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X-ray structure of antistasin at 1.9Aresolution and its modelled complex with blood coagulation factorXa

1997; Springer Nature; Volume: 16; Issue: 17 Linguagem: Inglês

10.1093/emboj/16.17.5151

1460-2075

Risto Lapatto,

Biochemical and Molecular Research

Article1 September 1997free access X-ray structure of antistasin at 1.9 Å resolution and its modelled complex with blood coagulation factor Xa Risto Lapatto Risto Lapatto Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Ute Krengel Ute Krengel Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Herman A. Schreuder Herman A. Schreuder Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Anita Arkema Anita Arkema Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Bijtske de Boer Bijtske de Boer Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Kor H. Kalk Kor H. Kalk Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Wim G.J. Hol Wim G.J. Hol Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Peter D.J. Grootenhuis Peter D.J. Grootenhuis Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author John W.M. Mulders John W.M. Mulders Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Rein Dijkema Rein Dijkema Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Henri J.M. Theunissen Henri J.M. Theunissen Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Bauke W. Dijkstra Corresponding Author Bauke W. Dijkstra Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Risto Lapatto Risto Lapatto Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Ute Krengel Ute Krengel Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Herman A. Schreuder Herman A. Schreuder Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Anita Arkema Anita Arkema Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Bijtske de Boer Bijtske de Boer Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Kor H. Kalk Kor H. Kalk Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Wim G.J. Hol Wim G.J. Hol Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Peter D.J. Grootenhuis Peter D.J. Grootenhuis Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author John W.M. Mulders John W.M. Mulders Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Rein Dijkema Rein Dijkema Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Henri J.M. Theunissen Henri J.M. Theunissen Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Bauke W. Dijkstra Corresponding Author Bauke W. Dijkstra Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands Search for more papers by this author Author Information Risto Lapatto1,2, Ute Krengel1, Herman A. Schreuder1,3, Anita Arkema1, Bijtske de Boer1, Kor H. Kalk1, Wim G.J. Hol1,4, Peter D.J. Grootenhuis5, John W.M. Mulders5, Rein Dijkema5, Henri J.M. Theunissen5 and Bauke W. Dijkstra 1,5 1Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands 2Children's Hospital and Institute of Biomedicine, University of Helsinki, 00014 Helsinki, Finland 3Hoechst Aktiengesellschaft, 65926 Frankfurt am Main, Germany 4Department of Biological Structure and Howard Hughes Medical Institute, University of Washington, Box 357742, Seattle, WA, 98195 USA 5Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands The EMBO Journal (1997)16:5151-5161https://doi.org/10.1093/emboj/16.17.5151 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Å resolution by X-ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two β-strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat. Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain. Introduction Blood coagulation proceeds through the sequential activation of a number of plasma serine proteases, ultimately resulting in the formation of fibrin, an insoluble protein that is a major component of blood clots. Coagulation factor Xa plays an important role in this cascade of events, since it converts the zymogen prothrombin into thrombin, the enzyme that catalyses the formation of fibrin from fibrinogen. Furthermore, factor Xa cleaves and hence activates other components of the coagulation cascade, including factors V, VII, VIII and IX (see Padmanabhan et al., 1993 for references). Inhibitors of factor Xa might be used as drugs for the treatment and prevention of thrombosis, a pathological condition in which blood coagulation occurs in an uncontrolled manner (Mao, 1993). It has been reported that antistasin, a potent reversible inhibitor of factor Xa, displays antithrombotic activity in various in vivo models of venous and arterial thrombosis (Nutt et al., 1991; Vlasuk et al., 1991; Dunwiddie et al., 1992a; Mellott et al., 1992; Hauptmann and Kaiser, 1993). Apart from its anticoagulant activity, antistasin is able to suppress metastasis, possibly also via inhibition of factor Xa (Tuszinsky et al., 1987). It is this antimetastatic property from which antistasin obtained its name. Originally, antistasin was isolated from the salivary glands of the Mexican leech, Haementeria officinalis, but since then, it has been cloned into eukaryotic expression vectors and overexpressed (Han et al., 1989; Nutt et al., 1991; Theunissen et al., 1994). The purified protein has an inhibition constant for factor Xa of approximately 5×10−10 M and displays competitive, slow-tight binding inhibition characteristics (Dunwiddie et al., 1989). Antistasin is rather selective for factor Xa: it hardly inhibits other serine proteases such as thrombin, chymotrypsin, pancreatic elastase and leukocyte elastase (Dunwiddie et al., 1989). Antistasin is a small, disulfide cross-linked protein of 119 amino acid residues (Mr = 15 kDa). Figure 1 shows a comparison of its amino acid sequence with the sequences of the related proteinase inhibitors ghilanten and hirustasin. Ghilanten is a factor Xa inhibitor isolated from the salivary glands of the Amazonian leech, Haementeria ghiliani (Condra et al., 1989; Blankenship et al., 1990; Brankamp et al., 1990); hirustasin was identified in the salivary glands of the European medicinal leech, Hirudo medicinalis (Süllner et al., 1994). The latter protein is an inhibitor of tissue kallikrein, trypsin and chymotrypsin, but it does not inhibit factor Xa. Figure 1.Amino acid sequences of antistasin-type inhibitors. The sequences of antistasin (Nutt et al., 1988; Han et al., 1989; Theunissen et al., 1994), ghilanten (Blankenship et al., 1990) and hirustasin (Süllner et al., 1994) are aligned. For antistasin, the sequence of the recombinant protein, of which the structure is presently discussed, is given in the upper line. Underneath, amino acid substitutions with respect to this sequence are indicated for ghilanten and antistasin isoforms. Cysteines are highlighted by asterisks; a dash indicates a gap in the protein sequence. The position of the reactive site arginine is marked by an arrow. Download figure Download PowerPoint Alignment of the 20 cysteines present in antistasin reveals a 2-fold internal repeat, suggesting that the protein has evolved via gene duplication (Nutt et al., 1988). The two repeats are very similar: the N-terminal domain (amino acid residues 1-55) and the C-terminal domain (amino acid residues 56-119) display ∼40% identity and ∼56% homology in their amino acid sequences (Dunwiddie et al., 1989). Between cysteines 82 and 88 in the C-terminal domain, there is one insertion of a single amino acid residue compared with the corresponding cysteines in the N-terminal domain. Furthermore, the C-terminal domain has a nine-residue extension with four positively charged amino acids. Mutation studies have shown that only the N-terminal domain of antistasin is necessary for the inhibition of factor Xa; the C-terminal domain does not contribute to its inhibitory activity (O'Neill Palladino et al., 1991; Theunissen et al., 1994). The reactive site of antistasin is formed by Arg34 (the P1-residue) and Val35 (the P1′-residue) in the N-terminal domain (Dunwiddie et al., 1989). Factor Xa slowly cleaves the peptide bond between these residues. The C-terminal residues equivalent to Arg34 and Val35 are Arg89 and Lys90, respectively. A Lys90→Val substitution, however, did not restore the inhibitory activity towards factor Xa (Hofmann et al., 1992; Theunissen et al., 1994), indicating that there are other factors causing the inactivity of the C-terminal domain. To investigate the mechanism of action of antistasin and the atomic details of its interaction with factor Xa, we initiated the crystal structure determination of antistasin. A preliminary crystallographic analysis has been published previously (Schreuder et al., 1993). Here, we report the 3D-structure of antistasin at 1.9 Å resolution and suggest a model of the antistasin-factor Xa complex, based on the antistasin and factor Xa crystal structures. Our structural results indicate that antistasin uses reactive site as well as exosite interactions to bind to factor Xa. Results Electron density map and quality of the model Table I summarizes the final results of the crystallographic refinement of antistasin. Two antistasin structures have been determined, one at room temperature (2.3 Å resolution) and one at 100 K to a resolution of 1.9 Å. A superposition of the two structures revealed root mean square (r.m.s.) differences for Cαs of 0.28 Å, a value close to the coordinate error of the two structures as determined from a Luzzati plot (not shown). The crystallographic R-factors are 19.4% and 21.7%, respectively, for the 2.3 Å and 1.9 Å structures. In both cases, the final model consists of amino acid residues 7-110. For the six N-terminal and the nine C-terminal residues, the density is too weak to define their positions with confidence. Residues 9-11, 94 and some side chains developed temperature factors of ∼50 Å2 and higher during refinement. These residues are disordered in the crystal structure. Several other amino acids, for example Phe50, Glu56, Ser65, Met71 and Arg75, may adopt two different conformations of which only the most prominent has been modelled. The overall geometry of both antistasin structures is good, as can be judged from the r.m.s. deviations from ideal geometry given in Table I and from the absence of outliers in the Ramachandran plot (not shown). The electron density map (Figure 2) is in good agreement with the published sequence and, in addition, establishes the so far unknown disulfide bond connectivities. Figure 2.Representative part of the σA-weighted (Read, 1986) (2Fo-Fc) OMIT map (Bhat, 1988; Vellieux and Dijkstra, 1997), centred at Arg34 and contoured at 1σ, showing the hydrophobic interaction network which stabilizes the reactive site loop of antistasin. Cysteines 33 and 51 and cysteines 37 and 53 are connected by disulfide bonds, respectively. The density for the side chain of Arg32 is somewhat weak. This residue is positioned close to a crystallographic 2-fold axis and has rather high B-values. Download figure Download PowerPoint Table 1. Refinement statistics Temperature (K) 293 100 Resolution range (Å) 5.0−2.3 5.0−1.9 Reflections used [Fo > 0σ(Fo)] 5711 9103 Final R-factor (%)a 19.4 21.7 Average real-space correlation coefficient (%) σA-weighted (2Fo − Fc) map 0.91 0.91 σA-weighted (2Fo − Fc) OMIT map 0.86 0.87 Residues included 7-110 7-110 Number of non-H atoms Protein 790 790 Non-protein 62 89 R.m.s. deviations from ideality for bond lengths (Å) 0.012 0.015 R.m.s. deviations from ideality for bond angles (°) 2.53 1.49 Secondary structure analysis according to PROCHECK (Laskowski et al., 1993) Residues in most favoured regions (%) 87.6 87.6 Residues in additional allowed regions (%) 12.4 12.4 Residues in generously allowed regions (%) 0.0 0.0 Residues in disallowed regions (%) 0.0 0.0 Average B-factors (Å2) Protein atoms 29.5 25.5 Solvent 40.3 30.3 Protein Data Bank code 1SKZ a R-factor = (Σ||Fo|−|Fc||/Σ|Fo|). Overall structure Figure 3 shows the 1.9 Å 3D-structure of antistasin as a simplified ribbon model. As could be expected on the basis of the internal sequence homology, antistasin consists of two structurally similar domains, the N-terminal domain (residues 1-55) and the C-terminal domain (residues 56-119), in agreement with the prediction of Nutt et al. (1988). The domains are spatially distant with no inter-domain backbone-backbone interactions and only few side chain-main chain and side chain-side chain interactions. They can further be subdivided into four similarly sized subdomains, two in each domain. The subdomains are linked by two hinge regions, consisting of Val31 and Arg32 in the N-terminal domain, and Asp85, Ile86 and Asn87 in the C-terminal domain. Since the hinge is more open in the C-terminal domain, this domain adopts a flat shape, whereas the N-terminal domain is wedge-shaped (see Figure 4). The difference in linker angle is caused by the insertion of Asp85 and by the special φ,ψ angles of Gly30 (91° and 160°). In the C-terminal domain, the residue corresponding to Gly30 is an isoleucine. However, apart from the differences in the hinge regions, the individual subdomains superimpose very well, with r.m.s. differences of Cα atoms being only 0.60 Å for residues 33-55 and 88-110 and 0.77 Å for residues 13-30 and 67-84. Figure 3.Stereo view of the overall structure of antistasin from H.officinalis [produced with Molscript (Kraulis, 1991)]. The P1 and P1′ residues Arg34 and Val35, as well as Glu15 of the putative exosite binding region of antistasin are indicated. Download figure Download PowerPoint Figure 4.Stereo view showing the superposition of the N- and C-terminal domains of antistasin, based on the Cα coordinates of the second subdomains, respectively [using the program O (Jones et al., 1991); figure produced with Molscript (Kraulis, 1991)]. The N-terminal domain (black) adopts a wedge shape, while the C-terminal domain (grey) is relatively flat. Download figure Download PowerPoint Folding pattern of the domains in antistasin Antistasin appears to exhibit mainly random coil structure (see Figure 3). Although two short antiparallel β-strands can be identified in each of the two domains on the basis of φ,ψ angles (involving residues 41-43 and 49-53 in the N-terminal domain and residues 96-98 and 104-108 in the C-terminal domain, respectively), they lack the appropriate hydrogen bonding interactions and thus do not form real sheets. Also in the rest of the structure, main chain hydrogen bonding interactions are very limited. Instead, side chain contacts dominate the antistasin structure. There are no α-helices present in antistasin, only one single α-helical turn. This turn involves residues 8-11 and is positioned in a region with very high temperature factors. In addition, several reverse turns exist. A remarkable feature of the antistasin structure is the absence of a proper core, which is also reflected by the fact that only 1144 Å2 of the total accessible surface of antistasin (7394 Å2) are buried. Instead, disulfide bridges seem to stabilize the fold. Antistasin contains 20 cysteine residues, 10 in each domain, all of which are involved in disulfide bridges. They are usually flanked by hydrophobic residues forming four small hydrophobic clusters, one in each subdomain, at topologically identical positions. In the N-terminal domain, disulfide bonds are observed between residues 8-19, 13-26, 28-48, 33-51 and 37-53 and in the C-terminal domain between residues 62-73, 67-80, 82-103, 88-106 and 92-108 (see Figure 5). Hence, all the disulfide bridges are within the individual domains, and two of them (involving Cys28/48 and Cys82/103) cross-bridge the subdomains. Figure 5.Schematic representation of the antistasin fold. The orientation of antistasin is similar to the one chosen in Figure 3. Disulfide connectivities and the linker residues connecting the two subdomains within each domain are indicated. The scissile bond is marked by an arrow. β-strands involve amino acid residues 41-43 and 49-53 in the N-terminal domain and residues 96-98 and 104-108 in the C-terminal domain, respectively. Download figure Download PowerPoint The reactive site region The reactive site of antistasin is formed by Arg34 (the P1-residue) and Val35 (the P1′-residue) in the N-terminal domain (Dunwiddie et al., 1989). Arg34 is positioned at the tip of an exposed loop at the protein surface (see Figure 3). Its side chain has adopted an extended conformation. In the crystal, this residue is involved in contacts with a symmetry-related molecule. Val35 is pointing into the opposite direction. Compared with Arg34, the Val35 side chain is considerably less exposed. The reactive site loop is connected to the rest of the protein by two disulfide bonds involving Cys33 and Cys37 and is further stabilized by a network of hydrophobic interactions between these two disulfides and the Val31, Phe41 and Val35 side chains (see Figure 2). As a consequence of the various interactions, the temperature factors in the reactive site region are rather low, with values of ∼15 Å2 for most residues. For Cys33 and Arg34, temperature factors are somewhat higher, with values close to the average temperature factor of antistasin, which is 26 Å2. Temperature factors are highest for Arg32 (up to 50 Å2 for side chain atoms). Thus, although the reactive site loop is highly exposed, it has a well-defined conformation. Only the side chains of Arg32 and Arg34 are somewhat more flexible and may adapt their conformation to fit optimally into the active site of the target protease. In the C-terminal domain of antistasin, the corresponding part of the reactive site (residues 87-92) also forms an exposed loop, positioned at the opposite end of the protein molecule. Its overall structure is very similar to the equivalent N-terminal region, as is reflected by the low r.m.s. differences in Cα positions of 0.43 Å for residues 88-92 compared with residues 33-37. As in the N-terminal domain, an arginine residue (Arg89) is at the position corresponding to P1, and also the network of hydrophobic interactions has a counterpart in the C-terminal domain, however, with Val31, Val35 and Phe41 being replaced by Ile86, the aliphatic part of the Lys90 side chain and Leu96, respectively. Although the reactive site and its C-terminal counterpart appear very similar at first sight, some differences exist. Most obvious is the substitution of Val35 by Lys90. Other differences include a different side chain conformation of Arg89 compared with Arg34, the increased temperature factors for residues 90-97 compared with residues 35-42, and the differences in φ,ψ angles, especially for residues N-terminal of residue 88 and 33, respectively (see Table II). Table 2. Main chain conformational angles φ,ψ of reactive site loops P3 P2 P1 P1′ P2′ P3′ Antistasin domain 1 Arg32 Cys33 Arg34 Val35 His36 Cys37 φ/ψ (°) −93/39 −156/178 −129/29 −59/132 −71/112 −113/110 Antistasin domain 2 Asn87 Cys88 Arg89 Lys90 Thr91 Cys92 φ/ψ (°) 33/70 −125/125 −119/27 −77/146 −105/131 −136/138 Canonical values (from Bode and Huber, 1992) 140°<φ<−120° −100°<φ<−60° −120°<φ<−95° −100°<φ<−60° −140°<φ<−99° −140°<φ<−99° 140°<ψ<170° (except BPTI) 139°<ψ<180° 9°<ψ<50° 139°<ψ<180° 70°<ψ<120° 70°<ψ<120° Modelling of the factor Xa-antistasin complex As we have not obtained crystals of a complex of antistasin with factor Xa, we performed docking studies in order to analyse possible binding modes of antistasin with its target protease. Antistasin was docked with its binding loop into the active site cleft of human factor Xa (Padmanabhan et al., 1993; Brandstetter et al., 1996) as described in Materials and methods (see Figure 6A). Docking was guided by the structures of the kallikrein A-BPTI (Chen and Bode, 1983) and trypsin-BPTI (Marquart et al., 1983) complexes. Only the side chain of the P3 residue Arg32 had to be manually reoriented to optimize its fit in the active site. The resulting energy-minimized structure of antistasin exhibits r.m.s. differences for Cαs of 0.40 Å compared with the X-ray structure. For residues 32-39, r.m.s. differences are even smaller (0.35 Å), indicating that only minor changes were required for complex formation with factor Xa. Two major sites of antistasin were found to interact with factor Xa, consisting of residues 15-17 and 32-39, respectively. Additional contact zones may include the backbone carbonyl oxygen atoms of antistasin residues 8, 9 and 51, which are likely to interact with the side-chain amino groups of Lys148 and Lys96 of factor Xa, respectively. In the following, the two major interaction sites will be discussed in detail. Figure 6.Representative views of the modelled complex of antistasin and factor Xa. (A) Stereo view of the Cα trace [produced with Molscript (Kraulis, 1991) and Raster3D (Merritt and Murphy, 1994)]. Antistasin residues Glu15, Arg32 (P3), Arg34 (P1) and Val35 (P1′) are highlighted. (B) Reactive site area. Possible interactions of antistasin residues Arg32 and Arg34 with factor Xa are indicated. Ser195 of the factor Xa catalytic triad is perfectly positioned for nucleophilic attack by the protease. Download figure Download PowerPoint The reactive site region (residues 32-39). As in the uncomplexed structure of antistasin, the side chain of the P1 residue Arg34 adopts an extended conformation in the modelled complex with factor Xa. It forms a salt bridge with twin-twin geometry to Asp189 at the bottom of the S1 specificity pocket of factor Xa (see Figure 6B). The interaction is similar to that of Arg439 from a symmetry-related molecule in the native factor Xa crystal structure (Padmanabhan et al., 1993). In addition to the ionic interactions, the Arg34 side chain is likely to be involved in hydrogen bonding interactions with the backbone carbonyl oxygen atoms of factor Xa residues 190, 216 and 218. The main chain carbonyl oxygen of Arg34 points into the oxyanion hole of factor Xa and probably forms hydrogen bonds with Ser195 N and Gly193 N. In the modelled complex, the scissile bond linking Arg34 and Val35 is sandwiched between the side chains of factor Xa residues Ser195 and Gln192, respectively, with the Arg34 carbonyl carbon being positioned only 2.9 Å from the serine Oγ of the factor Xa catalytic triad (His57, Asp102 and Ser195). This inhibitor atom thus seems perfectly positioned for nucleophilic attack by the protease. The majority of serine proteases of the trypsin/chymotrypsin family form an antiparallel β-sheet with their substrates or inhibitors which involves residues 214-216 from the protease. However, in the modelled complex with antistasin, these residues seem not to contribute to hydrogen bonding interactions. This finding is in agreement with the results from Brandstetter et al. (1996) who determined the crystal structure of factor Xa with the synthetic inhibitor DX-9065a. The lack of β-sheet interactions is compensated for by two other major interactions between antistasin and factor Xa. One has already been described and involves the P1 residue Arg34. The second important interaction between antistasin and factor Xa concerns the P3 residue Arg32. In the modelled complex, the Arg32 guanidinium group binds to the cation hole of factor Xa, which is formed by the carbonyl oxygens of Lys96 and Glu97 as well as the Glu97 carboxylate (Brandstetter et al., 1996) (see Figure 6B). The aliphatic part of the Arg32 side chain partly occupies the aryl binding site (S4) of factor Xa. The P4 residue Val31 is positioned far away from this binding site and seems not to interact with factor Xa. Thus, two residues, Arg32 and Arg34, are likely to be particularly important for the interaction of antistasin with factor Xa, with most of the interaction potential of the side chains of these residues satisfied. The exosite binding region (residues 15-17). An unprecedented feature of the antistasin structure is the region comprising residues 15-17. In our model, the Glu15 side chain, which interacts with Arg25 from a symmetry-related molecule in the crystal structure, is reoriented towards a cluster of three positively charged residues, Arg222, Lys223 and Lys224, on the surface of factor Xa. We will refer to this contact area as the ‘exosite’. Evidence exists that this putative exosite may not be unique for factor Xa, but may also be present in other proteases, since many factor Xa-related proteases like thrombin and trypsin also have positively charged amino acid residues at corresponding positions (Jackson and Nemerson, 1980). In addition to the charge-charge interactions, antistasin and factor Xa may form contacts via two hydrogen bonds involving the main chain carbonyl of Ser17 and the side chain of Arg222, respectively. In view of the fact that only the N-terminal domain of antistasin can inhibit factor Xa, even though the C-terminal domain is highly homologous, it seemed very interesting also to perform docking studies of factor Xa with the C-terminal domain of antistasin. As a result, the inability of the C-terminal domain to inhibit factor Xa seems mainly to be caused by sites other than the region corresponding to the antistasin reactive site. Although at first sight, clashes of this part of antistasin with factor Xa seem inevitable, at second sight, they appear resolvable. For instance Arg89, which corr