Structural prediction and analysis of endothelial cell protein C/activated protein C receptor

Bruno O. Villoutreix1, Anna M. Blom and Björn Dahlbäck

Lund University, The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The endothelial cell receptor (EPCR) for protein C (PC)/activated protein C (APC) is a 221 amino-acid residues long transmembrane glycoprotein with unclear physiological function. To facilitate future studies and to rationalize recently reported experimental data about this protein, we have constructed three-dimensional models of human, bovine and mouse EPCR using threading and comparative model building. EPCR is homologous to CD1/MHC class I molecules. It consists of two domains, which are similar to the {alpha}1 and {alpha}2 domains of MHC class I molecules, whereas the {alpha}3 domain of MHC is replaced in EPCR by a transmembrane region followed by a short cytosolic tail. The {alpha}1 and {alpha}2 domains of CD1/MHC proteins form a groove, which binds short peptides. These domains are composed of an eight-stranded antiparallel ß-pleated sheet with two long antiparallel {alpha}-helices. The distance between the helical segments dictates the width of the groove. The cleft in EPCR appears to be relatively narrow and it is lined with hydrophobic/aromatic and polar residues with a few charged amino acids. Analysis of the human EPCR model predicts that (a) the protein does not contain any calcium binding pockets; (b) C101 and C169 form a buried disulphide bridge, while C97 is free, and buried in the core of the molecule; and (c) four potential glycosylation sites are solvent exposed.

Keywords: activated protein C receptor/endothelial cell protein C/structural prediction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Human endothelial cells have been shown to express a membrane protein that binds both zymogen (PC) and activated (APC) forms of protein C, in a specific, saturable and calcium-dependent manner (Fukudome and Esmon, 1994Go). The physiological function of this molecule, which was named endothelial cell protein C/activated protein C receptor (EPCR), is not yet fully understood. PC/APC is a multi-domain glycoprotein, which consists of a calcium-dependent membrane binding domain (the Gla domain), two EGF-like modules and a serine protease (SP) domain. Protein C is activated by thrombin bound to thrombomodulin (TM), a transmembrane protein expressed at the surface of endothelial cells (reviewed in Esmon et al., 1997). APC is an anticoagulant that inactivates coagulation factors V/Va and VIII/VIIIa, thereby down regulating the coagulation system (Dahlbäck, 1995Go; Di Cera et al., 1997Go; Kalafatis et al., 1997Go). The physiological importance of the PC anticoagulant pathway is well established (Aiach et al., 1997Go; Dahlbäck, 1997Go; Jalbert et al., 1998Go; Koeleman et al., 1997Go) and PC/APC has gained recognition as an antithrombotic molecule of clinical interest [e.g. in the treatment of meningococcemia (Smith et al., 1997Go)].

EPCR is homologous to the antigen presenting MHC class I/CD1 molecules (28% amino acid sequence identity when comparing the entire sequence of mature EPCR with CD1d) (Fukudome and Esmon, 1994Go). Human EPCR has even higher sequence similarity (62% amino acid sequence identity when comparing the entire sequence of mature EPCR) with murine CCD41 (centrocyclin) which is a centrosome-associated protein involved in the regulation of the cell cycle. Indeed, it has been suggested that murine CCD41 and murine EPCR could be identical proteins (Fukudome and Esmon, 1995Go). MHC class I molecules are type 1 integral membrane proteins associated non-covalently with ß2-microglobulin (ß2m). The extracellular part of these MHC molecules is composed of three domains designated as {alpha}1, {alpha}2 and {alpha}3. Both the {alpha}3 domain and ß2m have immunoglobulin-like folds. The {alpha}1 and {alpha}2 domains form a binding groove and are composed of an eight-stranded antiparallel ß-pleated sheet lined by two long antiparallel {alpha}-helices. The width of the binding groove is dictated by the distance between the H1, H2a and H2b helical segments of the {alpha}2 domain and H2 helix of the {alpha}1 domain. ß2m has many atomic contacts with the underside of the floor of the binding groove as well as with the {alpha}3 domain (Chelvanayagam et al., 1997Go). The {alpha}3 domain of MHC class I/CD1 molecules is followed by a transmembrane segment and a short intra-cytoplasmic tail.

Expression of EPCR is restricted to the endothelium (Fukudome and Esmon, 1995Go) and it is mainly localized to the surface of large vessels (Fukudome et al., 1996Go; Fukudome et al., 1998Go). Mature human EPCR consists of 221 amino acids with 25 residues at the C-terminus predicted to form a transmembrane anchoring {alpha}-helical structure. Soluble EPCR (residues 1 to about 194) has been detected in plasma, but it is not known if this form results from alternative splicing or proteolytic cleavage (Kurosawa et al., 1997Go). The soluble form of EPCR binds PC/APC with the same affinity as the transmembrane receptor (Fukudome et al., 1996Go). This result suggests that the interaction between PC/APC and EPCR is mainly due to direct protein–protein interaction and that the phospholipid membrane is not involved in this process (Fukudome et al., 1996Go). It has been suggested that PC/APC binds to EPCR via its Gla domain because recombinant protein C lacking this domain fails to displace protein C from EPCR (Fukudome and Esmon, 1994Go). Upon binding to soluble EPCR, APC looses its anticoagulant activity due to inhibition of proteolytic degradation of factor Va (Regan et al., 1996Go). However, in the presence of EPCR, APC retains its enzymatic activity against small synthetic substrates. The PC/APC–EPCR interaction neither prevents the activation of PC by the thrombin–TM complex nor the inactivation of APC by its serpin inhibitors (Regan et al., 1996Go). These results suggest that EPCR does not sterically block the active site cleft of APC, but modulates selectively its enzymatic specificity (Regan et al., 1996Go). Membrane bound EPCR has been shown to enhance activation of PC by the thrombin–TM complex and it seems that EPCR does not contact TM in this process (Xu et al., 1999Go; Stearns-Kurosawa et al., 1996Go). This reaction was suggested to be of particular importance in large arteries since EPCR is expressed preferentially in large vessels where the concentration of TM is low (Fukudome et al., 1998Go). However, soluble EPCR could be present locally at concentrations high enough to attenuate such augmentation of PC activation by membrane-bound EPCR (Kurosawa et al., 1997Go). It has been shown that EPCR expression increases in response to endotoxin infusion, but it is not yet known if this receptor plays a role during inflammation (Esmon et al., 1997Go).

The numerous molecular events taking place during the coagulation cascade are still not well understood. In such complex biological systems, careful matching of structural, biochemical, clinical and theoretical data is essential. We have therefore developed several 3D models which provide valuable insights into the coagulation and PC pathways and which help to design and interpret experiments (Villoutreix et al., 1997Go, 1998Go; Villoutreix and Dahlbäck, 1998aGo,bGo; Blom et al., 1998Go; Knobe et al., 1999Go). Since the physiological function of EPCR and the regions involved in PC/APC interactions are incompletely characterized, we have developed in the present study, 3D models of human, bovine and murine EPCR molecules using threading and comparative model building. Structure–function relationships are discussed based upon the analysis of our models.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A structural template suitable for the creation of a model for human EPCR was identified using threading (to align an amino acid sequence with known 3D structures) via the UCLA-DOE fold recognition server (Fisher and Eisenberg, 1996Go). Coordinate files of the possible X-ray templates were obtained from the Protein Data Bank (PDB) (Bernstein et al., 1977Go). The top Z-score (10.28) resulting from the threading search for human EPCR identified mouse CD1 (Zenget al., 1997; PDB entry 1cd1) as the most similar structure, followed by a MHC class I protein (Z = 9.51; Collins et al., 1995; PDB entry 1tmc) and neonatal Fc receptor (Z = 8.96; Burmeister et al., 1994; PDB entry 1frt). Since the Z-scores were clearly above the confidence threshold of the method (Z-score = 4.8 ± 1) (Fisher and Eisenberg, 1996Go) and because EPCR has been shown to be related to this family of proteins by direct amino acid sequence alignments (Fukudome and Esmon, 1994Go), structural prediction of EPCR based on the X-ray structure of CD1/MHC class I molecules is clearly appropriate. Murine and bovine EPCR were evaluated in a similar fashion. The potential X-ray templates were also selected from threading searches using the murine and bovine EPCR sequences as query input. The top Z-scores found the mouse CD1 X-ray structure as the most probable template with Z-scores of 17.21 for mouse EPCR and 15.38 for bovine EPCR, followed by neonatal Fc receptor (Z = 11.69 for murine EPCR and Z = 12.92 for bovine EPCR) and MHC class I molecule (1tmc; Z = 10.77 for murine EPCR and Z = 10.78 for bovine EPCR). Therefore, the X-ray structure of mouse CD1 was selected as the starting conformation to build EPCR. The amino-acid sequences of human, bovine and murine EPCR molecules were aligned to the mouse CD1 sequence according to threading information as well as structural analysis of test EPCR theoretical models and of the mouse CD1 X-ray structure. The final alignment is presented in Figure 1Go.



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Fig. 1. Alignment of mouse CD1, human, bovine and mouse EPCR sequences. Only the amino acid sequence of the {alpha}1 and {alpha}2 domains of mouse CD1 is presented in this figure while the sequence of its {alpha}3 domain, transmembrane segment and intra-cytoplasmic region is omitted for clarity. Thus after residue 192, mouse CD1 is further continued by the {alpha}3 domain which has no counterpart in EPCR. There is no X-ray structure for the transmembrane segment and intra-cytoplasmic region of mouse CD1 and therefore aligning this part of its sequence with the ones of EPCR cannot help in building this region of EPCR. The CD1 secondary structure is shown above the sequences (h, helix; b, beta strand). Potential N-glycosylation sites in EPCR are denoted with a `*' and free cysteine residues in EPCR by a `@'. The predicted transmembrane helix is shown by the `h?h?' symbols below the sequences. A dashed line connects the two Cys residues that are involved in a disulphide bond.

 
A Silicon Graphics workstation was employed for the construction and analysis of the models together with the Biosym-MSI molecular modelling package (San Diego, USA). The coordinates of mouse CD1 were used as the initial framework to build the EPCR structure. Conservative side-chain replacements were modelled in conformations similar to the ones that were present in the CD1 structure. Other residue replacements were initially modelled using the CD1 coordinates and further optimized using low-energy rotamer conformations (Ponder and Richards, 1987Go). Low-energy rotamers were calculated using an 8 Å cut-off distance for non-bonded interactions. The positions of these non-identical side chains were selected not only based on low non-bonded energy value terms but also such that they occupy, as closely as possible, the overall position of the original CD1 side chains. The insertion regions were built from a search among a high-resolution protein structure database (Hobohm and Sander, 1994Go). At least three residues prior (preflex) and after (postflex) the loop (flex) to be built were selected for the root mean square (r.m.s.) calculations (i.e. calculations between the {alpha}-carbon distance matrix database extracted from the selected set of high resolution structures and the preflex and postflex residues of the segment to build). Deletion regions in EPCR, as compared with CD1, were subjected to short rounds of energy minimization. Disulphide bonds were created interactively between the appropriate cysteine residues. As this bridge is conserved in the template, the procedure was straightforward. The first four to seven EPCR N-terminal residues (Figure 1Go) had no counterpart in the mouse X-ray structure and were built in an extended conformation. The segment running from S185 to S190 (in the human EPCR sequence) was built in an extended conformation, while the region between Y191 and T216 was given an {alpha}-helical structure. Delineation of this transmembrane helix resulted from the theoretical prediction according to the hidden Markov model method developed by Sonnhammer and co-workers (Sonnhammer et al., 1998Go). Although the exact angle between the segment S185–S190 and the {alpha}1 and {alpha}2 domains of EPCR is not known, an orientation making the transmembrane helical segment begin at a position similar to the one expected for CD1 was judged appropriate. The short predicted intra-cytoplasmic segment was built in an extended conformation and then subjected to a short (20 ps) molecular dynamic simulation keeping the remaining part of the structure fixed. The average conformation was generated and the region briefly energy minimized.

The EPCR model structures were then energy minimized using Discover (Biosym-MSI). All calculations were carried out using the CVFF force field parameters, a dielectric constant of 1 and a 20 Å cut-off distance for non-bonded interactions (reviewed by (Mackay et al., 1989Go)). Energy minimization was carried out in a stepwise fashion (initially the heavy atoms were slightly tethered to their original positions and subsequently relaxed). The stereochemistry was analysed using ProStat (Biosym-MSI). Electrostatic calculations were performed with DelPhi using a standard set of formal charges (Gilson et al., 1987Go). The dielectric constants were set to 80 and 4 for the extra-molecular region and protein interior, respectively. Calculations were performed either assuming His residues to be neutral or to bear a charge of 0.5e. Only these last results are shown, as the overall observations would remain essentially unchanged. A theoretical model of full-length human APC (Knobe et al., 1999Go) was used in order to gain information about the relative shape and size of this enzyme with regard to EPCR.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Because the sequence identity between EPCR and CD1/MHC class I molecules of known structure is relatively low (between 20 and 27%), we decided to use threading experiments to help in the selection of a starting conformation. The mouse CD1 X-ray structure was identified as the most appropriate template and was therefore used to predict the structure of human, bovine and mouse EPCR molecules. Structural analysis of mouse CD1 performed by Zeng and co-workers (Zeng et al., 1997Go) indicated that this protein is more closely related to MHC class Ia and Ib than to class II. In fact, mouse CD1 has a strikingly similar 3D structure with those of MHC and related molecules, despite the fact that they have relatively low sequence identity. For example, the sequence identity between mouse CD1 and neonatal Fc receptor is only 25%, whereas the 3D structures, apart from certain loop regions, can be superimposed with an r.m.s. deviation for the C{alpha} trace of about 1.7 Å. Classical MHC Ia molecules present short peptides via their binding groove to CD8+ cytotoxic T lymphocytes. The mouse CD1 structure consists of three domains ({alpha}1, {alpha}2 and {alpha}3) and is associated with ß2m (Figure 2Go). Its binding groove is narrower than one of the other MHC molecules and is predominantly lined by hydrophobic residues, suggesting a possible role in presenting lipid or glycolipid instead of a short peptide (Porcelli et al., 1998Go).



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Fig. 2. Ribbon diagram of mouse CD1, human EPCR and human APC. The X-ray structures of mouse CD1 and ß2m are shown (left) with the secondary structure elements coloured. The CD1 helices are in yellow and the strands are white (orange in ß2m). The structure of the CD1 transmembrane segment is not known and is shown here as a dashed line (blue). Human EPCR model is presented in the middle with the Cys residues involved in a disulphide bond yellow (C101–C169). The free Cys (C97) is buried and is part of a ß-strand. The N- and C-terminal residues in CD1 and EPCR are noted N and C, respectively. Potentially glycosylated Asn residues in EPCR are shown in magenta. An average conformation is presented for the last few C-terminal residues of EPCR but the white arrow illustrates the fact that several possibilities can be considered. The conformation of this region is depending on the environment. The model of human full-length APC is presented (right) in order to show the relative size of the molecules. The Gla domain and the two EGF-like modules are in yellow with the seven calcium ions shown as blue spheres. The serine protease (SP) domain is in white with some key surface loops being labelled and coloured for orientation. The catalytic triad residues are in red (filled circles), and from left to right, they correspond to Asp102, His57 and Ser195. The 60 and 39 areas of APC are rich in positively charged residues. The loop 70 is involved in calcium binding whereas loop 220 is proposed to bind sodium ions (Di Cera et al., 1997Go; He and Rezaie, 1999Go). The numbering for SP domain residues and loops follows the chymotrypsinogen nomenclature. The overall orientation of these molecules with respect to the membrane plane has not been experimentally proven yet but is reasonable. A virtual membrane with a width of about 40 Å is displayed. Two phospholipid molecules were extracted from a lipid bilayer (Heller et al., 1993Go) to help the reading of the figure. Phosphorus atoms are in red.

 
Structure description and evaluation of the EPCR models

EPCR consists of the {alpha}1 and {alpha}2 domains, a transmembrane region, which is predicted to be 23–25 amino acids long and have a short cytoplasmic tail of about five residues, most likely partially embedded within the phospholipid bilayer (Figure 2Go). Several structures of MHC class I molecules are known as well as the structures of MHC class I molecules lacking, due to proteolytic cleavage, the {alpha}3 domain. There are no major structural changes within the {alpha}1 and {alpha}2 domains in these proteins when the {alpha}3 domain is removed, indicating that this region has little contact with the {alpha}1/{alpha}2 domains (Collins et al., 1995Go). It is thus possible to predict the structure of the {alpha}1 and {alpha}2 domains of EPCR using the fold of CD1/MHC class I molecules despite the fact that EPCR lacks the {alpha}3 region. Based on known structures of transmembrane proteins (Sakai and Tsukihara, 1998Go) we concluded that the transmembrane region of EPCR forms an {alpha}-helix. Indeed, the high content in hydrophobic residues and the amino acid composition of the transmembrane segment of EPCR is in accordance with other proteins presenting such a transmembrane helix (Arkin and Brunger, 1998Go). Consistent with the 3D structures of other bitopic proteins, the intracellular tail of EPCR has a few positively charged amino acids (`positive inside' rule) and the length of the transmembrane segment (23–25 amino acids) is sufficient to span the hydrophobic core of a phospholipid bilayer.

There are very few insertions/deletions, located outside the well-conserved secondary structure elements, in the EPCR sequences as compared with mouse CD1 (Figure 1Go). This, together with the conservation of key residues (e.g. the disulphide bond) and the possibility of reproducing the hydrophobic core of the {alpha}1/{alpha}2 domains (generally conserved in this family of molecules) support our choice of template and indicate that the overall structure of the EPCR models is correct. Thus, it was found possible to build the ß-strands and the {alpha}-helices for EPCR without observing any severe steric clashes. Indeed, all EPCR side chains fit very well into the CD1 fold. Furthermore, the unpaired cysteines of EPCR are either buried into the hydrophobic core of the molecule or embedded in the membrane (Table IGo). Extracellular free -SH groups are relatively uncommon because they can react with many external agents. Therefore, such free -SH groups are usually found in regular secondary structure elements, most often on ß strands, and pointing to the inner core of the protein. This is seen in the EPCR models and supports further our structural prediction. A free Cys is also found at position 12 of CD1 and it is buried within a ß-strand. There is one intra-cytoplasmic cysteine that is expected to be free in EPCR. This residue should however be located at the membrane–solvent interface and could make hydrogen bonds with the surrounding polar or charged groups. All Asn residues of EPCR that can be potentially N-glycosylated are fully solvent exposed (Table IGo). Moreover, no charged amino acids were found buried in the hydrophobic core without being appropriately counterbalanced.


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Table I. Asn and Cys residues of potential importance in EPCR
 
As calcium-binding sites in proteins are characterized by the presence of negatively charged side chains (and often Gln/Asn supplemented by backbone atoms and sometime water molecules) close in space in which the coordinating oxygen ligands defines an approximate pentagonal bipyramid (McPhalen et al., 1991Go), we investigated if EPCR had such structural characteristics. No calcium-binding pockets or calcium-binding loops were detected in the human and bovine EPCR structures while the electronegative 106-loop in the mouse could eventually interact with metal ions. However, such structural feature is usually conserved between species and we suggest that EPCR molecules (even in the mouse) do not bind calcium. Yet the very negatively charged 106-loop, in mouse EPCR, is striking and may have a specific function for protein–protein interactions that may not occur in the human and bovine species.

Collectively, the above discussed structural data, together with the fact that the stereochemistry of the models was found to be reasonable after evaluation with ProStat, supports the conclusion that EPCR adopts the CD1 fold. Thus the predicted 3D structures can be used for further analysis and as a basis for the interpretation of previously reported biochemical studies. However, there are three regions in the models that have to be considered with caution. These are the most N-terminal region, the linker region between the {alpha}2 domain and the transmembrane segment and the short intra-cytoplasmic tail. The first six to seven N-terminal residues of human and bovine EPCR molecules can adopt different conformations. We have built these residues in a somewhat extended conformation and introduced a small bend, which positions them close to the protein surface. The linker region running from the last helix of the {alpha}2 EPCR domain and the transmembrane segment may also have different conformations. In CD1, this region (around CD1 residues 184–189) is extended and is followed by a ß-strand (see after the CD1 H2b helix in Figure 2Go). It is likely that this region in EPCR adopts a similar structure, which would suggest that the transmembrane segment of EPCR starts at about the same position as the one that prolongs the {alpha}3 domain of CD1. A consequence of such a conformation is that the binding grooves of both CD1 and EPCR point away from the membrane surface (Figure 2Go) and are free for potential interaction with other molecules. Finally, the structure of the short intra-cytoplasmic tail cannot be predicted with precision at present and we have generated an average conformation.

The putative binding groove

The binding groove of mouse CD1 is more narrow than the corresponding region in other MHC molecules and it is predominantly lined by hydrophobic residues. It does not contain charged groups on its floor (`anchor residues'), which in other MHC molecules are involved in the interaction with the short peptide ligands. The groove in EPCR seems to be narrow not only because EPCR is structurally related to mouse CD1 but also because the side chains of the amino acids lining the groove are longer than the ones of CD1. The width of the CD1 groove is on average 14 Å (when measuring the distance between C{alpha} atoms of helices lining the cleft) while in the energy-minimized EPCR models the corresponding distance is about 12 Å. Regardless of the exact width of the EPCR groove, it is possible to propose a list of residues that line the cleft (Table IIGo). There are some variations in the residues which line one side of the groove within the {alpha}1 domain between mouse, bovine and human EPCR whereas, on the other side of the cleft, amino acids are highly conserved. In contrast to the groove of mouse CD1, the putative EPCR binding cleft does not present an outstanding aromatic/hydrophobic surface. Similar to CD1, the floor of the binding groove in EPCR does not contain the charged (anchor) residues that stabilize the interaction with short peptides. Although, it is tempting to propose that APC/PC could contact the EPCR groove, it is important to note that the Fc portion of immunoglobulin-G interacts with the side of the neonatal Fc receptor (a protein homologous to MHC class I) but not directly with the cleft (Burmeister et al., 1994Go).


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Table II. Key residues lining the groove of EPCR
 
Solvent exposed surfaces and electrostatic potential

Sites for recognition and binding of other molecules are key elements connected to protein function. A cluster of solvent exposed residues displaying hydrophobic/aromatic properties or a patch of charged amino acids is often part of a binding site. The surface of the EPCR models were scanned for unusual features such as solvent exposed hydrophobic residues and outstanding electrostatic potential (Figures 3 and 4GoGo). Some regions presented clusters of solvent exposed hydrophobic/aromatic residues (Figure 3Go). For instance, in human EPCR, one such site contains about eight residues (the patch is marked on figure 3Go and begins with W26), located relatively close to one another. Interestingly, this EPCR site corresponds approximately to a cluster of residues of similar nature, found in mouse CD1, which is involved in ß2m binding. However, in EPCR, the {alpha}3 domain is missing and because this hydrophobic /aromatic cluster is presumably located close to the membrane surface, it is unlikely that EPCR interacts with ß2m. It is not known if EPCR interacts with ß2m but it has been observed that human EPCR co-immunoprecipitates with an as yet unknown endothelial cell protein (Fukudome et al., 1996Go). This molecule may bind to the area where some hydrophobic/charged clusters are exposed or may interact with the binding groove.



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Fig. 3. Solvent accessible surface of human, bovine and mouse EPCR. In (A), the models are shown with the same overall orientation as the one used in Figure 2Go while in (B), they are seen from the opposite face of the binding groove. The solvent accessible solid surface is presented. The backbone atoms, Gly residues and the side chains of polar residues (N, Q, T and S) were coloured in orange, positively charged amino acids (K, R and H) in blue, negatively charged (E and D) in red and hydrophobic/aromatic residues in white. Two exposed hydrophobic clusters of possible importance are labelled in human EPCR.

 


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Fig. 4. Electrostatic potential isosurfaces of the EPCR models. The model structures are shown with the same overall orientation as the one used in Figures 2 and 3AGoGo. The electrostatic isosurfaces are displayed at a level of –1 (red) and +1 (blue) kcal/mol/e with some of the key residues contributing to the electric field being listed for human EPCR. In mouse EPCR, a region presenting a strikingly negative potential is observed. This arises essentially from the negatively charged insertion 106-loop. The region comprising R127, 130, 156, 158 and 162 was expected to display an electropositive surface but all these residues were found to be in part neutralized. The transmembrane segment and part of the binding groove are neutral. The dipolar distribution of the electrostatic potential within the putative binding groove is noteworthy and could be of functional importance. The intra-cytoplasmic tail contains two to three Arg residues that could interact with negative groups present in the inner part of the phospholipid bilayer.

 
The transmembrane helix of EPCR is clearly hydrophobic (Figures 3 and 4GoGo) while the intra-cytoplasmic tail is positively charged. The electrostatic potential of EPCR has a dipole-like distribution with the {alpha}1 domain being more electropositive and the {alpha}2 region more electronegative (Figure 4Go). Several areas in and around the binding groove are neutral. A very negative region in mouse EPCR is observed and is in part due to the presence of residues in the unique 106-loop (Figure 1Go). Such a difference between the species would suggest that APC/PC does not bind to this region of EPCR. Indeed, it seems that the segment above the transmembrane domain (thus the area nearby the 106-loop) is not involved in protein C binding (Fukudome et al., 1996Go).

Correlation between EPCR model and previously reported biochemical studies

The mechanisms by which EPCR affect the activation of PC by the thrombin–TM complex are incompletely understood. The availability of structural information for several of the components of the activation complex allows for critical evaluation of proposed hypotheses. The Gla domain of PC/APC and an electropositive region in the serine protease domain including loops 39 and 60, have been proposed to interact with EPCR (Regan et al., 1996Go; Esmon et al., 1997Go). The Gla domain of PC has also been suggested to interact with TM and/or thrombin (Olsen et al., 1992Go; Rezaie, 1998Go) and with the phospholipid membrane (Freysinnet et al., 1986Go; Horie et al., 1994Go). Presumably, the activation of PC is precisely regulated and it seems unlikely that the Gla module interacts specifically with four different molecular species (EPCR, thrombin, TM and the membrane). Taking these data together with the size differences between PC and EPCR and the most likely Gla–membrane interaction into account, our structural analysis suggests that part of the Gla module of PC/APC could contact one side of EPCR while at the same time interacting with the membrane surface.

The topology of a serine protease active site (here thrombin) imposes a very precise orientation for the reactive loop of an approaching substrate (here PC). From this, it follows that the orientation of the PC molecule in relation to the thrombin–TM complex should be dictated essentially by the activation peptide–thrombin contact. It has been shown by fluorescence energy transfer that the active site of thrombin, when bound to TM, is about 65 Å above the membrane (Lu et al., 1989Go) and similar experiments have proposed the active site of APC to be located about 94 Å above the phospholipid surface (Yegneswaran et al., 1997Go). Since the scissile bond in the activation peptide of membrane-bound PC has to be located about 65 Å away from the membrane, this suggests that PC, like APC, has a rod-like structure. These observations together with the structural analysis of the model for the PC–thrombin–TM ternary complex, are compatible with an orientation of PC in which the Gla domain points toward the membrane surface, but away from TM or thrombin, during activation (Knobe et al., 1999Go). In the same study, we proposed that loops 70, 60 and 39 of PC contact TM, but not thrombin, during the activation process. Because the activation peptide of PC is located in the vicinity of this electropositive region and that EPCR interacts equally well with PC and APC, it is unlikely that this region in APC/PC binds to EPCR. Also, because of the size differences between PC and EPCR, it would seem unlikely that EPCR has direct contact with this electropositive region during activation. Our analysis is further supported by the observation that binding of fluorescently labelled APC to full-length EPCR is not affected by heparin (Regan et al., 1996Go) and it has been recently shown that this highly negatively charged molecule binds with at least loops 60 and 39 of APC (Neese et al., 1997Go; Shen et al., 1999Go).

It has been shown that EPCR inhibits inactivation of FVa by APC although the active site of APC is still free to cleave small synthetic substrates (similar Km and kcat with and without EPCR) and to interact with two serpins, protein C inhibitor and {alpha}1-antitrypsin (same second order rate constant with or without EPCR) (Regan et al., 1996Go). As the active site of APC is about 94 Å above the membrane, it is still unclear how EPCR could change APC specificity. Possibly EPCR interacts with one side of APC (e.g. underneath the SP domain) resulting in sterical interference during the encounter between APC and factor Va.

Conclusion

We have built molecular models of human, bovine and murine EPCR molecules. Structural analysis and investigation of the amino acid distribution suggest the models to be correct. Some key regions are highlighted and candidate areas for site-directed mutagenesis, that could probe the EPCR–PC/APC interactions, are proposed.


    Acknowledgments
 
This project was supported by grants from Swedish Medical Research Council (project No. 07143 and 11793), research grants from the University Hospital in Malmö, the Louis Jeantet Foundation and the Swedish National Network for Cardiovascular and Inflammation Research.


    Notes
 
1 To whom correspondence should be addressed; email: bruno.villoutreix{at}klkemi.mas.lu.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received March 15, 1999; revised June 17, 1999; accepted July 28, 1999.