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Allosteric Regulation of the Cofactor-Dependent Serine Protease Coagulation Factor VIIa
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PII S1050-1738(98)00030-9
TCM
Allosteric Regulation of the CofactorDependent Serine Protease Coagulation Factor VIIa Wolfram Ruf* and Craig D. Dickinson
The integration of structure and function analysis of the tissue factor– factor VIIa complex has provided a detailed view of the functional surface of the extrinsic activation complex. An incomplete zymogen to enzyme transition is responsible for the strict cofactor dependence of catalytic function of factor VIIa. The mutational analysis demonstrates that factor VIIa is allosterically regulated by specific conformational linkages that involve the cofactor binding site, the catalytic cleft, and the macromolecular substrate exosite. Regions of the flexible activation domain appear to play an important role in the allosteric regulation of this cofactor-dependent coagulation serine protease. (Trends Cardiovasc Med 1998;8:350–356) © 1998, Elsevier Science Inc.
The coagulation pathways regulate essential cellular functions in hemostasis, wound healing, and immune response, as well as play important pathophysiologic roles in cancer metastasis, inflammation, and thrombosis (Ruf and Edgington 1994, Ruf and Mueller 1996). Central to the assembly of the coagulaWolfram Ruf and Craig D. Dickinson are at the Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California. * Address correspondence to: Wolfram Ruf, MD, Departments of Immunology and Vascular Biology, IMM-17, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. ©1998 Elsevier Science Inc. All rights reserved. 1050-1738/98/$–see front matter
tion cascade are macromolecular catalytic cofactors that direct the serine proteases to cell surfaces, the preferred location for the interaction with the downstream macromolecular substrates. Following the first structure determination of a coagulation serine protease, that is, thrombin (Bode et al. 1989), the last decade has seen a rapid progress in the structural knowledge of this homologous class of enzymes. Another major breakthrough was the recent crystallization of tissue factor (TF) in complex with factor VIIa (VIIa). The TF–VIIa complex has provided the structure solution of the first enzyme–cofactor complex of the coagulation cascade (Banner et al. 1996). We review here recent progress from functional and mutational analysis that TCM Vol. 8, No. 8, 1998
has elucidated the details of function and allosteric regulation of the serine protease VIIa. The interpretation of these functional studies in the context of the TF–VIIa structure may provide a more general paradigm for coagulation serine proteases that depend on assembly with a cell-associated cofactor. VIIa is a modular enzyme consisting of a trypsinlike protease domain and an amino terminal light chain with a gcarboxyglutamic acid-rich domain and two epidermal growth factor (EGF)–like modules. The crystal structure of the TF– VIIa complex demonstrated that each of
the structural modules of VIIa makes extensive contacts with the cofactor TF. TF is a member of the cytokine receptor family with two typical tightly associated b-strand, fibronectin type III repeat modules (Figure 1A). The Gla-domain of VIIa binds to the carboxyl-terminal module of TF in a position that would allow the interaction of the hydrophobic membrane binding portion of this domain with the cell surface. The interface of the Gla-domain with TF is hydrophobic, involving one face of the aromatic stack helix of VIIa and an unusual surface exposed disulfide bond in the car-
boxyl-terminal cytokine module of TF. The EGF-1 domain packs into a groove formed by the two modules of TF. This interface is the largest and energetically most important of the contacts, providing the anchor for tight binding of the protease with cofactor. The EGF-2 and protease domain form an extended interface with the amino-terminal module of TF. This interaction makes little contributions to the free energy of binding, but rather, serves the allosteric regulation of the VIIa protease domain, as discussed in detail later here. The structure of the TF–VIIa complex thus defined the
Figure 1. Overview of the tissue factor (TF)–VIIa structure. (A) shows the domain organization of the TF–VIIa complex with TF’s amino terminal (N) and carboxyl terminal (C) module shown in maroon and VIIa’s protease domain, EGF-1 and Gla shown in gray. The epidermal growth factor (EGF)-2 domain is located behind the protease domain and is not visible in this projection. The active site inhibitor D-Phe-L-Phe-Arg chloromethylketone (blue) is visible below catalytic triad residues His 57 and Ser 195 (red). Epitopes of antibodies that are competitive with factor X binding are highlighted. These antibodies are directed to the TF carboxyl module ( green: 5G9 epitope residues Tyr 156, Lys 166, Lys 169, Arg 200, Lys 201) (Huang et al. 1998) or to the VIIa protease domain ( yellow: F3-3.2A epitope residues Lys 20, Val 21, Lys 24, Glu 75, Met 156) (Dickinson et al. 1998). (B) gives an overview of residue side chains that are important for factor X activation, based on mutagenesis of TF (green: Lys 165, Lys 166) (Ruf et al. 1992) or VIIa ( yellow: Asp 72, Glu 75, Arg 148, Glu 154, Met 156, Asp 185B) (Dickinson et al. 1996). The amino terminus of the VIIa protease domain and catalytic triad residues His 57 and Ser 195 are red, the active site inhibitor in the TF–VIIa complex structure (Banner et al. 1996) is blue. Illustrations were created using the programs Molscript (Kraulis 1991), RASTER 3D (Merritt and Murphy 1994), and MIDAS-plus (Ferrin et al. 1988).
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molecular details of the TF–VIIa interaction and, furthermore, facilitated the interpretation of studies that delineated the regions of the complex that are involved in recognition of macromolecular substrates, in particular factor X. • The Macromolecular Substrate Docking Site of the Enzyme-Cofactor Complex It is well appreciated for thrombin, the ultimate protease of the coagulation cascade, that macromolecular substrate recognition involves both determinants in the catalytic cleft and within exosite I, a basic region in the protease domain that interacts with fibrinogen and the carboxyl-terminus of hirudin (Bode et al. 1992). The analysis of factor X activation by TF–VIIa suggests that extended recognition of macromolecular substrate by this cofactor-dependent protease is not restricted to the enzyme’s catalytic domain but involves the VIIa light chain and specific regions of TF as well. Concordant results from antibody mapping and mutagenesis suggest a mode of docking of macromolecular substrate with TF–VIIa. We have identified monoclonal antibodies to TF (Ruf and Edgington 1991) and the VIIa protease domain (Dickinson et al. 1998) that act as competitive inhibitors of factor X activation. The mapping of the antibody epitopes (Huang et al. 1998, Dickinson et al. 1998) indicates that steric interference with factor X docking must occur at one extended face of the TF– VIIa complex that is oriented toward the viewer in the projection of Figure 1A. Mutagenesis has further identified surface exposed residues that contribute to substrate activation on the same face of the complex (Figure 1B). First, basic residues in the TF carboxyl module, in particular Lys 165 and Lys 166 (Ruf et al. 1992), likely cooperate with the Gladomain of VIIa (Ruf et al. 1991a) in the recognition of factor X’s Gla-domain (Watzke et al. 1990, Huang et al. 1996). It is conceivable that the factor X Gladomain is docked with TF in an orientation similar to the VIIa Gla-domain, allowing membrane binding as well as interaction with the Gla-domain of VIIa. Mutagenesis has also identified residues in the VIIa protease domain that contribute to factor X activation (Dickinson et al. 1996). This cluster of mostly
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acidic residue is found on the same face of the TF–VIIa complex, facing the viewer in Figure 1B. Most of these residues, that is, Asp 72, Glu 75, Glu 154, are within the epitope of anti-VII antibody F3-3.2A mentioned earlier and in a position corresponding to thrombin’s exosite I. [Numbering of the residue positions of the VIIa protease domain is based on the chymotrypsin numbering scheme (Dickinson et al. 1996).] This site must be involved in interaction with the protease domain of substrate factor X, based on positional constraints by docking factor X’s scissile bond into the active site (Dickinson et al. 1996). The lower rim of the active site cleft makes also contributions to macromolecular substrate activation, in particular the basic Arg 148 (Ruf 1994) that is located in a surface loop referred to as the gautolysis loop in thrombin. This loop is generally flexible and often protease sensitive, and it contributes to efficient activation of macromolecular substrate, as demonstrated by the reduced function of g-thrombin. The available data thus emphasize the importance of extended recognition determinants for substrate specificity as a recurring theme in cofactor-dependent serine proteases. • A Unique Labile Zymogen to Enzyme Transition of VIIa The presence of cofactor markedly accelerates macromolecular substrate activation for each example of enzyme–cofactor complexes of the coagulation cascade. The amidolytic activities of the homologous proteases factor Xa, IXa, and VIIa, however, are influenced by cofactor interactions in specific ways: Factor IXa has very low amidolytic activity that is not enhanced by cofactor interactions (Castillo et al. 1983), whereas factor Xa, independently of cofactor, displays high activity toward small substrate mimics (Walker and Krishnaswamy 1993). VIIa has also very low catalytic activity toward peptidyl substrates, but the rate of hydrolysis is enhanced .10-fold by binding of cofactor (Ruf et al. 1991b). The precise structural basis for the low catalytic activity of free VIIa is unknown. Following zymogen activation, the amino terminus of most serine proteases moves .10Å to form a salt bridge with Asp 194 (Vijayalakshmi et al. 1994). These structural rearrangements of the
primary specificity site are necessary for catalytic activity and also insert the amino-terminal Ile 16 into the protein interior, where Ile 16’s a-amino group becomes protected from chemical modification. The amino terminal Ile 16 of the VIIa protease domain is susceptible to chemical modification, indicating solvent accessibility and a labile enzyme conformation of free VIIa. Binding of TF protects the amino terminus from chemical modification, consistent with stabilized insertion of the amino terminus (Higashi et al. 1994). The amino terminus is part of the activation domain, a larger region of increased flexibility or of alternative conformations in serine protease zymogens in comparison with the active enzymes (Huber and Bode 1978). In principle, free VIIa thus appears to make an incomplete zymogento-enzyme transition, and the cofactor TF is required to switch and maintain the protease domain in a catalytically active conformation. The following sections discuss results that demonstrate that the conformational transitions of VIIa involve discrete thermodynamic linkages that can be assigned to specific residue side chains in the VIIa protease domain. These studies suggest that the regulation of VIIa cannot be reduced to a simple global conformational transition, but rather that the inherent flexibility of the activation domain is utilized in this cofactor-dependent serine protease to allosterically influence catalytic function. • Specific Conformational Linkages in the VIIa Protease Domain Three interdependent regions of the VIIa protease domain are relevant for function: the cofactor binding site, the catalytic cleft, and the macromolecular substrate exosite (Figure 2). The cofactor binding site must transmit the allosteric changes by which TF influences VIIa’s catalytic function and the interface of VIIa with TF contributes to the affinity of enzyme–cofactor binding. The conformational state of the catalytic center can be monitored with small peptidyl substrates that provide adequate mimics for the active site docking of the scissile bond, in particular the P1 residue (Schechter and Berger 1967). The active site can further be probed by tight binding or covalent inhibitors that lock TCM Vol. 8, No. 8, 1998
Figure 2. Overview of known conformational linkages in the VIIa protease domain.
the protease domain into a conformation with restricted flexibility (Bode et al. 1978). The macromolecular substrate exosite is a major contributor to substrate specificity of these enzymes. Alterations in this region can be measured by changes in the kinetics of macromolecular substrate activation. Conformational changes in this exosite can also be detected by a unique monoclonal antibody with a conformation sensitive epitope that overlaps with the factor X docking site. Site-directed mutants of individual amino acid side chains in the VIIa protease domain have defined the following specific linkages between these functional regions of the VIIa protease domain.
Ruf 1997). Among the mutants in VIIa’s interface with TF, only the Met 164 replacement fails to dissociate slower from cofactor following inhibitor binding, establishing the crucial role of this residue in the thermodynamic coupling to the active site. The effect of interactions of Met 164
to the catalytic cleft may be transmitted through buried loop segments that are adjacent to the TF binding interface (the TF amino-terminal module is to the left in Figure 3). Importance of these buried segments is demonstrated by the loss of cofactor binding following selective reduction of the 168–182 disulfide bond (visible below Met 164 in Figure 3) that links the Met 164 helix to the 182–189 loop (Higashi et al. 1997). Moreover, Ala scanning mutations of residues in this loop (Dickinson et al. 1996), as well as hereditary mutations in adjacent buried positions (Bharadwaj et al. 1996, Matsushita et al. 1994, O’Brien et al. 1991), influence the catalytic function of VIIa. The 182–189 loop plays also an important role in the allosteric regulation of thrombin. This loop participates together with the 220–225 loop in the formation of a single Na1 ion binding site. Na1 binding is critical for the allosteric switch from the slow, anticoagulant conformer of the enzyme to the fast, procoagulant form (Di Cera et al. 1995). An aromatic amino acid at position 225
Allosteric Activation of VII’s Catalytic Function by Cofactor Binding Mutations in the interface of the protease domain of VIIa with TF influence the catalytic function of the TF–VIIa complex. The Ala replacement for Met 164 results in a concordant reduction in proteolytic and amidolytic function (Dickinson et al. 1996), indicating that the interaction at this residue position is directly linked to the catalytic cleft to which the small peptidyl substrates dock. The direct interdependence of the active site with this specific residue position in the interface with TF is further reinforced by analysis of the effect of active site occupancy on TF binding. Binding of covalent or tight binding inhibitors to the active site of VIIa increases the affinity for TF by significantly decreasing the dissociation rate of the enzyme from cofactor (Dickinson and TCM Vol. 8, No. 8, 1998
Figure 3. A potential conformational link from Met 164 to the active site of VIIa may occur through the Cys 168-Cys 182 disulfide bond into the 182-189 loop to Asp 189, the S1 subsite specificity determinant. Phe 225 is a signature aromatic residue for serine proteases that are allosterically regulated by sodium ion binding. The 182–189 loop is part of the activation domain, other loop segments in this flexible region (16–19, 142–152, 182–194, 216–223) are highlighted by shading.
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(Figure 3) is the hallmark for this class of allosterically regulated serine proteases to which VIIa also belongs (Dang and Di Cera 1996). We propose that cofactor interactions utilize the inherent flexibility of this segment of the activation domain (these loops are shaded dark in Figure 3) to allosterically regulate the catalytic activity of VIIa. Subtle changes in the conformation of the 182– 189 loop may influence the position of Asp 189 that is the specificity-determining residue at the bottom of the S1 binding pocket and may thus account for the cofactor effect on amidolytic function of VIIa. Consistently, the cofactor was found to facilitate small substrate binding, rather than to accelerate the deacylation step during substrate hydrolysis (Payne et al. 1996). Specific Influence of the Cofactor on Macromolecular Substrate Activation Mutations of TF residue Asp 44 in the interface with the VIIa protease (Figure 4) have only subtle effects on amidolytic function of VIIa, but factor X activation is severely reduced (Kelly et al. 1996). A similar specific effect on macromolecular substrate recognition followed mutation of residue Arg 134 in the corresponding interface in the VIIa protease domain (Dickinson et al. 1996), indicating that specific residues in the TF–VIIa interface are conformationally linked to the macromolecular substrate binding site. An attractive hypothesis is that the TF-mediated stabilization of the aminoterminal insertion (visible below the Arg 148 in Figure 4) also orders specific surface exposed side chains for optimal interaction with macromolecular substrate. Certain residues, that is, Leu 144, Glu 154, and Met 156 (visible to the right of the amino terminus in Figure 4), flank the amino-terminal insertion site and are indeed important for proteolytic function of VIIa (Dickinson et al. 1996). These residues also form the base of the flexible autolysis loop (colored dark in Figure 4) and may influence residues critical for macromolecular substrate recognition, such as Arg 148 (Ruf 1994). Moreover, Glu 154 and Met 156 are located within the epitope boundaries of monoclonal antibody F3-3.2A (for epitope location, see Figure 1), which reports a change in affinity for VIIa when TF is bound to the protease domain of VIIa
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Figure 4. A potential link from tissue factor (TF) to regions for macromolecular substrate interaction may involve a critical contact is the TF–VIIa interface marked by TF Asp 44 and VIIa Arg 134. A key residue that is specific for factor X activation is Arg 148, located in front of Lys 192, which contributes to formation of the S1 subsite. Arg 148 is part of the autolysis loop (residues 142–152, shaded) that was originally assigned to the activation domain. Residues at the base of the autolysis loop, that is, Leu 144, Glu 154, and Met 156, surround the amino-terminal insertion and are important for proteolytic function.
(Dickinson et al. 1998). This influence of cofactor on the antibody epitope conformation and the cofactor effect on factor X activation thus indicate a linkage from the TF binding site to the macromolecular substrate exosite in the VIIa protease domain. The cofactor binding site in VIIa thus makes distinct conformational linkages to the catalytic cleft and the macromolecular substrate binding exosite. Most conformational transitions in thrombin appear to be coupled to the single Na1 ion binding site. This may be attributable to specific salt bridges that connect the autolysis loop residue Glu 146 to Arg 221A and the active site cleft residue Glu 217 to Lys 224, resulting in global coupling to the Na1- binding 220–225 loop (Di Cera et al. 1997). There are no equivalent ionic interactions in VIIa that may account for uncoupling of certain conformation linkages, such as the cofactor effect on the active site and the macromolecular substrate exosite.
Crosstalk Between the Active Site and the Macromolecular Substrate Exosite in VIIa The concept that the conformational linkages in VIIa are discrete and not globally coupled is further supported by the characterization of the effect of active site occupancy on the binding kinetics of VIIa to antibody F3-3.2A (Dickinson et al. 1998). Active site modification decreases the association rate of VIIa with the antibody, an effect that is qualitatively different from the effect of TF binding to VIIa that accelerates the dissociation from the antibody. TF binding and active site inhibition similarly lower the susceptibility of the amino-terminal Ile 16 to chemical modification (Higashi et al. 1996), which may suggest that the conformational changes induced by each interaction are similar. Furthermore, active site modification of VIIa increases the affinity for TF, indicating that the same conformation of VIIa is preferred for TF binding and the interaction of acTCM Vol. 8, No. 8, 1998
tive site inhibitors. The response of the antibody epitope, however, demonstrates that the conformational changes in the macromolecular substrate exosite following TF or inhibitor binding to VIIa are not identical, providing clear evidence for an uncoupling of these conformational linkages in the VIIa protease domain. The linkage from the macromolecular substrate exosite to the active site is reciprocal, as demonstrated by the following two lines of evidence. First, the amidolytic function of VIIa is inhibited by antibody F3-3.2A to the macromolecular substrate exosite of the VIIa protease domain (Dickinson et al. 1998). Because the epitope of this antibody is localized far from the active site cleft and also from the cofactor binding site (Figure 1A), the inhibition of small substrate cleavage by this antibody must be an allosteric effect, rather than direct competition with cofactor or substrate. Second, the association of TF pathway inhibitor (TFPI) to the active site of VIIa is greatly enhanced by the simultaneous docking of factor Xa. This is not simply a facilitated presentation of the inhibitor by factor Xa but, rather, involves conformational changes. The mutant at Arg 148 that is located adjacent to the 192 position at the edge of the catalytic cleft (Figure 4) is inhibited slower by free TFPI, but the rate of inhibition is indistinguishable from wild-type VIIa when factor Xa is present (Rao and Ruf 1995). These data are consistent with the notion that docking of factor Xa changes the catalytic cleft environment to which the inhibitor binds. These data thus reiterate the finding in the analysis of thrombin that demonstrate a profound effect of exosite interactions on the function of the catalytic cleft. Binding of thrombomodulin or the fibrinogen-like sequence of hirudin to the macromolecular substrate exosite I in thrombin influences the active site environment (Ye et al. 1991, Liu et al. 1991), in particular residue 192, which is a key residue to allosterically regulate the substrate specificity of thrombin’s active site (Le Bonniec and Esmon 1991, Vijayalakshmi et al. 1994). Exosite docking is a major determinant for substrate specificity in the prothrombinase complex, and these exosite interactions appear to precede scissile bond docking and cleavage (Krishnaswamy and Betz TCM Vol. 8, No. 8, 1998
1997). The conformational flexibility of the macromolecular substrate docking site in VIIa may similarly be of critical importance to orchestrate the sequential steps of substrate docking with, and product release from, the TF–VIIa complex. • Perspective This review emphasized the importance of conformational flexibility for serine protease function, an aspect that cannot be readily resolved by the snapshots of selected conformational states, as provided by crystallographic structure solutions. Regions with increased flexibility, such as the loop segments of the activation domain (Figures 3 and 4) (Huber and Bode 1978), appear to be preferred for the allosteric regulation of serine protease domains. It is noteworthy that most segments of the activation domain are largely buried, with the notable exception of the autolysis loop that indeed may be more appropriately classified as a flexible segment independent of the activation domain (Vijayalakshmi et al. 1994). The importance of linkage through the more buried or more surface-exposed segments can be addressed by mutagenesis experiments that will continue to clarify the general rules of function for the serine protease family.
• Acknowledgments This work was supported by National Institute of Health grants HL-16411 and HL-48752 and performed during the tenure of an Established Investigator Award from the American Heart Association to W.R.
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the protease domain is essential for interaction with tissue factor. J Biol Chem 272:25,724–25,730. Huang M, Syed R, Stura EA, et al.: 1998. The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF-5G9 complex. J Mol Biol 275:873–894. Huang QL, Neuenschwander PF, Rezaie AR, Morrissey JH: 1996. Substrate recognition by tissue factor-factor VIIa—evidence for interaction of residues Lys165 and Lys166 of tissue factor with the 4-carboxyglutamaterich domain of factor X. J Biol Chem 271:21,752–21,757. Huber R, Bode W: 1978. Structural basis of the activation and action of trypsin. Acc Chem Res 11:114–122. Kelly CR, Schullek JR, Ruf W, Edgington TS: 1996. Tissue factor residue Asp44 regulates catalytic function of the bound protease factor VIIa. Biochem J 315:145–151. Kraulis PJ: 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24:946– 950. Krishnaswamy S, Betz A: 1997. Exosites determine macromolecular substrate recognition by prothrombinase. Biochemistry 36:12,080–12,086. Le Bonniec BF, Esmon CT: 1991. Glu-192– .Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. Proc Natl Acad Sci USA 88:7371–7375. Liu L-W, Vu T-KH, Esmon CT, Coughlin SR: 1991. The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J Biol Chem 266:16,977–16,980. Matsushita T, Kojima T, Emi N, Takahashi I, Saito H: 1994. Impaired human tissue factor-
mediated activity in blood clotting factor VIINagoya (Arg304–.Trp). Evidence that a region in the catalytic domain of factor VII is important for the association with tissue factor. J Biol Chem 269:7355–7363. Merritt EA, Murphy MEP: 1994. RASTER3D version 2.0: a program for photorealistic molecular graphics. Acta Cryst D50:869–873. O’Brien DP, Gale, KM, Anderson, JS, et al.: 1991. Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male. Blood 78:132–140. Payne MA, Neuenschwander PF, Johnson AE, Morrissey JH: 1996. Effect of soluble tissue factor on the kinetic mechanism of factor VIIa: Enhancement of p-guanidinobenzoate substrate hydrolysis. Biochemistry 35:7100–7106. Rao LVM, Ruf W: 1995. Tissue factor residues Lys165 and Lys166 are essential for rapid formation of the quaternary complex of tissue factor-VIIa with Xa-tissue factor pathway inhibitor. Biochemistry 34:10,867– 10,871. Ruf W: 1994. Factor VIIa residue Arg290 is required for efficient activation of the macromolecular substrate factor X. Biochemistry 33:11,631–11,636. Ruf W, Edgington TS: 1991. An anti-tissue factor monoclonal antibody which inhibits TF: VIIa complex is a potent anticoagulant in plasma. Thromb Haemost 66:529–533. Ruf W, Edgington TS: 1994. Structural biology of tissue factor, the initiator of thrombogenesis in vivo. FASEB J 8:385–390. Ruf W, Mueller BM: 1996. Tissue factor in cancer angiogenesis and metastasis. Curr Opin Hematol 3:379-384. Ruf W, Kalnik MW, Lund-Hansen T, Edgington TS: 1991a. Characterization of factor VII association with tissue factor in solu-
tion. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions. J Biol Chem 266: 15,719–15,725. Ruf W, Rehemtulla A, Morrissey JH, Edgington TS: 1991b. Phospholipid independent and dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem 266:2158–2166. Ruf W, Miles DJ, Rehemtulla A, Edgington TS: 1992. Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor–factor VIIa protease complex. J Biol Chem 267:6375–6381. Schechter I, Berger A: 1967. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27:157–162. Vijayalakshmi J, Padmanabhan KP, Mann KG, Tulinsky A. 1994. The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin. Protein Sci 3:2254–2271. Walker RK, Krishnaswamy S: 1993. The influence of factor Va on the active site of factor Xa. J Biol Chem 268:13,920–13,929. Watzke HH, Lechner, K, Roberts, HR, et al.: 1990. Molecular defect (Gla114➝Lys) and its functional consequences in a hereditary factor X deficiency (factor X “Vorarlberg”). J Biol Chem 265:11,982–11,989. Ye J, Esmon NL, Esmon CT, Johnson AE: 1991. The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation. J Biol Chem 266: 23,016–23,021.
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thrombin receptor interactions. Nature 353: 674–677. Wells CM, Di Cera E: 1992. Thrombin is a Na1-activated enzyme. Biochemistry 31: 11,721–11,730. Wu Q, Sheehan JP, Tsiang M, Lentz SR, Birktoft JJ, Sadler JE: 1991. Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin. Proc Natl Acad Sci USA 88:6775–6779.
Yan SB, Grinnell BW: 1994. Recombinant human protein C, protein S and thrombomodulin as antithrombotics. Perspect Drug Disc Des 1:503–520. Zhang E, Tulinksy A: 1997. The molecular environment of the Na1 binding site of thrombin. Biophys Chem 63:185–200.
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Allosteric Regulation of the CofactorDependent Serine Protease Coagulation Factor VIIa Wolfram Ruf* and Craig D. Dickinson
The integration of structure and function analysis of the tissue factor– factor VIIa complex has provided a detailed view of the functional surface of the extrinsic activation complex. An incomplete zymogen to enzyme transition is responsible for the strict cofactor dependence of catalytic function of factor VIIa. The mutational analysis demonstrates that factor VIIa is allosterically regulated by specific conformational linkages that involve the cofactor binding site, the catalytic cleft, and the macromolecular substrate exosite. Regions of the flexible activation domain appear to play an important role in the allosteric regulation of this cofactor-dependent coagulation serine protease. (Trends Cardiovasc Med 1998;8:350–356) © 1998, Elsevier Science Inc.
The coagulation pathways regulate essential cellular functions in hemostasis, wound healing, and immune response, as well as play important pathophysiologic roles in cancer metastasis, inflammation, and thrombosis (Ruf and Edgington 1994, Ruf and Mueller 1996). Central to the assembly of the coagulaWolfram Ruf and Craig D. Dickinson are at the Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California. * Address correspondence to: Wolfram Ruf, MD, Departments of Immunology and Vascular Biology, IMM-17, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. ©1998 Elsevier Science Inc. All rights reserved. 1050-1738/98/$–see front matter
tion cascade are macromolecular catalytic cofactors that direct the serine proteases to cell surfaces, the preferred location for the interaction with the downstream macromolecular substrates. Following the first structure determination of a coagulation serine protease, that is, thrombin (Bode et al. 1989), the last decade has seen a rapid progress in the structural knowledge of this homologous class of enzymes. Another major breakthrough was the recent crystallization of tissue factor (TF) in complex with factor VIIa (VIIa). The TF–VIIa complex has provided the structure solution of the first enzyme–cofactor complex of the coagulation cascade (Banner et al. 1996). We review here recent progress from functional and mutational analysis that TCM Vol. 8, No. 8, 1998
has elucidated the details of function and allosteric regulation of the serine protease VIIa. The interpretation of these functional studies in the context of the TF–VIIa structure may provide a more general paradigm for coagulation serine proteases that depend on assembly with a cell-associated cofactor. VIIa is a modular enzyme consisting of a trypsinlike protease domain and an amino terminal light chain with a gcarboxyglutamic acid-rich domain and two epidermal growth factor (EGF)–like modules. The crystal structure of the TF– VIIa complex demonstrated that each of
the structural modules of VIIa makes extensive contacts with the cofactor TF. TF is a member of the cytokine receptor family with two typical tightly associated b-strand, fibronectin type III repeat modules (Figure 1A). The Gla-domain of VIIa binds to the carboxyl-terminal module of TF in a position that would allow the interaction of the hydrophobic membrane binding portion of this domain with the cell surface. The interface of the Gla-domain with TF is hydrophobic, involving one face of the aromatic stack helix of VIIa and an unusual surface exposed disulfide bond in the car-
boxyl-terminal cytokine module of TF. The EGF-1 domain packs into a groove formed by the two modules of TF. This interface is the largest and energetically most important of the contacts, providing the anchor for tight binding of the protease with cofactor. The EGF-2 and protease domain form an extended interface with the amino-terminal module of TF. This interaction makes little contributions to the free energy of binding, but rather, serves the allosteric regulation of the VIIa protease domain, as discussed in detail later here. The structure of the TF–VIIa complex thus defined the
Figure 1. Overview of the tissue factor (TF)–VIIa structure. (A) shows the domain organization of the TF–VIIa complex with TF’s amino terminal (N) and carboxyl terminal (C) module shown in maroon and VIIa’s protease domain, EGF-1 and Gla shown in gray. The epidermal growth factor (EGF)-2 domain is located behind the protease domain and is not visible in this projection. The active site inhibitor D-Phe-L-Phe-Arg chloromethylketone (blue) is visible below catalytic triad residues His 57 and Ser 195 (red). Epitopes of antibodies that are competitive with factor X binding are highlighted. These antibodies are directed to the TF carboxyl module ( green: 5G9 epitope residues Tyr 156, Lys 166, Lys 169, Arg 200, Lys 201) (Huang et al. 1998) or to the VIIa protease domain ( yellow: F3-3.2A epitope residues Lys 20, Val 21, Lys 24, Glu 75, Met 156) (Dickinson et al. 1998). (B) gives an overview of residue side chains that are important for factor X activation, based on mutagenesis of TF (green: Lys 165, Lys 166) (Ruf et al. 1992) or VIIa ( yellow: Asp 72, Glu 75, Arg 148, Glu 154, Met 156, Asp 185B) (Dickinson et al. 1996). The amino terminus of the VIIa protease domain and catalytic triad residues His 57 and Ser 195 are red, the active site inhibitor in the TF–VIIa complex structure (Banner et al. 1996) is blue. Illustrations were created using the programs Molscript (Kraulis 1991), RASTER 3D (Merritt and Murphy 1994), and MIDAS-plus (Ferrin et al. 1988).
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molecular details of the TF–VIIa interaction and, furthermore, facilitated the interpretation of studies that delineated the regions of the complex that are involved in recognition of macromolecular substrates, in particular factor X. • The Macromolecular Substrate Docking Site of the Enzyme-Cofactor Complex It is well appreciated for thrombin, the ultimate protease of the coagulation cascade, that macromolecular substrate recognition involves both determinants in the catalytic cleft and within exosite I, a basic region in the protease domain that interacts with fibrinogen and the carboxyl-terminus of hirudin (Bode et al. 1992). The analysis of factor X activation by TF–VIIa suggests that extended recognition of macromolecular substrate by this cofactor-dependent protease is not restricted to the enzyme’s catalytic domain but involves the VIIa light chain and specific regions of TF as well. Concordant results from antibody mapping and mutagenesis suggest a mode of docking of macromolecular substrate with TF–VIIa. We have identified monoclonal antibodies to TF (Ruf and Edgington 1991) and the VIIa protease domain (Dickinson et al. 1998) that act as competitive inhibitors of factor X activation. The mapping of the antibody epitopes (Huang et al. 1998, Dickinson et al. 1998) indicates that steric interference with factor X docking must occur at one extended face of the TF– VIIa complex that is oriented toward the viewer in the projection of Figure 1A. Mutagenesis has further identified surface exposed residues that contribute to substrate activation on the same face of the complex (Figure 1B). First, basic residues in the TF carboxyl module, in particular Lys 165 and Lys 166 (Ruf et al. 1992), likely cooperate with the Gladomain of VIIa (Ruf et al. 1991a) in the recognition of factor X’s Gla-domain (Watzke et al. 1990, Huang et al. 1996). It is conceivable that the factor X Gladomain is docked with TF in an orientation similar to the VIIa Gla-domain, allowing membrane binding as well as interaction with the Gla-domain of VIIa. Mutagenesis has also identified residues in the VIIa protease domain that contribute to factor X activation (Dickinson et al. 1996). This cluster of mostly
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acidic residue is found on the same face of the TF–VIIa complex, facing the viewer in Figure 1B. Most of these residues, that is, Asp 72, Glu 75, Glu 154, are within the epitope of anti-VII antibody F3-3.2A mentioned earlier and in a position corresponding to thrombin’s exosite I. [Numbering of the residue positions of the VIIa protease domain is based on the chymotrypsin numbering scheme (Dickinson et al. 1996).] This site must be involved in interaction with the protease domain of substrate factor X, based on positional constraints by docking factor X’s scissile bond into the active site (Dickinson et al. 1996). The lower rim of the active site cleft makes also contributions to macromolecular substrate activation, in particular the basic Arg 148 (Ruf 1994) that is located in a surface loop referred to as the gautolysis loop in thrombin. This loop is generally flexible and often protease sensitive, and it contributes to efficient activation of macromolecular substrate, as demonstrated by the reduced function of g-thrombin. The available data thus emphasize the importance of extended recognition determinants for substrate specificity as a recurring theme in cofactor-dependent serine proteases. • A Unique Labile Zymogen to Enzyme Transition of VIIa The presence of cofactor markedly accelerates macromolecular substrate activation for each example of enzyme–cofactor complexes of the coagulation cascade. The amidolytic activities of the homologous proteases factor Xa, IXa, and VIIa, however, are influenced by cofactor interactions in specific ways: Factor IXa has very low amidolytic activity that is not enhanced by cofactor interactions (Castillo et al. 1983), whereas factor Xa, independently of cofactor, displays high activity toward small substrate mimics (Walker and Krishnaswamy 1993). VIIa has also very low catalytic activity toward peptidyl substrates, but the rate of hydrolysis is enhanced .10-fold by binding of cofactor (Ruf et al. 1991b). The precise structural basis for the low catalytic activity of free VIIa is unknown. Following zymogen activation, the amino terminus of most serine proteases moves .10Å to form a salt bridge with Asp 194 (Vijayalakshmi et al. 1994). These structural rearrangements of the
primary specificity site are necessary for catalytic activity and also insert the amino-terminal Ile 16 into the protein interior, where Ile 16’s a-amino group becomes protected from chemical modification. The amino terminal Ile 16 of the VIIa protease domain is susceptible to chemical modification, indicating solvent accessibility and a labile enzyme conformation of free VIIa. Binding of TF protects the amino terminus from chemical modification, consistent with stabilized insertion of the amino terminus (Higashi et al. 1994). The amino terminus is part of the activation domain, a larger region of increased flexibility or of alternative conformations in serine protease zymogens in comparison with the active enzymes (Huber and Bode 1978). In principle, free VIIa thus appears to make an incomplete zymogento-enzyme transition, and the cofactor TF is required to switch and maintain the protease domain in a catalytically active conformation. The following sections discuss results that demonstrate that the conformational transitions of VIIa involve discrete thermodynamic linkages that can be assigned to specific residue side chains in the VIIa protease domain. These studies suggest that the regulation of VIIa cannot be reduced to a simple global conformational transition, but rather that the inherent flexibility of the activation domain is utilized in this cofactor-dependent serine protease to allosterically influence catalytic function. • Specific Conformational Linkages in the VIIa Protease Domain Three interdependent regions of the VIIa protease domain are relevant for function: the cofactor binding site, the catalytic cleft, and the macromolecular substrate exosite (Figure 2). The cofactor binding site must transmit the allosteric changes by which TF influences VIIa’s catalytic function and the interface of VIIa with TF contributes to the affinity of enzyme–cofactor binding. The conformational state of the catalytic center can be monitored with small peptidyl substrates that provide adequate mimics for the active site docking of the scissile bond, in particular the P1 residue (Schechter and Berger 1967). The active site can further be probed by tight binding or covalent inhibitors that lock TCM Vol. 8, No. 8, 1998
Figure 2. Overview of known conformational linkages in the VIIa protease domain.
the protease domain into a conformation with restricted flexibility (Bode et al. 1978). The macromolecular substrate exosite is a major contributor to substrate specificity of these enzymes. Alterations in this region can be measured by changes in the kinetics of macromolecular substrate activation. Conformational changes in this exosite can also be detected by a unique monoclonal antibody with a conformation sensitive epitope that overlaps with the factor X docking site. Site-directed mutants of individual amino acid side chains in the VIIa protease domain have defined the following specific linkages between these functional regions of the VIIa protease domain.
Ruf 1997). Among the mutants in VIIa’s interface with TF, only the Met 164 replacement fails to dissociate slower from cofactor following inhibitor binding, establishing the crucial role of this residue in the thermodynamic coupling to the active site. The effect of interactions of Met 164
to the catalytic cleft may be transmitted through buried loop segments that are adjacent to the TF binding interface (the TF amino-terminal module is to the left in Figure 3). Importance of these buried segments is demonstrated by the loss of cofactor binding following selective reduction of the 168–182 disulfide bond (visible below Met 164 in Figure 3) that links the Met 164 helix to the 182–189 loop (Higashi et al. 1997). Moreover, Ala scanning mutations of residues in this loop (Dickinson et al. 1996), as well as hereditary mutations in adjacent buried positions (Bharadwaj et al. 1996, Matsushita et al. 1994, O’Brien et al. 1991), influence the catalytic function of VIIa. The 182–189 loop plays also an important role in the allosteric regulation of thrombin. This loop participates together with the 220–225 loop in the formation of a single Na1 ion binding site. Na1 binding is critical for the allosteric switch from the slow, anticoagulant conformer of the enzyme to the fast, procoagulant form (Di Cera et al. 1995). An aromatic amino acid at position 225
Allosteric Activation of VII’s Catalytic Function by Cofactor Binding Mutations in the interface of the protease domain of VIIa with TF influence the catalytic function of the TF–VIIa complex. The Ala replacement for Met 164 results in a concordant reduction in proteolytic and amidolytic function (Dickinson et al. 1996), indicating that the interaction at this residue position is directly linked to the catalytic cleft to which the small peptidyl substrates dock. The direct interdependence of the active site with this specific residue position in the interface with TF is further reinforced by analysis of the effect of active site occupancy on TF binding. Binding of covalent or tight binding inhibitors to the active site of VIIa increases the affinity for TF by significantly decreasing the dissociation rate of the enzyme from cofactor (Dickinson and TCM Vol. 8, No. 8, 1998
Figure 3. A potential conformational link from Met 164 to the active site of VIIa may occur through the Cys 168-Cys 182 disulfide bond into the 182-189 loop to Asp 189, the S1 subsite specificity determinant. Phe 225 is a signature aromatic residue for serine proteases that are allosterically regulated by sodium ion binding. The 182–189 loop is part of the activation domain, other loop segments in this flexible region (16–19, 142–152, 182–194, 216–223) are highlighted by shading.
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(Figure 3) is the hallmark for this class of allosterically regulated serine proteases to which VIIa also belongs (Dang and Di Cera 1996). We propose that cofactor interactions utilize the inherent flexibility of this segment of the activation domain (these loops are shaded dark in Figure 3) to allosterically regulate the catalytic activity of VIIa. Subtle changes in the conformation of the 182– 189 loop may influence the position of Asp 189 that is the specificity-determining residue at the bottom of the S1 binding pocket and may thus account for the cofactor effect on amidolytic function of VIIa. Consistently, the cofactor was found to facilitate small substrate binding, rather than to accelerate the deacylation step during substrate hydrolysis (Payne et al. 1996). Specific Influence of the Cofactor on Macromolecular Substrate Activation Mutations of TF residue Asp 44 in the interface with the VIIa protease (Figure 4) have only subtle effects on amidolytic function of VIIa, but factor X activation is severely reduced (Kelly et al. 1996). A similar specific effect on macromolecular substrate recognition followed mutation of residue Arg 134 in the corresponding interface in the VIIa protease domain (Dickinson et al. 1996), indicating that specific residues in the TF–VIIa interface are conformationally linked to the macromolecular substrate binding site. An attractive hypothesis is that the TF-mediated stabilization of the aminoterminal insertion (visible below the Arg 148 in Figure 4) also orders specific surface exposed side chains for optimal interaction with macromolecular substrate. Certain residues, that is, Leu 144, Glu 154, and Met 156 (visible to the right of the amino terminus in Figure 4), flank the amino-terminal insertion site and are indeed important for proteolytic function of VIIa (Dickinson et al. 1996). These residues also form the base of the flexible autolysis loop (colored dark in Figure 4) and may influence residues critical for macromolecular substrate recognition, such as Arg 148 (Ruf 1994). Moreover, Glu 154 and Met 156 are located within the epitope boundaries of monoclonal antibody F3-3.2A (for epitope location, see Figure 1), which reports a change in affinity for VIIa when TF is bound to the protease domain of VIIa
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Figure 4. A potential link from tissue factor (TF) to regions for macromolecular substrate interaction may involve a critical contact is the TF–VIIa interface marked by TF Asp 44 and VIIa Arg 134. A key residue that is specific for factor X activation is Arg 148, located in front of Lys 192, which contributes to formation of the S1 subsite. Arg 148 is part of the autolysis loop (residues 142–152, shaded) that was originally assigned to the activation domain. Residues at the base of the autolysis loop, that is, Leu 144, Glu 154, and Met 156, surround the amino-terminal insertion and are important for proteolytic function.
(Dickinson et al. 1998). This influence of cofactor on the antibody epitope conformation and the cofactor effect on factor X activation thus indicate a linkage from the TF binding site to the macromolecular substrate exosite in the VIIa protease domain. The cofactor binding site in VIIa thus makes distinct conformational linkages to the catalytic cleft and the macromolecular substrate binding exosite. Most conformational transitions in thrombin appear to be coupled to the single Na1 ion binding site. This may be attributable to specific salt bridges that connect the autolysis loop residue Glu 146 to Arg 221A and the active site cleft residue Glu 217 to Lys 224, resulting in global coupling to the Na1- binding 220–225 loop (Di Cera et al. 1997). There are no equivalent ionic interactions in VIIa that may account for uncoupling of certain conformation linkages, such as the cofactor effect on the active site and the macromolecular substrate exosite.
Crosstalk Between the Active Site and the Macromolecular Substrate Exosite in VIIa The concept that the conformational linkages in VIIa are discrete and not globally coupled is further supported by the characterization of the effect of active site occupancy on the binding kinetics of VIIa to antibody F3-3.2A (Dickinson et al. 1998). Active site modification decreases the association rate of VIIa with the antibody, an effect that is qualitatively different from the effect of TF binding to VIIa that accelerates the dissociation from the antibody. TF binding and active site inhibition similarly lower the susceptibility of the amino-terminal Ile 16 to chemical modification (Higashi et al. 1996), which may suggest that the conformational changes induced by each interaction are similar. Furthermore, active site modification of VIIa increases the affinity for TF, indicating that the same conformation of VIIa is preferred for TF binding and the interaction of acTCM Vol. 8, No. 8, 1998
tive site inhibitors. The response of the antibody epitope, however, demonstrates that the conformational changes in the macromolecular substrate exosite following TF or inhibitor binding to VIIa are not identical, providing clear evidence for an uncoupling of these conformational linkages in the VIIa protease domain. The linkage from the macromolecular substrate exosite to the active site is reciprocal, as demonstrated by the following two lines of evidence. First, the amidolytic function of VIIa is inhibited by antibody F3-3.2A to the macromolecular substrate exosite of the VIIa protease domain (Dickinson et al. 1998). Because the epitope of this antibody is localized far from the active site cleft and also from the cofactor binding site (Figure 1A), the inhibition of small substrate cleavage by this antibody must be an allosteric effect, rather than direct competition with cofactor or substrate. Second, the association of TF pathway inhibitor (TFPI) to the active site of VIIa is greatly enhanced by the simultaneous docking of factor Xa. This is not simply a facilitated presentation of the inhibitor by factor Xa but, rather, involves conformational changes. The mutant at Arg 148 that is located adjacent to the 192 position at the edge of the catalytic cleft (Figure 4) is inhibited slower by free TFPI, but the rate of inhibition is indistinguishable from wild-type VIIa when factor Xa is present (Rao and Ruf 1995). These data are consistent with the notion that docking of factor Xa changes the catalytic cleft environment to which the inhibitor binds. These data thus reiterate the finding in the analysis of thrombin that demonstrate a profound effect of exosite interactions on the function of the catalytic cleft. Binding of thrombomodulin or the fibrinogen-like sequence of hirudin to the macromolecular substrate exosite I in thrombin influences the active site environment (Ye et al. 1991, Liu et al. 1991), in particular residue 192, which is a key residue to allosterically regulate the substrate specificity of thrombin’s active site (Le Bonniec and Esmon 1991, Vijayalakshmi et al. 1994). Exosite docking is a major determinant for substrate specificity in the prothrombinase complex, and these exosite interactions appear to precede scissile bond docking and cleavage (Krishnaswamy and Betz TCM Vol. 8, No. 8, 1998
1997). The conformational flexibility of the macromolecular substrate docking site in VIIa may similarly be of critical importance to orchestrate the sequential steps of substrate docking with, and product release from, the TF–VIIa complex. • Perspective This review emphasized the importance of conformational flexibility for serine protease function, an aspect that cannot be readily resolved by the snapshots of selected conformational states, as provided by crystallographic structure solutions. Regions with increased flexibility, such as the loop segments of the activation domain (Figures 3 and 4) (Huber and Bode 1978), appear to be preferred for the allosteric regulation of serine protease domains. It is noteworthy that most segments of the activation domain are largely buried, with the notable exception of the autolysis loop that indeed may be more appropriately classified as a flexible segment independent of the activation domain (Vijayalakshmi et al. 1994). The importance of linkage through the more buried or more surface-exposed segments can be addressed by mutagenesis experiments that will continue to clarify the general rules of function for the serine protease family.
• Acknowledgments This work was supported by National Institute of Health grants HL-16411 and HL-48752 and performed during the tenure of an Established Investigator Award from the American Heart Association to W.R.
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