similarly, based upon the Na⁺ site in thrombin (35) and factor Xa (36), we added Na⁺ and appropriate solvent water molecules in that region of the (2F_obs  F_cal) electron density map. The structure was refined using XPLOR (37) and iterative use of computer graphics using the program O (38). In all, three rounds of refinement were performed. The resultant structure has an R factor of 17.8% compared with the starting R factor of 18.4%. The final structure has 149 water molecules, one calcium ion, and one sodium ion. All of the water molecules had significant electron density when the (2F_obs  F_cal) electron density maps were contoured at the 1 level. The coordinates are being deposited with the Research Collaboratory for Structural Bioinformatics.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

Na⁺ Potentiation of S-2288 Hydrolysis by APC in the Absence of Ca²⁺-- In earlier studies, Na⁺ has been shown to be the physiologically relevant and an effective monovalent ion in potentiating the amidolytic activity of APC (13-15, 18). To further investigate the mechanism of Na⁺-mediated potentiation of S-2288 hydrolysis, we determined K_m app and V'_max at several concentrations of NaCl. We used 200 mM NaCl as the highest salt concentration in these experiments. When the concentration of Na⁺ was less than 200 mM, Ch⁺ was used as a compensating ion to keep the ionic strength constant to 0.2 M. One mM EDTA was present in each buffer to eliminate the effect of divalent cations. These data are presented in Fig. 1A. The values of K_m app and V'_max were calculated for each salt concentration using the Enzyme Kinetics program from Erithacus Software. The results indicate that Na⁺ affects both the K_m app and V'_max of this reaction (Fig. 1A). The K_m app and V'_max values obtained at each Na⁺ concentration are given in Table I. When V'_max are plotted as a function of Na⁺ concentration (Fig. 1B), the midpoint of the curve should yield K_d app of interaction of Na⁺ with APC saturated with S-2288 (APC(S)) in the absence of Ca²⁺; this value was calculated to be 23.9 ± 2 mM.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Sodium-mediated potentiation of S-2288 hydrolysis by APC in the absence of calcium. A, effect of Na+ on the Km app and Vmax of S-2288 hydrolysis. The monovalent ion concentrations are: 0 mM Na+, 200 mM Ch+ (closed circles); 4 mM Na+, 196 mM Ch+ (open circles); 10 mM Na+, 190 mM Ch+ (closed squares); 20 mM Na+, 180 mM Ch+ (open squares); 50 mM Na+, 150 mM Ch+ (closed triangles); 100 mM Na+, 100 mM Ch+ (open triangles); 150 mM Na+, 50 mM Ch+ (closed diamonds); and 200 mM Na+, 0 mM Ch+ (open diamonds). The Km app and V'max values were calculated using the enzyme kinetic program GraFit from Erithacus Software and are given in Table I. The concentration of APC used was from 4 to 40 nM; for consistency, the data were normalized to a 40 nM enzyme concentration. The pNA release was measured as outlined under "Experimental Procedures." For these experiments, two stock buffers were made. One buffer was 50 mM Trizma base, 0.1% PEG, 1 mM EDTA (acid form), and 200 mM ChCl adjusted to pH 7.4 with HCl. In the second buffer, 200 mM ChCl was replaced by 200 mM NaCl, and the pH was again adjusted to 7.4 with HCl. These two buffers were mixed in appropriate proportions to yield the desired concentrations of Na+ and Ch+. Thus, the monovalent ion concentration was always 200 mM, and the buffer containing 200 mM Ch+ had no added source of Na+. B, V'max as a function of Na+. The data were fitted to Equation 1. The Kd of Na+ for APC(S) was calculated to be 24 ± 2 mM. The values of b and V (converted to kcat) are given in Table II.

Ca²⁺ Potentiation of S-2288 Hydrolysis by APC in the Absence of Na⁺-- These data are presented in Fig. 2. The kinetic data indicate that Ca²⁺ does not change the K_m app but increases the k_cat ~10-fold (Table II). From the data of Fig. 2B, we calculate the K_d app of Ca²⁺ in its interactions with APC(S) to be 438 ± 31 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Calcium-mediated potentiation of S-2288 hydrolysis by APC in the absence of sodium. A, effect of Ca2+ on the Km app and Vmax of S-2288 hydrolysis. The Ca2+ concentrations are: 0 mM (closed circles), 0.1 mM (open circles), 0.5 mM (closed squares), 1 mM (open squares), 3 mM (closed triangles), and 5 mM (open triangles). The ionic strength was kept constant in each reaction mixture by adding 185-200 mM Ch+. The Km app and V'max values were calculated using the enzyme kinetic program GraFit from Erithacus Software. As in Fig. 1, the data were normalized to a 40 nM APC concentration. Ca2+ did not affect the Km app, and its value ranged from 1723 to 1705 µM (Table II). B, V'max as a function of Ca2+. As in Fig. 1, the data were fitted to Equation 1. The Kd app of Ca2+ for APC(S) was calculated to be 438 ± 47 µM. The correlation coefficient for each curve was >0.95.

Na⁺ Potentiation of S-2288 Hydrolysis by APC in the Presence of Ca²⁺-- These data are presented in Fig. 3. The kinetic data indicate that Na⁺ decreases the K_m app ~3.7-fold and increases the k_cat ~2.8-fold. Importantly, Ca²⁺ decreases the K_d app of Na⁺ interaction with APC(S) to 6.2 ± 1 mM as compared with ~24 mM (Fig. 1) when Ca²⁺ is absent. Thus, Ca²⁺ site is thermodynamically linked to the Na⁺ site.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Sodium-mediated potentiation of S-2288 hydrolysis by APC in the presence of calcium. A, effect of Na+ on the Km app and Vmax of S-2288 hydrolysis. The Na+ concentrations are: 0 mM Na+ (closed circles), 2 mM Na+ (open circles), 4 mM Na+ (closed squares), 7 mM Na+ (open squares), 10 mM Na+ (closed triangles), 50 mM Na+ (open triangles), 100 mM Na+ (closed diamonds), and 185 mM Na+ (open diamonds). Each reaction mixture contained 5 mM Ca2+ and appropriate concentrations of Ch+ to maintain constant ionic strength. Thus, at 0 mM Na+, the Ch+ concentration was 185 mM. As in Fig. 1, the data were normalized to a 40 nM APC concentration. The Km app values ranged from 466 µM (in the presence of Na+) to 1705 µM (in the absence of Na+) and are given in Table II. B, V'max as a function of Na+. The Kd app of Na+ in binding to APC(S) in the presence of Ca2+ was calculated to be 6.2 ± 1 mM. The correlation coefficient for each curve was >0.95.

Ca²⁺ Potentiation of S-2288 Hydrolysis by APC in the Presence of Na⁺-- These data are presented in Fig. 4. As noted previously (Fig. 2), Ca²⁺ does not change the K_m app but increases the k_cat slightly (1.2 ± 0.1-fold) in the presence of Na⁺. Because the change in V'_max is very small, these data could not be used to obtain the K_d app of Ca²⁺ interaction with APC(S). However, we later show that in the presence of Na⁺ this K_d app is ~105 µM as compared with ~438 µM (Fig. 2) when Na⁺ is absent. Thus, as noted above, the Na⁺ site is linked to the Ca²⁺ site.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Calcium-mediated potentiation of S-2288 hydrolysis by APC in the presence of sodium. The Ca2+ concentrations are: 0 mM (closed circles), 0.5 mM (open circles), 1 mM (closed squares), 3 mM (open squares), and 5 mM (closed triangles). The concentration of Na+ in each case was 185 mM, and the Ch+ concentration was varied to keep the ionic strength constant. As in Fig. 1, the data were normalized to a 40 nM APC concentration. The Km app values did not change and ranged from 415 ± 46 to 466 ± 53 µM. The correlation coefficient for each curve was >0.95.

The above experiments were repeated using Gla domainless APC. Potentiation of S-2288 hydrolytic activity at several subsaturating concentrations of Na⁺ (± 5 mM Ca²⁺) or Ca²⁺ (± 200 mM Na⁺) was indistinguishable from that obtained with APC. These data are consistent with extensive studies conducted by Hill and Castellino (17) using bovine APC and Gla domainless APC. Thus, the effects of Na⁺ and/or Ca²⁺ in potentiating the activity of both human and bovine APC and Gla domainless APC are comparable. Importantly, our results for the first time reveal that the substrate binding is allosterically linked to the binding of Na⁺.

Binding of Ca²⁺ to Human Decarboxylated FPRck-APC and Gla Domainless FPRck-APC-- Bovine Gla domainless APC has been reported to contain one high affinity Ca²⁺ site in the presence of Na⁺ (11). By mutagenesis experiments, a Ca²⁺-binding site in the protease domain of human APC has also been implicated (12). Here, we have performed direct Ca²⁺ binding studies with human decarboxylated FPRck-APC and Gla domainless FPRck-APC. These data are presented in Fig. 5 and reveal that the Gla domainless FPRck-APC contains a single Ca²⁺-binding site with a K_d app value of 105 ± 11 µM, whereas the decarboxylated FPRck-APC contains two Ca²⁺-binding sites with K_d app values of 120 ± 19 µM. Because Gla domainless FPRck-APC has only one high affinity Ca²⁺-binding site compared with the two in the decarboxylated FPRck-APC, it is likely that the EGF1 domain residues 42-49 containing Asp-46 and Gln-49 that are implicated in binding to Ca²⁺ (39) are flexibly disordered in the Gla domainless FPRck-APC and cannot participate in forming the high affinity site in this domain. Thus, the Ca²⁺ effects observed on the S-2288 amidolytic activity of APC are due to the Ca²⁺-binding site in the protease domain and not in the EGF1 domain.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Interaction of Ca2+ with human decarboxylated FPRck-APC and Gla domainless FPRck-APC as determined by a Ca2+-specific electrode. The free Ca2+ concentration (Caf) is plotted against the bound Ca2+ concentration (Cab). The concentration of each protein was 29 µM. The binding analysis was carried out by a nonlinear least squares fitting of the data to Equation 2, yielding a plateau value of 55 µM CaB for decarboxylated FPRck-APC and 28 µM CaB for Gla domainless FPRck-APC. Note that the active site in APC is blocked and that the Ca2+-binding data reflect interaction with APC(S). Open circles, decarboxylated FPRck-APC; closed circles, Gla domainless FPRck-APC.

The data presented thus far establish the interdependence of the binding of Na⁺ and Ca²⁺ in the protease domain of APC. APC(S) can be converted to the state with both ions bound (sodium-APC(S)-calcium) by acquiring either Na⁺ (via APC(S)-calcium) or Ca²⁺ (via sodium-APC(S)). The ratio of the apparent K_d Na in the presence and absence of Ca²⁺ is 3.9 and that of apparent K_d Ca in the presence and absence of Na⁺ is 4.2. Therefore, within experimental error, the net sum of binding energy over the cycle appears to be 0. This establishes an accurate thermodynamic linkage between the Na⁺ site and the protease domain Ca²⁺ site of APC as opposed to the 16-20-fold ratio proposed by He and Rezaie (19). Furthermore, because Na⁺ affects the K_m app of S-2288 hydrolysis, there appears to be a link between the substrate-binding and the Na⁺-binding site. Both in the absence and presence of Ca²⁺, Na⁺ decreases the K_m app of S-2288 by ~3.8-fold. Using K_m app as an approximation of substrate affinity, one can complete this part of the linkage using the thermodynamic principles (40). Therefore, apparent K_d Na and apparent K_d Ca to the free form of APC in the absence of substrate or the other cation can be calculated; these values are ~95.1 mM and ~443 µM, respectively.

Effects of Na⁺ and Ca²⁺ on the Interaction of PABA with APC-- We next investigated whether Na⁺ and/or Ca²⁺ affect the S1 part of the active site in APC. We used the S1 site probe PABA for these studies. The interaction of PABA with APC was determined under each of the four salt conditions. These data are presented in Fig. 6 and summarized in Table III. Under a saturating concentration of Na⁺, the affinity of PABA for APC was increased ~4.2-4.4-fold as compared with that in its absence. Further, Ca²⁺ had essentially no effect on the apparent K_d PABA in the absence or presence of Na⁺. These K_d PABA data agree well with the K_m data presented in the previous sections where Na⁺ decreased the K_m ~4-fold and Ca²⁺ had no effect. Our PABA data are similar to the PABA binding to bovine APC, where the K_d PABA is 13 µM in the presence and 95 µM in the absence of Na⁺ (17). However, the absolute apparent K_d PABA value for human APC is ~79 µM in the presence and ~333 µM in the absence of Na⁺ (Table III). Thus, it would appear that compared with human APC, bovine APC binds PABA with ~6- and ~3.5-fold higher affinity in the presence and absence of Na⁺, respectively. Importantly, our data establish that Na⁺ binding is linked to the substrate binding through its S1 site.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Ability of PABA to inhibit S-2288 hydrolysis by APC under various conditions. The six different experimental conditions are: 1) 0 mM Na+, 0 mM Ca2+ (closed circles); 2) 0 mM Na+, 5 mM Ca2+ (open circles); 3) 185 mM Na+, 0 mM Ca2+ (closed triangles); 4) 185 mM Na+, 5 mM Ca2+ (open triangles); 5) 485 mM Na+, 0 mM Ca2+ (closed squares); and 6) 485 mM Na+, 5 mM Ca2+ (open squares). To keep ionic strength constant, condition 1 contained 200 mM Ch+, condition 2 contained 185 mM Ch+, condition 3 contained 15 mM Ch+, and condition 4 contained 0 mM Ch+. To maintain constant ionic strength (500 mM monovalent ion concentration) between conditions 5 and 6, condition 5 contained 15 mM Ch+ and condition 6 contained 0 mM Ch+. The APC concentration used for conditions 1 and 2 was 40 nM, and the S-2288 concentration was 500 µM. The APC concentration used for conditions 3-6 was 4 nM, and the S-2288 concentration was 100 µM. The substrate S-2288 was mixed with varying concentrations of PABA before addition of APC to the reaction mixture. S-2288 hydrolysis was monitored by pNA release. The IC50 values were calculated using Equation 3. The apparent Kd PABA values were obtained using Equation 4 and are listed in Table III.

Location of the Protease Domain Ca²⁺ Site and the Na⁺ Site in the Gla Domainless FPRck-APC Crystal Structure-- To provide a structural basis for the linkage between cation-binding sites and the S1 site, we refined the APC structure after including Ca²⁺ and Na⁺ in the protease domain. A schematic representation of the protease domain of human APC depicting the Ca²⁺ site and the Na⁺ site are presented in Fig. 7A. Details of the Ca²⁺- and the Na⁺-binding sites are presented in Fig. 7 (B and C, respectively). The Ca²⁺-binding site is similar to that in trypsin (41) and involves the carboxylates of Glu-c70 and Glu-c80, carbonyl oxygens of c72 and c75, and two water molecules. The Na⁺-binding site is similar to that in factor Xa (36) and involves carbonyl oxygens of c184A, c185, c221A, and c224³ and two water molecules.

View larger version (43K): [in this window] [in a new window]   Fig. 7.   Location of the Na+ site as well as the Ca2+ site in the protease domain of APC. A, overall fold of the protease domain. -Sheets are in red, -helices are in blue, the Ca2+-binding loop is in white, the two Na+-binding loops are in yellow and white, and the autolysis loop is in magenta. The active site residues (Asp-c102, His-c57, and Ser-c195) are labeled D, H, and S, respectively. The N and C termini are marked N and C, respectively. The positions of residues that are important in binding to Na+ as well as to Ca2+ are labeled. The residue numbers are based upon chymotrypsin numbering as outlined by Mather et al. (25). B, details of the Ca2+-binding site in the protease domain. The main chain carbonyl groups of c72 and c75 and the carboxyl side chains of residues c70 and c80 along with two water molecules that coordinate to Ca2+ are depicted. The Ca2+-binding loop is colored by atom type. Ca2+ is shown as a white sphere, and water molecules are shown as red spheres. The dotted lines represent coordination of Ca2+ with its ligands. w, oxygen of water. C, details of the Na+-binding site and its relationship to the S1 site. The two Na+-binding loops, c183-c189 (colored red) and c221-c225 (colored magenta), are shown. The Na+ is coordinated by the carbonyl oxygen atoms of c184A, c185, c221A, c224, and two water molecules. Tyr-c225 residue, an important determinant for Na+ binding (13), and the S1 specificity binding pocket residue, Asp-c189, that is linked to the Na+ site as well as to the Tyr-c228, are also depicted. Na+ is shown as a blue sphere, and water molecules are shown as red spheres. The dotted lines represent hydrogen bonds as well as coordination of Na+ with its ligands. w, oxygen of water.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

The purpose of the present study was to structurally define the protease domain Ca²⁺ site as well as the Na⁺ site and their roles in regulating the catalytic activity of human APC. The spatial relationship of the Ca²⁺ site, the Na⁺ site, the autolysis loop, the Asp-c189 S1 site, and the catalytic triad is provided in Fig. 7A. Dang and Di Cera (13) recently reported that several serine proteases, including those in coagulation, possess a functional Na⁺ site. X-ray crystal structures of thrombin (35, 42) and factor Xa (35, 36) are reported where the Na⁺ site in these molecules is defined. The Na⁺ site in thrombin uses a single loop involving the carbonyl oxygen atoms of residues c221A and c224 as well as four water molecules, whereas the Na⁺ site in factor Xa uses two loops involving the carbonyl oxygen atoms of residues c185, c185A, c222, and c224 as well as two water molecules. The nature of the residue c225 plays an important role in orienting the carbonyl oxygen atom of c224 toward the Na⁺ coordination shell (35, 36, 42). As shown in Fig. 7C, the Na⁺ site in APC resembles that of factor Xa but not thrombin. This may be due to the insertion of three residues in the c183 loop of thrombin. As a result of this insertion, the carbonyl oxygens of this loop in thrombin are spatially distant and unable to coordinate with Na⁺. Instead, the cavity in thrombin is filled with water molecules, two of which are optimally situated to coordinate with Na⁺.

In the presence or absence of Ca²⁺, Na⁺ increased the affinity of S-2288 to APC (Table II) by ~4-fold and binding of PABA (Table III) by a similar fold. Because Ca²⁺ does not influence the K_m app value of S-2288 and has essentially no effect on the PABA binding, it would appear that these effects are primarily mediated through the S1 site in APC. These results can be readily rationalized, because the Na⁺ site in APC is directly linked to the S1 specificity pocket residue Asp-c189 (Fig. 7C). Occupancy of the Na⁺ site could rigidify the c189 side chain as well as the c183-c189 loop for optimal interaction with the basic P1 residue (Arg) of the substrate or the basic amino groups of the S1 probe, PABA.

In the absence of Na⁺, Ca²⁺ increased the k_cat of S-2288 hydrolysis ~10-fold (Table II). In the presence of Na⁺, however, Ca²⁺ had a minimal effect (~1.2-fold) on the hydrolysis of S-2288. Of interest, Ca²⁺ in the presence or absence of Na⁺ had no effect on the K_m app of S-2288 hydrolysis as well as on the PABA binding. Thus, Ca²⁺ does not affect the ground state binding of substrates/inhibitors to APC. The ~10-fold increase in k_cat for S-2288 hydrolysis thus appears to be due to a decrease in the transition state energy. All of the catalytic effects of Ca²⁺ on APC are remarkably similar to those observed earlier for factor Xa (43). This is not surprising because both proteases are structurally similar.

Of interest is the fact that the Na⁺ site and the protease domain Ca²⁺ site are thermodynamically linked. Thus, Ca²⁺ increases the affinity of Na⁺ by ~4.0-fold, and Na⁺ increases the affinity of protease domain Ca²⁺ binding by a similar fold. The thermodynamic linkage between the Na⁺ site and the protease domain Ca²⁺ site of APC is depicted in Fig. 8. This part of the linkage is supported by the data presented in Figs. 1, 2, 3, and 5. Thus, although the effects of Na⁺ and Ca²⁺ at individual steps of the thermodynamic cycle depicted in Fig. 8 are different, the overall change in apparent affinity for Na⁺ or Ca²⁺ is the same regardless of the pathway followed in going from APC(S) to the sodium-APC(S)-calcium state or the pathway followed in going from APC to the sodium-APC-calcium state.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Linkage relations in binding of S-2288, Na+, and Ca2+ to the protease domain of APC. In addition to the substrate-binding site, the protease domain of APC possesses a monovalent (Na+) as well as a divalent (Ca2+) cation-binding site. Thus, the protease domain can exist in eight forms: APC, APC(S), sodium-APC, APC-calcium, sodium-APC(S), APC(S)-calcium, sodium-APC-calcium, and sodium-APC(S)-calcium. The dissociation constant listed for each equilibrium was calculated using the data of Figs. 1, 2, 3, and 5. S, substrate. Note that the apparent Kd Na and apparent Kd Ca depicted on the left of the figure are not experimentally determined values but rather are calculated values based upon thermodynamic principles (40).

A major observation of significance in this paper is that the S1 site in APC is linked to the Na⁺ site. Hence, occupancy of the S1 site increases the affinity of Na⁺ ~4-fold, and Na⁺ increases the affinity (K_m) of the substrate by a similar fold. This part of the thermodynamic linkage between the S1 site and the Na⁺ site in APC is also depicted in Fig. 8 and is supported by the data presented in Figs. 1, 3, and 6. Consequently, although the effects of Na⁺ and substrate at individual steps of this part of the thermodynamic cycle are different, the overall change in apparent affinity for Na⁺ or substrate S-2288 is the same regardless of the pathway followed in going from APC to the sodium-APC(S) state or the pathway followed in going from APC-calcium to the sodium-APC(S)-calcium state. Note that Ca²⁺ is not thermodynamically linked to the S1 site. Thus, in going from APC to the APC(S)-calcium state, the affinity of Ca²⁺ or the substrate for APC is not altered regardless of the pathway followed in Fig. 8. Similarly, in going from sodium-APC to the sodium-APC(S)-calcium state, the affinity of Ca²⁺ or the substrate for sodium-APC is not altered regardless of the pathway followed in Fig. 8.

The plasma concentration of Ca²⁺ is ~1 mM and that of Na⁺ is ~150 mM. Under these conditions, ~85% of the formed APC will exist in the sodium-APC-calcium state. Binding of the substrate further drives this equilibrium in the direction that favors APC binding to Na⁺ and Ca²⁺. Thus, the overall effect of the linkage between the S1 site, the Na⁺ site, and the Ca²⁺ site is that during physiologic hemostasis, the APC formed will mostly exist in the sodium-APC-calcium state that has maximum anticoagulant activity. Notably, the linkage between Ca²⁺ and Na⁺ and the S1 site are essentially the same as proposed for factor Xa earlier (43). This similarity between the two proteases (APC and factor Xa) is consistent with the structural resemblance in the Ca²⁺ site, the Na⁺ site, and the S1 site.

The Ca²⁺ site in the APC protease domain (Fig. 7B) is typical of that found in trypsin-like proteases (41). It is of note that the c70-c80 calcium loop in APC contains three basic and two hydrophobic Trp residues (25). The serine proteases that bind Ca²⁺ contain primarily acidic and polar residues in this loop, which facilitate attraction of a positively charged divalent cation. Thus, trypsin contains four acidic and three polar residues (41), and elastase contains two acidic and seven polar residues (44). Similarly, human factor IXa contains five acidic and five polar residues and is thought to bind Ca²⁺ in this loop (45). Human factor VIIa contains six acidic and three polar residues and contains a Ca²⁺-binding site in this loop (46). Furthermore, the Gla domainless PC or APC was reported to contain only a single Ca²⁺-binding site (11), which was thought to be located in the EGF1 domain (39). Based upon the above considerations, Bajaj et al. (45) had initially proposed that PC or APC might not contain a Ca²⁺-binding site in the protease domain. However, recent mutagenesis experiments (12), direct binding studies (Fig. 5), and crystal structure data (Fig. 7B) clearly reveal the presence of a Ca²⁺-binding site in the c70-c80 loop of APC. Thus, the Ca²⁺ site in the Gla domainless PC or APC previously attributed to the EGF1 domain is most likely the Ca²⁺ site in the protease domain.

Structural features of the c70-c80 Ca²⁺-binding loop of APC are shown in Fig. 9. A unique feature of this loop appears to be that all three basic residues and the two hydrophobic Trp residues are exposed to the solvent. The other hydrophobic residue Leu-c73 is buried in a hydrophobic pocket involving spatially close neighbors. The Trp-c76 is stabilized by two hydrogen bonds and van der Waals' interactions involving the side chain carbon atoms of Lys-c78. Similarly, Trp-c79 is held in place by a hydrogen bond involving the carbonyl oxygen of Gly-c69. The hydroxyl group of Tyr-c71 in this loop is hydrogen-bonded to a water molecule as well as to the side chain of Thr-c22. Further, the phenyl ring of Tyr-c71 is stabilized through van der Waals' interactions with the side chain carbon atoms of Arg-c24. Thus, it is not unreasonable to conclude that the binding of Ca²⁺ to this loop provides favorable energy for the two Trp residues to stay exposed to the solvent. Interestingly, these two Trp resides have been implicated to be important for the activation of PC by the thrombin-thrombomodulin complex (8).

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Surface features of the APC protease domain Ca2+-binding loop. Location of Leu-c73 in a hydrophobic pocket comprised of Val-c32, Leu-c40, and Trp-c141 is shown in white. Two hydrophobic residues and three basic residues, namely, Trp-c76, Trp-c79, Arg-c74, Arg-c75, and Lys-c78 are exposed to the solvent. These residues are stabilized by hydrogen bonds and van der Waals' interactions and are discussed in the text. The dotted lines represent hydrogen bonds as well as coordination of Ca2+ with its ligands. Magenta sphere (Ca), calcium; red sphere (w), oxygen of water.

	CONCLUDING REMARKS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

In this report, we provide evidence that the Na⁺ site in APC is thermodynamically linked to the S1 site as well as to the protease domain Ca²⁺ site. Such information was critical in defining the accurate thermodynamic cycle for the linkage of the Na⁺ site to the Ca²⁺ site as well as to the S1 site (Fig. 8). Our data also indicate that most of the APC under physiologic conditions will exist as sodium-APC-calcium, which has maximal biologic activity. These conclusions with respect to function are supported by the structural data. The Ca²⁺ site in APC is similar to other trypsin-like proteases (41), and the Na⁺ site is similar to factor Xa (36) but not thrombin (35). Based upon the sequence comparison (47) and structural homology between factor Xa (36), factor IXa (48), and factor VIIa (46), we postulate that factors IXa and VIIa will have Na⁺ sites similar to that of factor Xa and APC. If so, then Na⁺ appears to play a structural role in maintaining the fully active conformer of the protease domain in these vitamin K-dependent proteins.

	ACKNOWLEDGEMENTS

We thank Dr. Tomasz Heyduk for useful discussions and Dana Oliver for help with the statistical analyses.

	Addendum

Fig. 2 originally present in Papers in Press has been deleted. In this figure the velocity curve as a function of substrate concentration showed weak sigmoidal behavior at subsaturating concentration (1 mM) of Na⁺. Subsequently, when several simulation experiments were conducted each at different subsaturating concentrations of Na⁺, linear curves were observed. Moreover, at each substrate concentration, the ratio of enzyme-substrate complex to the enzyme-Na⁺-substrate complex was also unchanged. Thus, Fig. 2 was removed, and the text has been adjusted appropriately.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL36365 and HL70369.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1aut) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^§ Supported in part by Predoctoral Fellowship 0110191Z from the American Heart Association Heartland Affiliate.

To whom correspondence should be addressed: Div. of Hematology and Oncology, St. Louis University Health Sciences Center, 3635 Vista Ave., P.O. Box 15250, St. Louis, MO 63110-0250. Tel.: 314-577-8499; Fax: 314-773-1167; E-mail: Bajajps@slu.edu.

Published, JBC Papers in Press, May 23, 2002, DOI 10.1074/jbc.M201892200

² For comparison, the chymotrypsin amino acid numbering system is used. Thus, the numbers with the prefix "c" (e.g. c225) refer to the chymotrypsin equivalents for the protease domain of APC. Where insertions occur, the chymotrypsin numbering is followed by a capital letter, such as A.

³ In APC (25), the residues in the c183 loop are numbered as c184, c184A, c185, c186, c186A, and c187. In factor Xa (36), the equivalent residues in this loop are numbered as c184, c185, c185A, c185B, c186, and c187. The residues in the c221 loop of APC are numbered as c221, c221A, c222, c223, c224, and c225. The equivalent residues in this loop of factor Xa are numbered as c221, c222, c222A, c223, c224, and c225. The residues involved in binding to Na⁺ in APC and factor Xa are given in italics in this footnote.

	ABBREVIATIONS

The abbreviations used are: APC, activated protein C; PC, protein C; S-2288, H-D-Ile-Pro-Arg-p-nitroanilide; PEG, polyethylene glycol 8000; Gla, -carboxyglutamic acid; Ch⁺, choline; FPRck, D-Phe-Pro-Arg-chloromethylketone; PABA, p-aminobenzamidine; pNA, p-nitroaniline; EGF, epidermal growth factor; APC(S), APC saturated with S-2288.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUDING REMARKS REFERENCES

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