Lund University, The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden
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Abstract |
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Keywords: activated protein C receptor/endothelial cell protein C/structural prediction
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Introduction |
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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, 1994). 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, 1995
). 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
1,
2 and
3. Both the
3 domain and ß2m have immunoglobulin-like folds. The
1 and
2 domains form a binding groove and are composed of an eight-stranded antiparallel ß-pleated sheet lined by two long antiparallel
-helices. The width of the binding groove is dictated by the distance between the H1, H2a and H2b helical segments of the
2 domain and H2 helix of the
1 domain. ß2m has many atomic contacts with the underside of the floor of the binding groove as well as with the
3 domain (Chelvanayagam et al., 1997
). The
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, 1995) and it is mainly localized to the surface of large vessels (Fukudome et al., 1996
; Fukudome et al., 1998
). Mature human EPCR consists of 221 amino acids with 25 residues at the C-terminus predicted to form a transmembrane anchoring
-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., 1997
). The soluble form of EPCR binds PC/APC with the same affinity as the transmembrane receptor (Fukudome et al., 1996
). This result suggests that the interaction between PC/APC and EPCR is mainly due to direct proteinprotein interaction and that the phospholipid membrane is not involved in this process (Fukudome et al., 1996
). 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, 1994
). Upon binding to soluble EPCR, APC looses its anticoagulant activity due to inhibition of proteolytic degradation of factor Va (Regan et al., 1996
). However, in the presence of EPCR, APC retains its enzymatic activity against small synthetic substrates. The PC/APCEPCR interaction neither prevents the activation of PC by the thrombinTM complex nor the inactivation of APC by its serpin inhibitors (Regan et al., 1996
). These results suggest that EPCR does not sterically block the active site cleft of APC, but modulates selectively its enzymatic specificity (Regan et al., 1996
). Membrane bound EPCR has been shown to enhance activation of PC by the thrombinTM complex and it seems that EPCR does not contact TM in this process (Xu et al., 1999
; Stearns-Kurosawa et al., 1996
). 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., 1998
). 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., 1997
). 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., 1997
).
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., 1997, 1998
; Villoutreix and Dahlbäck, 1998a
,b
; Blom et al., 1998
; Knobe et al., 1999
). 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. Structurefunction relationships are discussed based upon the analysis of our models.
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Materials and methods |
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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., 1989)). 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., 1987
). 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., 1999
) was used in order to gain information about the relative shape and size of this enzyme with regard to EPCR.
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Results and discussion |
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EPCR consists of the 1 and
2 domains, a transmembrane region, which is predicted to be 2325 amino acids long and have a short cytoplasmic tail of about five residues, most likely partially embedded within the phospholipid bilayer (Figure 2
). 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
3 domain. There are no major structural changes within the
1 and
2 domains in these proteins when the
3 domain is removed, indicating that this region has little contact with the
1/
2 domains (Collins et al., 1995
). It is thus possible to predict the structure of the
1 and
2 domains of EPCR using the fold of CD1/MHC class I molecules despite the fact that EPCR lacks the
3 region. Based on known structures of transmembrane proteins (Sakai and Tsukihara, 1998
) we concluded that the transmembrane region of EPCR forms an
-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, 1998
). 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 (2325 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 1). This, together with the conservation of key residues (e.g. the disulphide bond) and the possibility of reproducing the hydrophobic core of the
1/
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
-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 I
). 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 membranesolvent 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 I
). Moreover, no charged amino acids were found buried in the hydrophobic core without being appropriately counterbalanced.
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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 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
2 EPCR domain and the transmembrane segment may also have different conformations. In CD1, this region (around CD1 residues 184189) is extended and is followed by a ß-strand (see after the CD1 H2b helix in Figure 2
). 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
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 2
) 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 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 II
). There are some variations in the residues which line one side of the groove within the
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., 1994
).
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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 4). Some regions presented clusters of solvent exposed hydrophobic/aromatic residues (Figure 3
). For instance, in human EPCR, one such site contains about eight residues (the patch is marked on figure 3
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
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., 1996
). 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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Correlation between EPCR model and previously reported biochemical studies
The mechanisms by which EPCR affect the activation of PC by the thrombinTM 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., 1996; Esmon et al., 1997
). The Gla domain of PC has also been suggested to interact with TM and/or thrombin (Olsen et al., 1992
; Rezaie, 1998
) and with the phospholipid membrane (Freysinnet et al., 1986
; Horie et al., 1994
). 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 Glamembrane 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 thrombinTM complex should be dictated essentially by the activation peptidethrombin 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., 1989) and similar experiments have proposed the active site of APC to be located about 94 Å above the phospholipid surface (Yegneswaran et al., 1997
). 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 PCthrombinTM 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., 1999
). 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., 1996
) 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., 1997
; Shen et al., 1999
).
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 1-antitrypsin (same second order rate constant with or without EPCR) (Regan et al., 1996
). 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 EPCRPC/APC interactions, are proposed.
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Acknowledgments |
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Notes |
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References |
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Received March 15, 1999; revised June 17, 1999; accepted July 28, 1999.