Bmc Structural Biology Characterization of a Human Coagulation Factor Xa-binding Site on Viperidae Snake Venom Phospholipases a 2 by Affinity Binding Studies and Molecular Bioinformatics

Background: The snake venom group IIA secreted phospholipases A 2 (SVPLA 2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA 2-inhibitors from snake serum; and (iii) coagulation factors present in human blood.


Background
Haemostasis (vasoconstriction, platelet plug formation and blood clotting) is a defense mechanism that evolved to prevent the loss of blood after injury to the transporting vessels [1]. The intrinsic and the extrinsic alternate pathways initiate the blood clotting process. One of the com-mon steps in both pathways of coagulation is the activation of coagulation factor X (FX) to factor Xa (FXa).
FXa circulates in plasma as the light and the heavy chains connected by a single disulfide linkage. The N-terminal region of the light chain (residues 1-39) is the Gla domain, rich in post-translationaly modified γ-carboxyglutamic acid, which interacts with the phospholipid (PL) membrane [2]. The Gla domain is followed by a short stack of hydrophobic residues (residues [40][41][42][43][44][45], and two epidermal growth factor-like repeats -the EGF-like 1 domain (residues  and the EGF-like 2 domain (residues . The heavy chain of FXa contains the catalytically active serine proteinase domain (254 amino acids, residues 16-269 in chymotrypsinogen numbering system). As a serine proteinase of the chymotrypsin family [3], FXa consists of two subdomains of antiparallel β-barrel structure each comprising a sheet of six strands and four helices. Residues His57, Asp102, and Ser195 (chymotrypsinogen numbering) form a catalytic triad at the active site cleft between the two subdomains [4]. The fold contains a number of solvent-exposed loops, which determine S1 and subsite preferences in structurally homologous enzymes of the family. To the north of the active site cleft in the canonical view are the 60-and 99-loops; to the west are the 174 -and the 217-225 loops, restricting access to the active site. The autolysis loop 149-151 occupies the southern boundary of the active site cleft. Adjacent to it is the 70-loop. To the east is the 37-loop. Loop 185-188 is associated with S1 preference [5,6]. Upon binding to FVa in the presence of Ca 2+ ions on negatively charged membrane PL at the cellular surface, the prothrombinase complex is formed, resulting in the accelerated conversion of FII (prothrombin) to FIIa (thrombin) by FXa [7,8]. Afterwards, thrombin converts fibrinogen into fibrin, consolidating the primary plug.
In addition to the esterase activities, sPLA 2 s are also specific ligands that interact with different targets, such as membrane-bound PLA 2 receptors [16,17], anionic heparan sulfate proteoglycans (HSPG) [18,19], and with a cytoskeleton protein (vimentin) [20]. On the other hand, the soluble receptors of PLA 2 s, such as the natural inhibitors in the blood of snakes, the coagulation factors, and the PLA 2 binding protein have been also well characterized [21,22]. Hence, it has become clear that PLA 2 s exert physiological and patho-physiological effects through protein-protein interaction and/or protein-PL interactions [22,23]. These protein-protein mechanisms are sometimes dependent on, and other times independent of their enzymatic activity, playing important roles in determining the specific function of sPLA 2 s [24-26]. Viperidae snake venoms contain several toxic group IIA PLA 2 s (SVPLA 2 ), which may act as presynaptic neurotoxins [27] and may interfere with blood coagulation by possessing strong anticoagulant properties [28][29][30][31][32][33][34][35][36][37][38][39][40]. Viperidae SVPLA 2 s have high primary sequence identity with human group IIA PLA 2 (hsPLA 2 ; 30-60%) and overall structural homology [41,42]. Almost all anticoagulant snake venom PLA 2 are basic proteins and may inhibit coagulation by several mechanisms. A first mechanism involves the hydrolysis and destruction of procoagulant PL [43][44][45][46][47]. CM-I and CM-II, illustrate this mechanism in which the group I PLA 2 inhibits the extrinsic tenase complex [22,43], and does not bind to FXa. A second mechanism is based on the competition with clotting proteins for binding to the lipid surface due to the high affinity of PLA 2 toward PL the "antagonist effect" [28,48] [49]. A third mechanism is a non-enzymatic, PL-independent mechanism in which the PLA 2 interacts with FXa, inhibits the prothrombinase complex by preventing formation of the FXa/FVa complex and introduces a lag time in the generation of thrombin [22,32,34,35,39,50,51]. In addition, some snake venom PLA 2 s show a combination of enzymatic and non-enzymatic mechanisms, like the basic sPLA 2 isoform CM-IV from Naja nigricollis (Elapidae) venom [22,29].
The non-enzymatic inhibition mechanism of the prothrombinase complex was first demonstrated for CM-IV[32,34] and then for hsPLA 2 [50]. The catalytically inactive His48Gln mutant of hsPLA 2 possesses an identical anticoagulant effect and binds to FXa with the same kinetic constant as the wild-type enzyme, showing that in this mechanism the anticoagulant process is independent of the catalytic activity of the PLA 2 [51].
Kini and Evans first proposed that a "pharmacological site," in addition to the catalytic site, could explain the specific biological anticoagulant activity of snake venom group I PLA 2 s[33]. This anticoagulant region consists of the basic exposed loop located in the region 55-70, and the beginning of the β-sheet on Elapidae group I PLA 2 s [52,53]. The proposed region is positively charged in strong anticoagulant enzymes, but negatively charged in weak and non-anticoagulant enzymes [33]. More recently, Kini proposed that weakly anticoagulant enzymes, which lack the anticoagulant region, fail to bind specifically to FXa in the coagulation cascade [22].
In spite of all the studies, the corresponding site in Viperidae SVPLA 2 s is not yet clearly established. In addition, no distinction is usually made between the mechanisms by which the anticoagulant potency is exerted by the PLA 2 s. It seems that only the mechanisms that imply participation of PL have been taken into account in the past.
Anticoagulant snake venom PLA 2 represent a novel family of agents useful in identifying the sites of interaction of anticoagulants at the level of specific amino acid residues and thus have a potential in identifying new drug leads [54]. In order to characterize the FXa-binding site of SVPLA 2 from the Viperidae family, we have focused on the non-enzymatic, PL-independent, anticoagulant mode of action. Previous work from the Unité des Venins (Institut Pasteur, Paris) had shown the involvement of basic residues located around the IBS of hsPLA 2 [51], on one hand, and the possible involvement of the C-terminal and βwing regions of AtxA [39] in binding to FXa, on the other. We were henceforth interested in determining whether similar or different amino acid patterns were present in Viperidae SVPLA 2 s. Using SPR and a physiological test of inhibition of prothrombinase activity, we identified SVPLA 2 s that formed complexes with FXa and determined the apparent affinity constants of the complexes. With this experimental information at hand, we applied sequence analysis, molecular bioinformatics and docking procedures in order to define the anticoagulant region of the PLA 2 and the nature of the residues involved in the interaction with FXa.

Identification of SVPLA 2 s that bind to FXa and inhibit prothrombinase activity
As shown in Table 1, the SVPLA 2 s from Viperidae snake CBc and MtxII interact with FXa with very high affinity and constitute group VS (<K d app > 0.6 -2 nM). These enzymes strongly inhibit the formation of the prothrombinase complex (IC 50 1-3 nM). Under identical conditions, the <K d app > value determined for hsPLA 2 is 14 nM. CBa 2 , AtxA and CbII have high affinity values and anticoagulant potency, and form group S (20-50 nM). A third group of SVPLA 2 , group M, is formed by Vbb , VRV-PLVIII, and bAhp. In this group, the affinity with FXa is smaller (<K d app > 400 -830 nM) and the IC 50 ranges from 90 to 130 nM. AGTX (the neutral PLA 2 from A. halys pallas (blomhoffii)), reported previously as weakly anticoagulant in the presence of PL [38], does not interact with FXa and does not inhibit prothrombinase activity in the absence of PL. Neither CbI nor Catx inhibit prothrombinase activity in the absence of PL. These three SVPLA 2 s constitute group NB. Last, reduced and carboxymethylated CBc does not interact with FXa. Furthermore, we found a positive linear correlation between <K d app > and IC 50 (R = 0.995) for the three groups of SVPLA 2 that interact with FXa. Thus, the strongly anticoagulant SVPLA 2 s bind with high affinity to FXa, whereas the less efficient anticoagulant SVPLA 2 possess low affinity for FXa. Also, basic SVPLA 2 s (8.35<pI<9.10) bound to FXa and inhibited prothrombinase activity, whereas those with acidic pIs (4.60-5.43) did not (Table 1).
It is interesting to note that the isoenzymes CBc and CBa 2 , which differ by 8 amino acids (His1Ser, Ile18Val, Arg34Gln, Pro74Arg, Glu92Lys, Tyr115Asn, Gly116Glu, Gly128Glu) and associate with the acidic subunit CA to form two pharmacologically distinct classes of crotoxin complexes, present differences in toxicity, enzymatic activities and stability [67,68]. These two isoforms bind to FXa with different kinetics ( Table 1). The average rate of dissociation constant <k off > for the FXa-CBa 2 complex is about two orders of magnitude greater than that of the FXa-CBc complex, implying a more stable FXa-CBc complex (<K d app > 0.6 nM). Consequently, CBc strongly inhibits the prothrombinase complex (IC 50 0.7 nM), whereas the inhibition by CBa 2 is much weaker (IC 50 41 nM). Indeed, we had observed in the past that the difference in stability between crotoxin isoforms was due only to the CB subunit [55,68].
We also investigated by SPR the possibility of the formation of a ternary CA-CB-FXa complex. On one hand, CA binds to immobilized CB [69], whereas FXa does not interact with immobilized CB[23]. An anti-CA monoclonal antibody (mAb) A-73.13 [70] was covalently attached to the chip, as previously described [71]; CTX was then captured via this mAb before the injection of FXa (Fig. 1). FXa bound to CTX, as seen in the rise of the resonance signal. Fig. 1 also shows that after the injection of a specific anti-CB mAb (B32.13), the signal increased further, indicating the presence of CB on the chip and show-ing that the CB-FXa complex is stable and remained attached to CA.

Sequence analysis, comparisons and consensus residues of the anticoagulant SVPLA 2 s
In order to identify those residues or sequence patterns that differ between members of the SVPLA 2 s, we obtained Weblogo plots of the four groups VS (CBc, MtxII), S (CbII, AtxA, CBa 2 ), M (VRV-PLVIII, bAhp, Vbb) and NB (AGTX, Catx, CbI). Thereafter, we performed comparisons of all the sequences among themselves and of the four groups against each other.
Considering only segments of three or more residues, we observed three conserved regions for AGTX, Catx and CbI of the NB group (Tyr25-Gly30, Thr41-Gly53 and Cys96-Asp99; Fig. 2A), and six for the CBc-MtxII pair of the VS group (Tyr25-Cys27, Gly33-Gly35, Pro37-Cys45, Cys50-Tyr52, Cys96-Asp99 and Cys105-Arg107; Fig. 2B). A striking difference between the two groups is the presence of the acidic residue Glu at position 128 in the C-terminal region of all SVPLA 2 s of the NB group, as opposed to a Lys or a Gly residue in the VS group. For the other groups, only CBa 2 and Vbb contain a Glu at this position. With respect to the S group, the M group contains a conserved basic residue at position 115. Interestingly, the M group contains a conserved Lys in position 132 that is absent in the VS and NB groups. Group NB contains acidic side chains in the first strand of the β-wing and the adjacent β- The interaction between an isoform of CTX and FXa, as measured by SPR Figure 1 The interaction between an isoform of CTX and FXa, as measured by SPR. The anti-CA monoclonal antibody mAb A-73.13 [70] was covalently attached to the chip [71]. CTX (the CA 2 -CBc complex) was then captured via this mAb before injection of FXa (25 μg/ml). FXa bound to CTX, as seen in the rise of the resonance signal, showing that the CTX-FXa complex remained attached to the anti-CA mAb. After injection of a specific anti-CB MAb (B-32.13, 10 μg/ml), the signal increased further, confirming the presence of CB in the ternary CA-CB-FXa complex.
turn (residues [75][76][77][78][79][80][81][82]. For the VS and S groups, the tendency is towards a net balance of positive charges in the βwing. The NB group Catx presents three Glu and one Asp residue in the β-wing (residues 74-90); CbI presents two Asp residues. The β-wing of CBc of group VS contains two Lys residues; that of MtxII two Lys, one Asp and one Glu residues. For the S and M groups the tendency is towards net positive charges, except for bAhp, for which the total charge is neutral.

Tertiary structures and molecular electrostatic potentials of SVPLA 2 s
We obtained 3D homology models of CBc, CBa 2 , CbI, CbII, AtxA and Vbb. The models show the canonical struc-Weblogo representation of the multiple sequence alignment of the SVPLA 2 s Figure 2 Weblogo representation of the multiple sequence alignment of the SVPLA 2 s. A. Group NB (AGTX, Catx and CbI). Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. The symbols in each stack are arranged by alphabetical order from top to bottom and do not follow the order in which the sequences were fed. Arg, His and Lys residues are in blue; Asp and Glu in red; Ala, Ile, Leu, Met, Phe, Pro, Trp, and Val in black; Gly, Cys, Ser, Thr, and Tyr, in green; Asn, Gln in purple. Renetseder numbering system for all PLA 2 s throughout this work [15]. B. Group VS (CBc and MtxII).
tural features of the PLA 2 template molecules: a N-terminal α-helix (helix A), a short helix (helix B), a Ca 2+binding loop, a long α-helix (C), a loop preceding an antiparallel two-stranded sheet (β-wing), a long α-helix (D), anti-parallel to helix C, and a C-terminal extended fragment. All seven disulfide-bridges are at their expected positions. Fig. 3 shows the molecular model of AtxA in the canonical ("front") face orientation, i.e., presenting the catalytic hydrophobic channel. shows the MEP on the "back" face. Qualitatively, we observe that the MEP is positive in the front face of strong and mild anticoagulants (CBc, CBa 2 , CbII, and AtxA); approximately neutral for the weak anticoagulant Vbb, and negative for CbI (Fig. 4A). The back face is largely devoid of positive potential, except for Vbb (Fig. 4B). This difference in MEP leads to an electrostatic asymmetry. We observe the same trend for the crystallized SVPLA 2 (MtxII, VRV-PLVIII, bAhp, AGTX, Catx and hsPLA 2 ; not shown).

Molecular docking and intermolecular interfaces: mapping of the anticoagulant site and of the binding site on FXa
The output generated by the docking program PatchDock showed many complexes in contact with one of the binding site regions. Only the AtxA-FXa candidate complex (rank number 7) showed the best compatibility with the binding site derived from the mutagenesis data [39] (in this complex, position 150, which shows a Glu -> Arg sequence difference between the purchased FXa and the crystal structure (PDB entry 2BOH) is neither at the light/ heavy chains interface nor at the AtxA/FXa interface). Sev-eral residues belonging to the two regions identified by the mutagenesis of AtxA and defining part of the binding site (the "front" strand of the β-wing and the C-terminal fragment) are at the interface in this complex. In it, the relative orientation of FXa positions the N-terminus of the EGF-like 2 domain of the light chain of FXa towards the βwing of AtxA, allowing the 1-domain (not visible in the Xray structure) to enter in interaction with the β-wing and thus contact the remaining residues of the front edge of the β-wing. Fig. 5 shows the ribbon representation of the final complex between AtxA and the light and heavy chains of FXa. Fig. 6 shows the same complex in the form of a solvent-accessible surface. In both representations, the SVPLA 2 is in the "classical" orientation, i.e. that of Fig.  4A. Overall, we observe just small conformational changes after complex formation.
For the FXa strong-binding SVPLA 2 s CBc, MtxII, CbII and CBa 2 , the complexes that ranked 18, 10, 20 and 17, respectively, showed the same binding mode as the AtxA-FXa complex, i.e., the same relative orientation of FXa with respect to the SVPLA 2 . As measured by the FIT function of Pymol, these complexes showed Cα RMSD's with respect to the reference AtxA-FXa complex of 3.4, 2.2, 5.1 and 4.8 Å, respectively.
The percentage of surface residues at the interface of the complexes is of 24-36% for the five SVPLA 2 s and of 20-32% for FXa. The interface area for the five SVPLA 2 s is in the 860-1700 Å 2 range and 915-1470 Å 2 for FXa. The interface area of the complexes varies from 1610 to 1930 Å 2 . The values obtained for the solvent accessible area buried in the five characterized complexes fall between the small and the large interfaces categories in a study of 362 protein-protein interfaces [72]. Fig. 7 shows the mapping of the SVPLA 2 -FXa interaction surface of the complexes between the five SVPLA 2 s and FXa. The shadowed residues in that figure are those at the interface of the AtxA-FXa complex satisfying the mutagenesis data, and at the interface of the complexes of the other four SVPLA 2 s that show the same complexation mode as the AtxA-FXa complex. The consensus residues that participate to FXa binding are: solvent-exposed parts of helix A (positions 2, 3, 7) and helix B (positions 18, 19); positions 16, 23 and 24; a part of the Ca 2+ loop (positions 31-34); a part of the 69loop (between helix C and the β-wing; positions 53, 59, 60, 69 and 70); and the C-terminal segment (positions 118, 119, 121-124, 129-131, 133). By taking into account the type of atom-atom contacts as defined by Sobolev et al. [73], 36.5% of the contacts are hydrophobic at the AtxA-FXa light chain interface, as compared to 26.4% at the AtxA-FXa heavy chain interface. Overall, 29.7% of the contacts are hydrophobic at the AtxA-FXa interface. These interface regions are identified separately by the protein-protein interface prediction server PPI-Ribbon diagram of the molecular model of AtxA Pred. For example, for AtxA, the highest score PPI-Pred patch, which represents the most probable protein-protein binding site, includes helices A and B, the Ca 2+ -loop, part of the 69-loop, two residues from the front strand of the β-wing, and the four C-terminal residues (results not shown). Fig. 8 shows the regions mapped by docking simulations in the modeled structure of complexed AtxA.
The first line of Fig. 9A and 9B show the amino acid sequences of the parts of the heavy and light chains of the crystal structure of FXa (PDB 2BOH), detected at the surface respectively. The heavy chain (Fig. 9A) includes the serine proteinase catalytic domain of FXa (catalytic-site residues in bold), and the light chain (Fig. 9B) includes the EGF-like 2 domain. The following lines in Fig. 9A and 9B show the FXa residues at the interface of the docked FXa-SVPLA 2 complexes (residues appearing three or more times). The regions of FXa at the interface with CBc, MtxII, CbII, AtxA and CBa 2 in the resulting docked complexes include, in the light chain (Fig. 9B), the N-terminal region of the EGF-like 2 domain: Arg86-Ser90 and Cys100-Glu102, Gln104, Asn105. In the heavy chain (Fig. 9A), they include the following regions. Region I: Arg93; Phe101 (from the 99-loop). Region II: Arg125; Asp126; Glu129-Ser130; Thr134. Region III: Tyr162; Asp164-Asn166; Lys169; Leu170. Region IV: Gln178 and Asn179 (from the 174-loop). Region V: Lys230, Thr232, Ala233, Phe234 and Lys236 (all residues from the C-terminal helix). Fig. 10 shows these regions in the 3D model of complexed FXa. Some of these residues belong to loops surrounding the catalytic-site cleft.
We show in Fig. 11A and 11B the intermolecular residue contacts for the AtxA-FXa interface for which the contact area is equal to or greater than 10 Å 2 . We observe from this map that the heavy chain of FXa interacts with the following regions of the PLA 2 : a portion of the Ca 2+ loop, the helix C-β-wing loop, and the C-terminal segment (including Lys127, which has an effect in the binding to FXa) (Fig. 11A). The light chain of FXa establishes intermolecular contacts with the remaining regions: helices A and B, and the β-wing (Arg77) of AtxA (Fig. 11B). From the CSU analysis of interfaces, it follows that the contacts between SVPLA 2 and FXa are of diverse nature -aromatic-aromatic, hydrophobic-hydrophobic, salt bridges and H-bonds. There are also destabilizing hydrophobic-hydrophilic interactions. Few of the contacts take place between samenature residues, like Lys127 from AtxA and Arg93 from FXa's heavy chain (Fig. 11A). These contacts are between non-polar atoms or between non-polar and polar atoms of side chains at the surface of the molecules, where the presence of the aqueous solvent can buffer the electrostatic interactions.

Existence of different anticoagulant mechanisms
The classification of the anticoagulant potency (strong, weak, non-anticoagulant) of a SVPLA 2 depends on the anticoagulant assay used and on whether this assay leads to establishing the mechanism of action. Usually, the Solvent-accessible surface representation of the 3D molecu-lar model of the complex between AtxA and the light and heavy chains of FXa recalcification time assay, one of the simplest assays, is used. This method is very sensitive to the lipid levels in the plasma but does not show whether the mechanism is PLindependent or not. Our results deal with the interaction of anticoagulant PLA 2 s with FXa through the non-catalytic, PL-independent mechanism of action and are to be compared to studies performed under the same conditions. As mentioned before, only three PLA 2 s -CM-IV of N. nigricolis, AtxA of V. ammodytes ammodytes, and hsPLA 2have been studied under these conditions. In this work, we complete these studies by testing several other SVPLA 2 from Viperidae venom. Our results clearly show that CBc and MtxII interact with FXa with very high affinity and strongly inhibit the formation of the prothrombinase complex. CbII, AtxA and CBa 2 possess good affinity for FXa and good anticoagulant potency. VRV-PLVIII, bAhp and Vbb possess weak affinity for FXa and show weaker inhibition of prothrombinase activity. Some of these enzymes (CB, AtxA) also inhibit prothrombinase activity in the presence of PL (not shown). AGTX, the CA subunit of CTX, Catx and CbI do not interact with FXa and do not inhibit prothrombinase activity in the absence of PL. Therefore, AGTX, described previously as a weakly anticoagulant PLA 2 [38], and VRV-PLVIII, described previously as strongly anticoagulant [74], appear to inhibit blood coagulation through different mechanisms.
Viperidae SVPLA 2 interface amino acid residues Figure 7 Viperidae SVPLA 2 interface amino acid residues. Interface amino acid residues of the SVPLA 2 complexes with FXa (CBc, MtxII, CbII, AtxA and CBa 2 ). Underlined characters denote residues identified by mutagenesis to be critical for binding to FXa and inhibition of prothrombinase activity [39,51]. Bold characters denote residues defining the IBS of hsPLA 2 [13,14]. Cyanshadowed characters denote residues found at the interface of the selected complexes. The alignment reflects Renetseder's numbering system.

Search for an anticoagulant site
Possible location of an anticoagulant site common to Viperidae SVPLA 2 s that interact directly with FXa Reduced and carboxymethylated CBc did not interact with FXa ( Table 1), suggesting that the SVPLA 2 needs to adopt the proper conformation for the interaction to take place. In agreement and in the context of the PL-independent anticoagulant mechanism, our combined theoretical and experimental approach highlights the presence of an anticoagulant region composed of amino acid residues that come together in space to constitute a conformational epitope situated in the "front" face of the SVPLA 2 s. These are the solvent-exposed parts of helix A, helix B, the Ca 2+ loop, the helix C-β-wing loop, the front strand of the βwing, and the C-terminal segment of the PLA 2 (Fig. 8). Of course, the detailed distribution and composition of those residues varies for each of the SVPLA 2 s.
Carredano et al. [52] determined the 3D structure of group II A monomeric PLA 2 RVV-VD from Vipera r. russelli (PDB 1VIP), described as a strongly anticoagulant SVPLA 2 . The authors proposed a site responsible for the strong anticoagulant properties of the toxin, consisting of Glu53, together with a positively charged ridge of non H-bonded lysine residues free for intermolecular interactions in the 53-70 region (Lys60 in RVV-VD and in CBa 2 ). On FXa interface amino acid residues Figure 9 FXa interface amino acid residues. A. Amino acid residues of the heavy chain of FXa at the interface of the selected docked complexes for each SVPLA 2 . The sequence of FXa shows only residues detected in the X-ray experiment. The last line shows the experimentally reported residues of FXa involved in the binding to FVa [2,78,79,83]. We use Renetseder's notation for the SVPLA 2 s and chymotrypsinogen notation for FXa.  another hand, Zhao et al. [75] suggested that residues Trp70 and Glu53 in bAhp might play an important role in the anticoagulant activity of the basic SVPLA 2 s. The study of Zhong et al. [76], who tested the anticoagulant potency of bAhp mutants, revealed that the Glu53Gly and Trp70Met mutants lost their effects on blood clotting, while Thr56Lys and Asp67Lys had enhanced activity. The reported residues fall in the 53-70 interface region detected in our docked complexes of the strong FXa binders CBc, MtxII, CbII, AtxA and CBa 2 (Fig. 8). The possible contribution of Trp70 to the strong anticoagulant activity of PLA 2 s has also been proposed elsewhere [48]. Nevertheless, the anticoagulant region cannot be localized solely to the 53-70 segment, since enzymes that bind weakly or not at all to FXa contain also basic residues in this region.
The natural mutants CBc and CBa 2 present two Gly -> Glu mutations in the C-terminal region (Gly116Glu, Gly128Glu) leading to increases in the IC 50 values for inhibition of prothrombinase activity of CBa 2 with respect to CBc. This is consistent with the results of our sequence comparison analysis, in which we detect the acidic residue at position 128 as characteristic of the NB group, and with the docking results that point to this region as being at the interface of the complexes.
On the other hand, we localized in the crystal structure of hsPLA 2 (PDB 1BBC) the mutations that showed the major effects in the inhibition of prothrombinase activity and FXa-binding kinetic parameters [51]. Residues Arg7, Lys10 and Lys16 (helix A) are exposed to the solvent and form a cluster. Residue Lys38 (loop on N-terminus of helix B), and residues Lys123 and Arg126 form another cluster (underlined residues Fig. 7). As expected, the two clusters are situated on the front face and are oriented 180° about this convex surface. They act cooperatively in the binding to FXa. Lys86 carries with it an effect on IC 50 , Contact map for the AtxA-FXa complex Figure 11 Contact map for the AtxA-FXa complex. A. Heavychain FXa residues. Only residues for which the contact area is equal or greater than 10 Å 2 are shown.B. Light-chain FXa residues.
Ribbon diagram of the crystal structure of FXa (PDB 2BOH) showing the identified interface regions but is in the end of the second strand of the β-wing ("back" face) of hsPLA 2 , indicating that its effect on IC 50 is not due to the residue being at the interface. No effects are reported dealing with the Ca 2+ loop and the front strand of the β-wing does not appear in hsPLA 2 (Fig. 7); however, to our knowledge, these regions have not yet been probed.
Lastly, our experimental data suggest that the CB-FXa interaction site is different from the CB-CA interface and show that the interaction between FXa and CTX proceeds through a transient ternary (CA, CB, FXa) complex (Fig.  1).
The FXa binding region of PLA 2 involves also hydrophobic residues hsPLA 2 contains an unusually large number of prominent cationic patches on its molecular surface, some of which lie on the putative IBS [14], in contrast to bovine pancreatic PLA 2 and the SVPLA 2 from Agkistrodon p. piscivorus, which display only a limited number of such patches [10]. Given the charged nature of the residues critical for binding hsPLA 2 and AtxA to FXa, it is clear that electrostatic interactions play a role in the binding. Indeed, the electrostatic asymmetry showed by the MEP calculations must be enhanced by the presence of the essential Ca 2+ cofactor ion [77] and may be at the origin of the increased affinity of hsPLA 2 for FXa in the presence of Ca 2+ [50]. Thus, long distance electrostatic forces operating at the molecular surface are important and may optimally orient the molecules before binding to FXa. However, electrostatic interactions might not exclusively drive binding to FXa by the Viperidae SVPLA 2 s, as in hsPLA 2 . Indeed, in addition to basic residues, hydrophobic and aromatic residues play also key roles in optimizing the interaction between SVPLA 2 s and FXa, given that a ring of hydrophobic residues surrounds the opening to the catalytic site cavity. Thus, for AtxA, many hydrophobic residues (Leu2, 3, 10, 18 and 58; Pro17, 59, 68 and 121; Val31), most of which are located in the N-terminal region, and several aromatic residues (Phe24 and 123; Tyr52) are part of the surface presented to FXa (Fig. 7). Lastly, even though a Phe24Ala mutation in AtxA was not found to change the FXa-binding kinetic parameters [39], we emphasize the need to probe other residues belonging to the hydrophobic ring.

The PLA 2 binding region of FXa involves both, the light and heavy chains but not the catalytic site
On our results, the catalytic site of FXa is free and not involved in the interface, in agreement with the conservation of the serine-proteinase catalytic activity after binding by SVPLA 2 [51]. Our AtxA-FXa complex shows that one region of the light chain of FXa is involved in the binding to the SVPLA 2 and that the N-terminus of the EGF-like 2domain is pointing south (Fig. 5). Therefore, the EGF-like 1-domain, not visible in the crystal structure of FXa, can interact with the front edge of the β-wing of the PLA 2 . The Gla domain of FXa (bound to the N-terminus of the EGFlike 1-domain), is well beyond reach in space and not in contact with the PLA 2 . These two features are in agreement with experimental and biochemical data which support the conclusion that the Gla domain is needed rather for insertion into the PL membrane [51], and with a model of the entire FXa. Indeed, based on the crystal structure of the Gla domain of bovine prothrombin and the NMR coordinates of the bovine FX EGF1 domain, Bajaj and coworkers [2] proposed a model structure for the entire FXa molecule, based upon the crystal structure of porcine FIXa. The EGF domains in the model are oriented south and thus capable of establishing contacts with the β-wing of the PLA 2 .
Based on the ability of synthetic peptides from FXa to inhibit FXa-induced clotting, a number of authors have reported the FVa binding sites on FXa. The last line of Fig.  9A shows the experimentally reported residues of the heavy chain of FXa incontrovertibly involved in the binding to FVa. These residues are: FXa(His83-Lys96) [78]; FXa(Arg165, Lys169) [79][80][81]; the 185-189 loop, i.e., FXa(Lys186) [82]; FXa(Val231-Thr244) [83]; and FXa(Arg240) [81]. Three of four of the segments of the catalytic domain of FXa identified to interact with FVa overlap with those identified by us to interact with the Viperidae SVPLA 2 s, namely the segment about Lys90, the 162-loop, and the C-terminal part, about Lys237 (Fig.  9A). This suggests that several residues are shared by both, the SVPLA 2 -FXa and the FVa-FXa binding interfaces. Moreover, the experimentally identified FXa heavy chain residues involved in binding to FVa are located on the same 3D face of FXa, as in our SVPLA 2 -FXa complexes.
Arni and coworkers have recently reported the crystal structures of human Gla domainless FXa complexed with two small anticoagulant proteins from a hematophagous nematode [84,85]. The determined exosite from those complexes involves residues from one of the strands of the N-terminal seven-stranded β-barrel (strand β6, residues 80-93) and from the short C-terminal α-helix (residues 233-243) of the catalytic subunit of FXa. Several of these residues fall in regions I and V mapped in our complexes with the SVPLA 2 s (Fig. 9A and 10).

Several IBS residues are part of the PL-independent anticoagulant site of SVPLA 2
By combining the residues defining the IBS [13,14] with the site-directed mutagenesis experiments for probing the basic residues of hsPLA 2 involved in binding to FXa [51], we deduce that IBS residues Arg7 and Lys10 bind to FXa.
By homology with hsPLA 2 , the presumed IBS amino acid residues for AtxA are Leu3, Leu18, Phe24, Lys74 and Tyr113. On one hand, Lys74, experimentally found to be critical for binding of AtxA to FXa [39], belongs to the IBS. On the other hand, our simulations indicate that IBS residues Leu3, Leu18 and Phe24 are in contact with FXa. In conclusion, several IBS residues are part of the PL-independent anticoagulant site and participate in formation of the complex with FXa.

SVPLA 2 are multifunctional proteins with multiple pharmacological sites
The Viperidae SVPLA 2 s studied here are multifunctional proteins, raising the possibility of overlapping or multiple pharmacological sites distinct from the catalytic site [86]. One team [52] has suggested that the neurotoxic site of group II neurotoxic enzymes overlaps with the anticoagulant region. Thus, from the mutants used to test the anticoagulant potency [39] and the neurotoxic function [87] of AtxA, it appears that several residues in the C-terminus are clearly shared by both functions. The overlapping of pharmacological sites is most easily understood in terms of the small size of this family of proteins.

Conclusion
In this paper, we concentrated our efforts on identifying the anticoagulant Viperidae SVPLA 2 s that inhibit blood coagulation via a non-enzymatic, PL-independent mechanism through direct binding to human FXa. Using SPR technology, we showed that CBc and MtxII bind to FXa with the highest affinity and inhibit strongly the prothrombinase complex, whereas CbII, AtxA and CBa 2 bind with good, but lesser affinity.
Of the eight mutations that differentiate CBc from CBa 2 , Arg34Gln in the Ca 2+ loop and Gly128Glu in the C-terminal segment fall in our identified interface regions; Gly116Glu is just borderline to the C-terminal fragment. Thus, the disappearance of a positive charge and the appearance of two negative charges in this region account for the loss of affinity of CBa 2 for FXa with respect to CBc. This is consistent with our consensus sequence analysis, which had associated the presence of Glu at position 128 to a decrease in affinity.
The molecular electrostatic potential we calculate at the surface of the 3D molecular models shows a correlation with the anticoagulant potency of the SVPLA 2 s. However, since not all basic PLA 2 are strong anticoagulants [44], the basic character of the PLA 2 seems to be a necessary but not sufficient condition for its anticoagulant potency.
Mapping of the FXa-interface zone in the 3D structures of the SVPLA 2 s by binding-site directed docking simulations, allowed us to detect several FXa-binding regions that come together to form a conformational epitope on the "front" surface of the SVPLA 2 s. One of the regions maps to the 53-70 segment, proposed in the past to be the anticoagulant region. According to our findings, this region is to be extended, on one hand, to helices A and B and to the "front" strand of the β-wing, and to the Ca 2+ loop and the C-terminal direction, on the other. The FXa interface forms a novel exosite that involves both, the light and heavy chains.
Our work epitomizes the use of binding affinity and mutational experimental data in guiding molecular docking simulations by indicating which species associate and then outlining the possible interacting surface patches.
Finally, there has been intense interest in the development of FXa inhibitors for the treatment of thrombotic diseases. Anticoagulant Viperidae SVPLA 2 s, which interact with FXa via a non-catalytic, PL-independent mechanism, represent a novel family of selective FXa inhibitors. Structural information on the binding of these PLA 2 to FXa should be useful in the 3D structure-based design of therapeutic agents. The synthesis of peptides or peptidomimetics derived from our mapped regions could lead to the development of new antithrombotic molecules capable of delaying in vivo the activation stage of the prothrombinase complex and to their use as supplementary agents in antithrombotic therapy.

Methods
Reagents including Sensor Chips CM5, surfactant P20, the amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N'- (3- [88]. AtxA from Vipera ammodytes ammodytes was purchased from Latoxan. Recombinant hsPLA 2 was produced in our laboratory as described previously [89]. Isoforms of the CB subunit of crotoxin (CBa 2 and CBc), isoform CA 2 of the acidic subunit of crotoxin, and the VRV-PLVIII from Daboia russelli pulchella venom were purified in our laboratory as described previously [55,56,62,67]. CBc was reduced by dithiothreitol and alkylated with iodoacetic acid according to the procedure described by Faure

Determination of prothrombinase activity
We determined the anticoagulant potency of the SVPLA 2 s by measuring in vitro the inhibition of prothrombinase activity (IC 50 ). We used an in vitro biological test in which the prothrombinase complex was reconstituted at 37°C from purified human factors FVa and FXa in the presence of Ca 2+ , but without the addition of PL [39]. The purified prothrombinase components (FVa 10 nM, FXa 10 nM, and different concentrations of SVPLA 2 : 0, 10, 20, 50, 100, 200, 500 nM) were incubated 5 min at 37°C in Tris-buffered saline (0.05 M Tris/HCl, 0.1 M NaCl, 0.5% BSA, 5 mM CaCl 2 , pH 7.4). The reaction was then started with 110 nM prothrombin. We measured activated prothrombin activity every 20 min, as described previously [50], with small modifications. We determined the IC 50 value, which corresponds to 50% inhibition of thrombin generation for the different SVPLA 2 s in the absence of PL.

Surface Plasmon Resonance studies
We analyzed of the interaction between the SVPLA 2 s from the Viperidae family, and FXa by SPR using a BIACORE ® 2000 system (Biacore AB, Uppsala, Sweden). The running and dilution buffer in all experiments was Hepes (HBS; 10 mM Hepes, 150 mM NaCl, 5 mM CaCl 2 , 0.005% surfactant P20, pH 7.4). The experiments were conducted at 37°C. Human FXa was covalently coupled via primary amino groups on a CM5 sensor chip surface according to Prijatelj et al. [39]. One independent flow cell of the same sensor chip was used as a control flow cell and was subjected to a "blank immobilization," i.e., with no FXa added. We found the SPR signal for immobilized FXa on three different flow cells to be 1500 RU, 3000 RU and 5840 RU (1 RU corresponds to 1 pg/mm 2 of immobilized protein). We injected PLA 2 samples (0, 0.25, 0.5, 1, 2, 4, and 8 μg/ml) at 37°C with a flow rate of 20 μl/min on independent runs on the control and assay flow cells and their binding was monitored. Between each injection, we regenerated surfaces with twice 5 μL of 1 M NaCl. The apparent equilibrium constant, <K d app > = <k off >/<k on >, the average dissociation rate constant <k off > and the average association rate constant <k on > were calculated using Biacore's BIAevaluation 3 software. The kinetic models used to fit the data included the Langmuir association, heterogeneous analyte and conformational change. Only the first one showed the lowest closeness-of-fit value (χ 2 ). In addition, the other models resulted in affinities much lower (μM) than expected (nM).

Sequence homologies and alignments
The sequence databases and identifiers are the following: CBc (also known as CB1), Uniprot P62022; CBa 2 (also known as CB2 For sequence alignments, we used the LALIGN program [91,92], which finds multiple matching sub-segments in two sequences and shows the local sequence alignments. For representing sequences and their alignments, we used Weblogo, a web-based application designed to generate sequence logos [93,94].
After building the structurally conserved regions (SCR) and the structurally variable regions (SVR), we replaced the side chains of the template protein by the target protein's side chains in a predetermined library conformation. We then performed a rotamer search assignment in order to avoid atomic steric clashes and optimize the inter-residue energies. We included a structurally conserved catalytic water molecule during the modeling. We did not model the Ca 2+ ion at the Ca 2+ binding site, since it is absent in several X-ray structures. We subjected the obtained models to an overall internal energy minimization using the CFF91 force field. The protonation state of ionizable side chains and of the N-and C-termini was set for pH 7. Atomic partial charges were those of the CFF91 field. We used a distance-dependent dielectric function of 4r. We applied the cell multipole summation method for van der Waals and coulomb interactions. We used none of the cross terms of the force field. We applied the same conditions to the amino acid residues in the crystal structures used. We checked the stereo chemical quality of the models with the Struct_Check program, and the correctness of the folding with the Profiles3D Verify functionality (self-compatibility scores, insightII). The amino acid residue numbering is based on that of Renetseder et al. [15].

Molecular Electrostatic Potential
The MEP is generated by the combined presence of all partial charges residing on the atoms as a function of their positions. The potential was calculated with the DelPhi 2.5 program in insightII. The grid resolution was of 0.833 Å/grid point. The interior and exterior dielectric constants were 2 and 80, respectively. The value of the ionic strength was 0.145; the probe radius was 1.4 Å and the ionic radius 2.00 Å. The treatment of the grid points at the boundary used a full Coulomb approximation. The values of the potential are given in kT/e units at T = 298 K.

Molecular docking. Protein interfaces and interface contacts
We generated the molecular complexes with the Patch-Dock rigid body molecular docking procedure [101]. Given two molecules, the surfaces of the molecules to dock are divided by PatchDock into patches according to the surface shape. These patches correspond to patterns that visually distinguish between puzzle pieces. We used a clustering RMSD criterion of 4.0 Å. The output lists the rank of the complex and its approximate interface area. We selected PatchDock for the docking simulations since it allows a priori focusing on the vicinity of potential binding sites. In other words, it is possible to upload a receptor-binding site and a ligand-binding site. We took the SVPLA 2 s as the "receptor" molecules and FXa as the "ligand" molecule.
Thus, we generated molecular complexes only for those SVPLA 2 s for which we find experimental evidence of bio-physical interaction, i.e., in which the binding affinity between the PLA 2 and FXa, as measured by SPR, was high. In addition, we used the available mutagenesis data for AtxA [39] to filter the docked complexes so that AtxA residues that show a decrease in the binding affinity for FXa are at the interface of the complex and define a binding site. The residues defining the binding site on AtxA are Arg72, Lys74, His76 and Arg77 (front edge of the β-wing), and Arg118, Lys127, Lys128 and Lys132 (C-terminal fragment) [39]. We defined no ligand-binding site for FXa.
From the candidate complexes generated by PatchDock for AtxA, only one showed a binding mode compatible with the ensemble of the mutagenesis data -many complexes showed binding to either the β-wing or the C-terminal fragment of the SVPLA 2 or other regions of the AtxA. Thereafter, we selected those complexes for the other SVPLA 2 s that showed the same binding mode as AtxA and whose Cα RMSDs with respect to the AtxA-FXa complex were minimal. After generation of the complexes, we used the "move apart" option of PatchDock, which separates (by 1.6 Å) the receptor and ligand subunits in order to eliminate steric hindrances at the interface. We further improved the fitting of the complex by applying firstly the SCWRL3 side chain modeling procedure [102], in which we froze all disulfide bonds, as well as the side chains of heavy chain residues Asp70 and Glu80, which chelate the Ca 2+ ion in FXa. After the rotamer search, we applied additional energy minimizations in order to reach a minimal internal energy conformation. The entire approach assumes that no drastic conformational changes occur during complexation.
On another hand, we submitted the SVPLA 2 models to the Protein-Protein Interface Prediction (PPI-Pred) server [26,103] to predict their binding sites. PPI-Pred predicts protein-protein binding sites using a combination of surface patch analysis and a support vector machine trained on 180 proteins involved in both obligate and non-obligate interactions.
The interface between the two polypeptide chains of each of the complexes was characterized with the Protein interfaces, surfaces and assemblies service PISA [104,105]. The interface contacts were obtained through a contact map analysis and characterized with the SPACE bioinformatics tools CMA (Contact Map Analysis) and CSU (Contacts of Structural Units) [106,107]. We show only interface contacts for which the contact area is equal to or greater than 10 Å 2 .

Authors' contributions
GF and RCM conceived the study, analyzed the results and wrote the manuscript. All authors performed the research. In particular, GF carried out SPR affinity measurements, prothrombinase inhibition experiments, and purification and carboxymethylation of PLA 2 s; VTG assisted in purification of PLA 2 and participated in SPR studies and discussions; RCM carried out sequence analysis, molecular modeling and docking simulations.