High Resolution Structures of p-Aminobenzamidine- and Benzamidine-VIIa/Soluble Tissue Factor

Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8Å resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87Å resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Human factor VII (FVII) 3 is a vitamin K-dependent trace plasma protein that is synthesized by hepatocytes and secreted into the blood as a single chain molecule of M r ϳ50,000 (1,2). Gene structure, amino acid sequence, and the modular structure of FVII reveal that the protein is organized into several discrete domains (3,4). FVII consists of an N-terminal ␥-carboxyglutamic acid domain (Gla), a short hydrophobic segment, two epidermal growth factor (EGF)-like domains, and a C-terminal serine protease module, which consists of two ␤-barrel subdomains (3)(4)(5). Several coagulation enzymes, including FIXa, FXa, and FVIIa, have been shown to activate FVII (2,(5)(6)(7)(8)(9). In each case, the activation of FVII involves the cleavage of a single peptide bond between Arg 152 and Ile 153 , located in the connecting region between the EGF2 and the protease domains. This results in the formation of a two-chain FVIIa molecule that consists of a 152-residue light chain and a 254residue heavy chain held together by a single disulfide bridge. Characteristic of trypsin-like serine proteases (10), the heavy chain of FVIIa contains the catalytic triad, namely His 57 , Asp 102 , and Ser 195 , with Asp 189 (chymotrypsin numbering) at the bottom of the S1 specificity pocket (3,5).
Tissue factor (TF) (CD142) is a transmembrane protein belonging to the class 2 cytokine receptor family (11). Human TF consists of the following three regions: an N-terminal extracellular region of 219 residues, a transmembrane region of 23 residues, and a C-terminal cystoplasmic region of 19 residues. The extracellular region of TF consists of two fibronectin type III repeats. The crystal structure of the extracellular domain pair termed soluble TF (sTF, residues 1-219) is known (12)(13)(14)(15). The two fibronectin type III domains are connected end-to-end at an angle of 120° (12)(13)(14)(15). Similar to full-length TF, sTF binds FVIIa with high affinity and potentiates its enzymatic activity (16 -18).
Recent evidence indicates that the TF pathway (or the extrinsic pathway) plays a primary role in initiating blood coagulation during normal hemostasis as well as during many pathologic situations, including arteriosclerosis and septicemia (5). This pathway begins by exposure of blood to TF in the extravascular space at an injury site and formation of the complex between TF and plasma factor FVII/VIIa. The FVIIa/TF complex then activates FIX, FX, and FVII. The FXa thus formed with FVa, Ca 2ϩ , and phospholipid (PL) then activates prothrombin to thrombin, which cleaves fibrinogen to fibrin, which polymerizes to form a clot (5).
Banner et al. (19) first reported the 2.0 Å crystal structure of D-Phe-Phe-Arg-chloromethyl ketone (D-FFR) inhibited FVIIa/ sTF. The crystals were grown in the presence of Ca 2ϩ using chloromethyl ketone-inhibited active site FVIIa and two fragments of sTF obtained by subtilisin treatment. The structure revealed that part of the helix composed of residues 30 -40 of the Gla domain makes van der Waals contacts with sTF, and the EGF1 domain makes extensive contacts with sTF. The EGF2 and the protease domain also make contacts with sTF, which appears to be a complex interface region. Consistent with biochemical data (20 -22), FVIIa in the crystal structure has nine Ca 2ϩ ions bound, seven in the Gla domain, one in the EGF1 domain, and one in the protease domain (19).
Recently, it has been reported that Mg 2ϩ plays an important role in physiologic coagulation (23)(24)(25)(26). However, Mg 2ϩ sites in FVIIa have not been identified. Furthermore, it has been proposed that FVIIa contains a Na ϩ site (27), but the site has not been defined in any of the crystal structures (19, 28 -33). Notably, FVIIa also contains two Zn 2ϩ sites in its protease domain (34). Occupancy of the Zn 2ϩ sites inhibits FVIIa catalytic activity in the absence but poorly in the presence of Ca 2ϩ . This observation led to the hypothesis that Zn 2ϩ binds to the protease domain Ca 2ϩ loop and attenuates the activity of FVIIa (34,35). As is the case for Na ϩ , the Zn 2ϩ sites in FVIIa are not defined.
Here we report a 1.8 Å crystal structure of p-aminobenzamidine-FVIIa/sTF (pAB-VIIa/sTF), solved from crystals grown in the presence of Ca 2ϩ , Mg 2ϩ , Na ϩ , and Zn 2ϩ . The Gla domain has four Ca 2ϩ and three Mg 2ϩ sites, and the protease domain has two Zn 2ϩ sites unique to FVIIa. The Na ϩ site is structurally similar to the Na ϩ site in FXa (36,37), APC (38,39), and the proposed site in FIXa (40) but not to that in thrombin (37,41). Moreover, the main chain NH of 193 (chymotrypsin numbering) in FVIIa points away from the oxyanion hole in the pAB-VIIa/sTF and benzamidine-VIIa/sTF structures. This is an uncharacteristic feature of serine proteases (10), and inclusion of pAB (42,43) or benzamidine (44 -46) at the S1 site is expected not to disrupt the geometry of the oxyanion hole. Interestingly, soaking benzamidine-VIIa/sTF crystals with D-Phe-Pro-Arg-chloromethyl ketone (D-FPR-ck) resulted in benzamidine displacement, D-FPR incorporation, and induction of the oxyanion hole. From these data, we infer that TF binding restructures the activation domains in FVIIa (47), but it does not induce formation of the oxyanion hole. Instead, it is the binding of the substrate/inhibitor at the active site that induces oxyanion hole formation required for catalysis. The importance of this mechanism in attaining unique selectivity of the molecular substrates to initiate physiologic coagulation by FVIIa/TF is discussed.

MATERIALS AND METHODS
Reagents-Magnesium chloride, calcium chloride, and PEG 4000 were purchased from Hampton Research. 45 CaCl 2 was obtained from ICN Biochemicals. Phosphatidylcholine and phosphatidylserine, Trizma (Tris base), choline chloride (ChCl), zinc chloride, benzamidine, p-aminobenzamidine (pAB), and all other chemicals of the highest grade available were obtained from Sigma. D-FPR-ck was obtained from Calbiochem.
Proteins-Human FVII was expressed using pMon3360b expression vector in BHK/VP16 cells as described by Hippenmeyer and Highkin (48) and Zhong et al. (49). The FVII was purified using a Ca 2ϩ -dependent monoclonal antibody and FPLC Mono Q column chromatography (49). Gla analysis was performed by Commonwealth Biotechnologies, Inc. (Richmond, VA), using alkaline hydrolysis followed by high pressure liquid chromatography analysis. The amount of Gla was quantitated based upon Asp and Asn present per mol of FVII. Purified FVII contained nine Gla residues per mol. FVIIa was obtained using FXa-Sepharose as described previously, and the resin was removed by gentle centrifugation (2,49). The purified protein was concentrated to ϳ20 mg/ml and stored at Ϫ80°C until used. sTF (residues 1-219) was obtained from E. G. Tuddenham (Imperial College, London) and from Tom Girard Monsanto/Pharmacia. sTF was concentrated to ϳ10 mg/ml and stored at Ϫ80°C. Both proteins were ϳ98% pure as judged by SDS-gel electrophoresis (50). Human FX was purchased from ERL (South Bend, IN). Recombinant human TF containing the transmembrane domain (residues 1-243) was a gift from Genentech (San Francisco, CA).
Preparation of Relipidated (r)TF-Phospholipid (PL) vesicles (75% phosphatidylcholine, 25% phosphatidylserine) were prepared by a slight modification of the method of Husten et al. (51). Human TF was diluted to 30 g/ml in 50 mM Tris-HCl, 150 mM NaCl (TBS), pH 7.5, containing 10 mM CHAPS and mixed with an equal volume of 2 mM PL vesicles. The mixture was incubated for 2 h at 37°C. The rTF was extensively dialyzed at 4°C against TBS, pH 7.5, to remove CHAPS. The functional TF concentration was determined as described previously (52) using FX as a substrate. Approximately 60% of TF (calculated from the starting concentration) was available on the outside of the vesicles assuming a stoichiometry of 1:1 between FVIIa and TF.
Crystallization-The pAB-VIIa/sTF or the benzamidine-VIIa/sTF complex was crystallized using the sitting drop or the hanging drop vapor diffusion method. Specifically, the protein drop contained 4 mg/ml VIIa/sTF complex, 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl 2 Ϯ 20 M ZnCl 2 , and 10 mM pAB or 10 mM benzamidine, whereas the reservoir solution contained 16 -22% PEG 4000, 100 mM MgCl 2 , and 20 mM BisTris, pH 6.5. Drops were prepared by mixing 2 l of protein solution with 2 l of reservoir solution at 20°C. Crystals appeared within 7 days and were allowed to grow up to 14 -20 days before being flash-frozen without additional cryoprotectant.
For soaking experiments, 50 l of 100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 20 M ZnCl 2 , and 10 mM CaCl 2 was mixed with 50 l of the reservoir solution (22% PEG 4000, 100 mM MgCl 2 , pH 6.5). The above mixture was allowed to equilibrate with 1 ml of the reservoir solution for 1 week. Five-l volume of D-FPR-ck was added to the drop to achieve a final concentration of 250 M inhibitor. Two benzamidine-VIIa/sTF crystals grown in the presence of 20 M ZnCl 2 were then soaked in the drop for 48 h. During soaking, the drop was allowed to equilibrate with the reservoir solution. The crystals were flash-frozen without additional cryoprotectant.
X-ray Data Collection-The pAB-VIIa/sTF data set was collected at beamline X12C of the National Synchrotron Light Source in Upton, NY, using a Brandeis 4 CCD detector. One crystal containing 20 M ZnCl 2 diffracted to a resolution of 1.8 Å, the details of which are presented here. Three different data sets were collected for the benzamidine-VIIa/sTF crystals. One crystal had 20 M Zn 2ϩ , and the other two did not contain any added Zn 2ϩ . One of the crystals without added Zn 2ϩ diffracted to 1.87 Å, which is presented here. This data set was collected at the European Synchrotron Radiation Facility beam 14-2 at 0.93 . The other two benzamidine crystals, one with additional ZnCl 2 and one without additional ZnCl 2 , each diffracted to 2.2 Å, and the crystal (which contained ZnCl 2 ) soaked with D-FPR (subsequently referred to as D-FPR-VIIa/ sTF) diffracted to 2.0 Å. These three data sets were collected at beamline X12C of the National Synchrotron Light Source. All crystals belonged to the orthorhombic space group P2 1 2 1 2 1 with one molecule of the pAB-VIIa/sTF or benzamidine-VIIa/sTF or D-FPR-VIIa/sTF complex in the asymmetric unit. The data were processed and scaled using the programs DENZO and SCALEPACK (53).
Structure Determination and Refinement-First, the structure of pAB-VIIa/sTF was determined. Initial phases were calculated using the structure of Banner et al. (19) (PDB code 1DAN), as a starting model. This was followed by several rounds of positional, B-factor, and simulated annealing protocols using the program XPLOR (54). Ten percent of the data was kept out of refinement for cross-validation (55). During initial refinement stages, it became obvious that the Gla domain is tilted ϳ6°toward the TF2 domain, and that the loop has a slightly different conformation in comparison to the Ca 2ϩ only structure (19). The residues in the Gla domain were fitted using the program O (56). The R cryst dropped to 24.1 with R free of 30.2. Based upon previous information (57,58) as well as the distances and the positions of the Gla residues, Mg 2ϩ ions were placed at the 1-, 4-, and 7-position (59, Tulinsky numbering), 4 and Ca 2ϩ ions were placed at the 2-, 3-, 5-, and 6-positions based upon previous structures of Gla domains (19,59). The positions of these metals were refined and subsequently solvent molecules surrounding these metals as well as other metal ions (EGF1 and protease domain Ca 2ϩ , Na ϩ , and Zn 2ϩ ) were added to the model. At this point, it was also noted that the peptide bond between Lys 341 H (192) 5 and Gly 342 H (193) must be flipped to agree with the electron density maps. The structure was put through refinement, and the resulting R cryst was 22.4, and R free was 28.3. At this stage, additional solvent molecules were added in steps, and the structure was refined. The final R cryst was 19.8, and R free was 25.9, and the refinement statistics are given in Table 1. The coordinates are deposited into the RCSB Protein Data Bank with accession code 2A2Q.
Initial phases for the benzamidine-VIIa/sTF crystal were calculated using pAB-VIIa/sTF as a starting model; however, pAB was removed from the structure, and the Lys 341 H (192)-Gly 342 H (193) peptide bond was flipped such that a standard oxyanion hole conformation was attained prior to use for phasing. The refinement protocol was the same as described above for the pAB-VIIa/sTF crystal. Similar to the pAB-VIIa/sTF, it was again noted that in all three structures of benzamidine-VIIa/sTF, the peptide bond between Lys 341 H (192) and Gly 342 H (193) needed to be in the nonstandard oxyanion hole conformation in order to agree with the electron density maps. The refinement statistics for the benzamidine-VIIa/sTF crystal that diffracted to 1.87 Å are given in Table 1. The coordinates are deposited into the RCSB Protein Data Bank with accession code 2AER.
The refinement protocol for the D-FPR-VIIa/sTF crystal was similar to those described for the benzamidine-VIIa/sTF crystals. During refinement, it was noted that soaking of benzamidine-VIIa/sTF with D-FPR resulted in the D-FPR-VIIa/sTF structure having the Lys 341 H (192)-Gly 342 H (193) peptide bond in standard oxyanion hole conformation. The refinement statistics for D-FPR-VIIa/sTF are also given in Table 1. The coordinates are deposited into the RCSB Protein Data Bank with accession code 2FIR.
Ca 2ϩ Binding to Various Fragments of FVIIa or Prothrombin Fragment 1-Calcium binding was determined by the technique of equilibrium dialysis using 45 Ca 2ϩ as a probe. The specific procedure has been described earlier (20). Gla-domainless FVIIa and human prothrombin fragment 1 were obtained as described previously (20,60). Prior to Ca 2ϩ binding studies, full-length FVIIa and Gla-domainless FVIIa were inactivated using dansyl-Glu-Gly-Arg-chloromethyl ketone as described previously (20). Molecular weights of 50,000 for FVIIa (2), 23,000 for prothrombin fragment 1 (60), and 45,000 for Gladomainless FVIIa (20) were used. The concentration of protein ranged from 20 to 40 M. The buffer used was TBS, pH 7.5.
Activation of FX by FVIIa/sTF or FVIIa/rTF-FX was activated by either FVIIa/sTF or FVIIa/rTF in TBS, pH 7.5, containing 0.1% PEG 8000 using three different Ca 2ϩ /Mg 2ϩ conditions as follows: 1) 1.1 mM Ca 2ϩ , 0 mM Mg 2ϩ ; 2) 1.1 mM Ca 2ϩ , 0.6 mM Mg 2ϩ ; and 3) 1.7 mM Ca 2ϩ , 0 mM Mg 2ϩ . Each reaction contained 10 nM FX and either 1 nM FVIIa, 10 nM sTF, or 1 nM FVIIa, 10 pM rTF, 50 M PL. FVIIa was incubated with sTF or 4 The metals in the Gla domain of pAB-VIIa are numbered 1-7 according to the system of Tulinsky and co-workers (59) who first described the structure of the Gla domain of prothrombin. The EGF1 domain Ca 2ϩ is numbered 8, and the protease domain Ca 2ϩ is numbered 9. 5 To distinguish between the residues of the light chain and heavy chain of FVIIa and that of sTF as well as to coincide with the numbering system used previously for D-FFR-VIIa (19) and BPTI-VIIa (30), the residue number is followed by the chain identifier in boldface. Thus, the light chain residues are followed by L, heavy chain residues by H, and the TF residues by T. The heavy chain of FVIIa represents the serine protease domain and the standard chymotrypsin numbering is given in square brackets after the sequential FVIIa numbering. The insertions in the protease domain are followed by the letter A, B, C, etc. Water molecules are labeled with a prefix S for solvent. rTF for 5 min at 25°C to allow for complex formation. FX was then added, and reactions were carried out at 25°C. At various times, 95-l aliquots were removed and added to tubes containing 5 l of 0.5 M EDTA, pH 8.0 (final EDTA concentration of 20 mM), to stop the reaction. From this mixture, 90 l was removed and placed in a well on an Immulon 4 HBX flat bottom 96-well microtiter plate (Dynex Technologies), and 10 l of benzoyl-Ile-Glu-Gly-Arg-p-nitroaniline was added to yield a final concentration of 250 M. The p-nitroaniline formed was measured (⌬A 405 /min) for up to 30 min. The FXa generated was calculated from a standard curve constructed using FXa obtained from ERL (South Bend, IN).
FVIIa Binding to sTF Using Surface Plasmon Resonance-Binding of FVIIa to sTF was studied on a Biacore 3000 device (Biacore, Inc. Uppsala, Sweden). sTF was immobilized on a carboxymethyl dextran (CM5) flow cell (752 response units). Bovine serum albumin was immobilized on a separate flow cell as a control for nonspecific binding. FVIIa (2.8 -22.3 nM) was injected across the flow cell in 50 mM Tris-HCl, pH 7.4, containing 5 mM CaCl 2 and either 185 mM ChCl or 185 mM NaCl (flow rate 10 l min ؊1 ). Six-minute association and 10-min dissociation times were used. Dissociation was monitored after return to buffer flow, and the chip surface was regenerated by injecting 0.1 M EDTA followed by equilibration with 50 mM Tris-HCl, pH 7.4, containing 5 mM CaCl 2 and either 185 mM ChCl or 185 mM NaCl. Data were corrected for nonspecific binding by subtracting the signal for binding to bovine serum albumin. Data were analyzed with BIAevaluation 3.1 software (Biacore, Piscataway, NJ), and curve fitting was done with the assumption of one-to-one binding.

RESULTS
General Aspects of the pAB-VIIa/sTF, Benzamidine-VIIa/ sTF, and D-FPR-VIIa/sTF Structures-Although overall structures of pAB-VIIa/sTF, benzamidine-VIIa/sTF, and D-FPR-VIIa/sTF in the presence of Ca 2ϩ /Mg 2ϩ are similar to the D-FFR-VIIa/sTF structure (19), there is significant new information afforded by our structures. 1) The conformation of the Gla domain loop is different in our structures. 2) In the new structures, two Zn 2ϩ sites and one putative Na ϩ site are identified in the protease domain. 3) Three Ca 2ϩ sites in the Gla domain at positions 1, 4, and 7 were replaced by Mg 2ϩ in the new structures. Binding and kinetic data are presented that show that under physiologic conditions these three sites are occupied by Mg 2ϩ and play an important role in biologic coagulation. However, the EGF1 and protease domain Ca 2ϩ sites were not displaced by Mg 2ϩ . 4) Importantly, as compared with the D-FFR-VIIa (19), the oxyanion hole in the pAB-VIIa or benzamidine-VIIa structure is not preformed. This is in contrast to known structures of serine proteases with pAB (42,43) or benzamidine (44 -46). However, soaking benzamidine-VIIa crystals with D-FPR-ck resulted in the formation of the oxyanion hole in the D-FPR-VIIa. This finding has important implications as to FVIIa substrate specificity. Each of these points is elaborated below primarily using the pAB-VIIa/sTF structure.
R free was computed identically, except that 10% of the reflections was omitted as a test set. d In all structures Lys 32 L is in the disallowed region in the Ramachandran plot. This residue is also in the disallowed region in the Banner structure (19).
A ribbon diagram of the pAB-VIIa/sTF structure is presented in Fig. 1 (left panel). When the C␣ carbons of all residues except the first 46 residues of the light chain (i.e. Gla domain) were used for superpositioning of the Ca 2ϩ structure (19), the root mean square deviation was 0.7 Å. However, the Gla domain in the present Ca 2ϩ /Mg 2ϩ structure is tilted ϳ6°t oward the C-terminal TF2 domain of sTF. This tilt results in closer and tighter hydrophobic interactions involving Gla domain residues Leu 13 L, Phe 31 L, Arg 36 L, Leu 39 L, and Phe 40 L with C-terminal TF residues Tyr 156 T, Trp 158 T, Cys 186 T, Val 207 T, and Cys 209 T. An expanded view of these interactions is detailed in Fig. 1 (right panel). All of these residues are well ordered in the pAB-VIIa/sTF structure, and the van der Waals contacts are considerably shorter as compared with the Ca 2ϩ structure (19). For example, the distances between some of the residues in this region in the present structure and the 1DAN structure that differ by Ͼ0.5 Å are as follows: 3.86 versus 4.33 Å between Phe 31 L C⑀ 1 and Trp 158 T Cz 2 ; 4.18 Å versus 4.91 Å between Arg 36 L C␥ and Trp 158 T Cz 2 ; 4.75 Å versus 6.64 Å between Leu 39 L C␦ 1 and Tyr 156 T C D2 ; and 4.14 Å versus 4.65 Å between Leu 39 L C␥ and Trp 158 T C H2 . The more intimate hydrophobic interface was also observed in the FVIIa/sTF structure (PDB code 1Z6J) determined from a crystal grown in the presence of Mg 2ϩ and citrate. This is most likely because of the binding of Mg 2ϩ to Gla25 and Gla29 (see below) in the Ca 2ϩ /Mg 2ϩ structures.
Gla Domain-The conformation of the loop between the Ca 2ϩ / Mg 2ϩ structure (present structure) and the Ca 2ϩ structure (19) was also observed to be different. Here, residues 12L-46L of the Gla domain were used to superimpose the two structures. As shown in Fig. 2A, the main chain atoms and the side chain positions of the three hydrophobic residues (Phe 4 L, Leu 5 L, and Leu 8 L) as well as those of Gla6 and Gla7, differ in the two structures. The positions of the Ca 2ϩ /Mg 2ϩ ions in the Gla domain of the present structure and the positions of the Ca 2ϩ ions in the Banner structure (19) are also depicted in Fig. 2A. The hydrophobic residues as well as Gla6 and Gla7 are well ordered in the present structure. In the present structure (Fig. 2B), the side chain NH 2 of Arg 9 L makes an H-bond with O⑀ 1 of Gla6. The amide N of Phe 4 L makes an H-bond with O⑀ 3 of Gla7, and NH 2 of Arg 15 L makes an H-bond with the carbonyl O of Pro 10 L (not shown). In the Ca 2ϩ structure (19), the NH 2 groups of both Arg 9 L and Arg 15 L make H-bonds with Gla6. An additional H-bond (amide N of Ala 1 L and Gla26) and the coordination of the carbonyl O of Ala 1 L to Ca5 are common to both structures. Thus, there are sufficient interactions that allow for well ordered folding of the loop in the present structure.
The coordination of each Ca 2ϩ and Mg 2ϩ ion in the Ca 2ϩ / Mg 2ϩ structure to the Gla residues and water molecules is shown in Fig. 2B. All Ca 2ϩ , Mg 2ϩ , and water molecules are well ordered, and the coordinations are listed in Table 2. Mg 2ϩ at position 1 has similar coordination to the Ca 2ϩ in the D-FFR-VIIa structure except that the distances are shorter (Table 2), compatible with Mg 2ϩ and not Ca 2ϩ . There are also two additional water molecules coordinated to Mg1 in the present structure. Furthermore, the position and coordination of Mg1 is similar to that observed for the Gla domain of FIX in the pres-  (19) (PDB code 1DAN) in the presence of Ca 2ϩ is superimposed on the present Ca 2ϩ /Mg 2ϩ structure and is shown in magenta. The C␣ atoms of the Gla domain residues 1-46 were excluded in superpositioning. For orientation, Ser 195 (chymotrypsin numbering) is shown and colored by atom type where oxygens are red, nitrogens are blue, and carbons are green. Shown on the right is an extended view of the hydrophobic interactions between sTF and the FVIIa Gla domain. The ␣-helices are presented as ribbon cylinders and ␤-strands as thick arrows. FVIIa is in yellow, and sTF is in magenta. Residues Leu 13 L, Phe 31 L, Arg 36 L, Leu 39 L, and Phe 40 L of FVIIa that interact with residues Tyr 156 T, Trp 158 T, Cys 186 T, Val 207 T, and Cys 209 T of sTF are labeled without the chain identifier. Overall, these residues make shorter contacts than that seen in the Banner structure (19). ence of Ca 2ϩ /Mg 2ϩ (57) and of FXa in the presence of Mg 2ϩ only (58).
Ca 2ϩ at position 2 coordinates to Gla26, Gla29, and to a water molecule as in the structure of Banner et al. (19). However, coordination to Gla7 in the D-FFR-VIIa structure (19) is replaced by coordination to two water molecules (S508 and S602) that occupy the Gla7 position in the present structure. Ca 2ϩ at position 3 coordinates to Gla16, Gla26, and Gla29 as well as to three water molecules one of which, namely S602, occupies the same position as Gla7 O⑀ 2 in the D-FFR-VIIa structure (19).
Mg 2ϩ at position 4 coordinates to Gla16 and Gla26, as is the Ca 2ϩ in the Banner structure (19). Instead of direct coordination of Gla6 and Gla7 to Ca 2ϩ in the D-FFR-VIIa structure, Gla7 coordinates to Mg4 via two water molecules (S209 and S363). Furthermore, S209 coordinates to O␦ 1 of Gln2L as compared with its direct coordination with Ca4 in the structure of Banner et al. (19). Again, the distances for the Mg4 coordination are shorter (Table 2), and its position is compatible with that of FXa structure in the presence of Mg 2ϩ (58).
Ca 2ϩ at position 5 coordinates to Gla16, Gla20, and to the carbonyl O of Ala-1L similar to that in the D-FFR-VIIa structure (19). However, instead of coordination to Gla6, two water molecules are present in the present structure, one (S630) of which coordinates to Gla7. Ca 2ϩ at position 6 coordinates to Gla6, Gla20, and to a water molecule (S630), which is connected to Gla7. In the D-FFR-VIIa structure, coordination to only Gla20 is observed.
Mg 2ϩ at position 7 coordinates to Gla14 and Gla19, as is the case for Ca 2ϩ at this position in the D-FFR-VIIa structure (19). The coordination distances are short and are compatible with Mg 2ϩ and not Ca 2ϩ (Table 2). This position and coordination of Mg7 is consistent with a previous report of FVIIa/sTF in Mg 2ϩ /Ca 2ϩ /citrate (PDB code 1Z6J). However, the reported coor-  (19). The C␣ atoms used for superpositioning were residues 13L-46L. The Ca 2ϩ /Mg 2ϩ structure is in blue and the Ca 2ϩ structure is in magenta. Phe 4 , Leu 5 , Gla6, Gla7, and Leu 8 are depicted for both structures. Ca 2ϩ (blue) and Mg 2ϩ (cyan) ions for the present structure as well as Ca 2ϩ ions (magenta) for the PDB code 1DAN (19) are shown as spheres. In summary, in the present structure, two water molecules (S508 and S602) occupy the position of the side chain of Gla7 seen in the Banner structure and coordinate with Ca2 and Ca3. Furthermore, Gla7 occupies the position of Gla6 in the Banner structure and substitutes for it directly in coordinating to Ca5 and via a water molecule (S363) to Mg4 (Fig. 2B). The side chains of Gla6 and Gla7 are well ordered (Fig. 2B) and provide additional coordination ( Table 2) not seen in the Banner structure.
Ca 2ϩ Binding to FVIIa, Gla-domainless FVIIa, and Prothrombin Fragment 1-The goals of these experiments were 2-fold. The first goal was to determine the number of Ca 2ϩ sites that would be occupied by FVIIa and prothrombin fragment 1 in the presence of plasma concentrations of 0.6 mM Mg 2ϩ (65,66). The second goal was to determine the Ca 2ϩ sites that would be occupied by FVIIa under our crystallization conditions (5 mM Ca 2ϩ and 50 mM Mg 2ϩ ). Prothrombin fragment 1 was used as a substitute for the Gla domain of FVIIa because we were unable to obtain large enough amounts of the Gla domain of FVIIa that were suitable for direct Ca 2ϩ binding studies. The results of the Ca 2ϩ binding experiments are presented in Fig. 3 and summarized in Table 3. In agreement with previous data (20,67), full-length FVIIa bound nine, Gla-domainless FVIIa bound two, and prothrombin fragment 1 bound seven Ca 2ϩ ions in the absence of Mg 2ϩ (Fig. 3, A-C). The two Ca 2ϩ -binding sites in the Gla-domainless FVIIa could not be displaced by Mg 2ϩ (Fig. 3C). In full-length FVIIa and in prothrombin fragment 1, Mg 2ϩ displaced a maximum of three Ca 2ϩ sites under plasma (Fig. 3, A and B) or crystallographic concentrations of Mg 2ϩ (Fig. 3C). From these data, we conclude that in full-length FVIIa, Mg 2ϩ occupies three sites in the Gla domain. Based upon the Ca 2ϩ /Mg 2ϩ structure of FIX Gla domain (57) as well as the  (58), these three sites were identified at positions 1, 4, and 7. The Mg 2ϩ ions at these sites in the present structure coordinate to their respective ligands with distances compatible with Mg 2ϩ and not Ca 2ϩ ( Table 2). Thus, in systems with and without PL, plasma concentrations of Mg 2ϩ potentiate the activation of FX 5 to 8-fold at physiologic concentrations of Ca 2ϩ . Thus, the three sites in the FVIIa Gla domain that are predicted to be occupied by Mg 2ϩ (Fig. 3) in vivo are likely to play a significant role in FX activation (Fig. 4) during physiologic coagulation (25,26).

Role of Mg 2ϩ in Activation of FX by FVIIa/TF-We
EGF1 and EGF2 Domains-The Ca 2ϩ -binding site in the EGF1 domain is similar to that of Banner et al. (19) and Zhang et al. (30) except that the present structure has two water molecules as compared with one in the structure of Zhang et al. (30) and none in the structure of Banner et al. (19). The distances of ligands coordinating to Ca 2ϩ are ϳ2.4 Å ( Table 4) in agreement with the biochemical data presented in Fig. 3C, indicating that Mg 2ϩ cannot displace the EGF1 domain Ca 2ϩ site. As shown in Fig. 5A, the EGF1 domain Ca 2ϩ (Ca8) is coordinated to five protein ligands composed of the carbonyl O of Gln 64 L, the carbonyl O of Gly 47 L, the side chain carboxyl group of Asp 46 L, and both carboxyl groups of Asp 63 L as well as to two water molecules, which are well ordered. All major hydrophobic and polar interactions observed previously (19,30) involving the EGF1 and EGF2 domains with TF were observed in the present pAB-VIIa/sTF structure. For brevity, these are not listed here again.
Zn 2ϩ Sites and the Ca 2ϩ Site in the Protease Domain of FVIIa-The protease domain of FVIIa has been shown to bind two Zn 2ϩ ions (34). Zn 2ϩ has also been shown to inhibit the activity of FVIIa and reduce its affinity for TF (34,35). Based upon mutagenesis and modeling studies, it was proposed that the Zn 2ϩ sites involve His 216 H (76) and His 257 H (117) (34). This information was utilized in locating the Zn 2ϩ sites in the protease domain (Fig. 5B) (69), and two well ordered water molecules. The distances and geometry for the two Zn 2ϩ sites are given in Table 4. Zn1 is connected to the protease domain Ca 2ϩ site via the carboxylate side chain of Glu 220 H (80), whereas Zn2 is connected to the Ca 2ϩ site via water molecules. Except for Glu 220 H (80), all side chains involved in Zn1 and Zn2 coordination are unique to FVIIa (68) and are ideal for Zn 2ϩ coordination (see "Discussion"). Furthermore, refining the structure with water mole- cules at these two sites gave B values (crystallographic temperature factors) of ϳ12.0, indicating that these sites are occupied by ions with number of electrons much higher than water. These observations further validate assigning Zn 2ϩ ions to these sites.
The protease domain Ca 2ϩ -binding site in the pAB-VIIa/sTF structure is located between the two Zn 2ϩ sites and is similar to that described previously (19,30). Ca 2ϩ coordinates to the side chains of Glu 210 H (70) and Glu 220 H (80), to the carbonyl oxygens of Asp 212 H (72) and Glu 215 H (75), as well as to two well ordered water molecules (Fig. 5B). Moreover, in agreement with Zhang et al. (30), we were also unable to observe coordination of Asp 217 H (77) to Ca 2ϩ via a water molecule as initially observed by Banner et al. (19). However, Asp 217 H (77) links to Zn2 site via a water molecule (Fig. 5B and Table 3).
Putative Na ϩ Site in the Protease Domain of FVIIa-We recently observed that Na ϩ increases the catalytic activity of FVIIa in the absence of Ca 2ϩ and a mutant in which Phe 374 H

TABLE 4
The EGF1 domain Ca 2؉ and the protease domain Ca 2؉ , Na ؉ , and Zn 2؉ coordination in the pAB-VIIa/sTF crystal structure The * denotes ligands that are in the apical position in octahedral geometry. The remaining ligands occupy the square planar positions. Residue numbering system for Ca8 is that of light chain of FVIIa and that of Ca9, and Zn1, Zn2 and Na in the protease domain is that of chymotrypsin. S denotes solvent (water).

Ion
Ligand a The site occupancy for each Zn 2ϩ site appears to be 1. This is based upon the fact that the B-factors dropped from the low 40s to ϳ12 when the structure was refined with water molecules only. For Na ϩ , the site occupancy could not be determined because when a water molecule was substituted for Na ϩ , the B-factor dropped only slightly from 22 to 19.
(225) is mutated to Pro loses the Na ϩ potentiation effects (69). However, in the presence of Ca 2ϩ , physiologic concentrations of Na ϩ have minimal effect on the activity of FVIIa (69,70). Based upon homology, FVIIa might contain a Na ϩ site at a position similar to that proposed for FXa, FIXa, and APC (36 -40). In previous FVIIa structures, a water molecule has been placed at the proposed Na ϩ site (19, 29 -31). The location of this putative Na ϩ site in FVIIa and its ligands are shown in Fig. 6A and In previous studies it has been proposed that the Na ϩ site in FIXa is linked to the FVIIIa-binding site (40), and in FXa it is linked to the FVa-binding site (71,72). In the pAB-FVIIa/sTF structure, we observed that Phe 374 H (225), a key determinant of the Na ϩ -binding site, is located in a hydrophobic cavity con- We used surface plasmon resonance to experimentally determine the effect of Na ϩ on the binding of FVIIa to sTF. As shown in Fig. 7, in the presence of Na ϩ , the k on for binding was 4.6 Ϯ 0.6 ϫ 10 5 M Ϫ1 s Ϫ1 ; k off was 2.4 Ϯ 0.2 ϫ 10 Ϫ3 s Ϫ1 , and K d was 5.2 Ϯ 0.3 nM. The K d value in the presence of Na ϩ is similar to that observed by Petrovan and Ruf (73). In the presence of Ch ϩ , the k on was 1.8 Ϯ 0.3 ϫ 10 5 M Ϫ1 s Ϫ1 ; k off was 2.5 Ϯ 0.2 ϫ 10 Ϫ3 s Ϫ1 , and K d was 13.9 Ϯ 0.7 nM. Thus, Na ϩ affects the binding of FVIIa to sTF by ϳ3-fold, which is primarily via an effect on k on . This may be due to stabilization of the interactions noted in the preceding paragraph.
Atypical Conformation of the Lys 341 H-Gly 342 H (192)(193) Peptide Bond in the pAB-VIIa/sTF Structure-During intermediate stages of refinement, it was observed that the carbonyl O of Lys 341 H (192), which normally points away from the oxyanion hole in serine proteases, was positioned in negative electron density (F obs Ϫ F calc ) contoured at Ϫ2.5 (Fig. 8A). At the same Electron density (2F obs Ϫ F calc ) contoured at 1 (gray) for Ca 2ϩ (magenta sphere) and two water molecules (red spheres) is shown. Electron density contoured at 5 for Ca 2ϩ is shown in blue. Note that in the presence of Ca 2ϩ , Mg 2ϩ will not occupy this site. All eight coordination ligands (black dashed lines) are shown, and the residues labeled are those of the light chain of FVIIa. B, location of the Zn 2ϩ sites and their linkage to the protease domain Ca 2ϩ site. Electron density (2F obs Ϫ F calc ) contoured at 1 (gray) of Zn 2ϩ (cyan spheres), Ca 2ϩ (magenta sphere), and water molecules (red spheres) is shown. The electron density contoured at 3 for Zn 2ϩ and Ca 2ϩ ions is shown in blue. Note the linkage between the Zn 2ϩ sites and the Ca 2ϩ site. The metal ion coordination to its ligands and H-bonds between water molecules is shown with black dotted lines. Residue numbering used in the figure is that of chymotrypsin.
time, it was also noted that the side chain of Gln 286 H (143) was located in negative density as well. Moreover, there was significant positive density (F obs Ϫ F calc ) contoured at 4 (Fig. 8A) where Structures of Benzamidine-VIIa/sTF and D-FPR-VIIa/sTF-Both the benzamidine-VIIa/sTF and DFPR-VIIa/sTF structures are comparable with the pAB-VIIa/sTF structure with respect to the Gla domain loop placement as well as Na ϩ , Ca 2ϩ , and Mg 2ϩ binding. However, the benzamidine-VIIa/sTF crystals, which were grown in the absence of added Zn 2ϩ , have water molecules at the two Zn 2ϩ positions. In the benzamidine-VIIa/ sTF structure, included in this report, the Zn 2ϩ sites were initially refined with water molecules and had B-factors of 19 and 22; however, when the water molecules were replaced with Zn 2ϩ , the B-factors increased to 56 and 63. For consistency, the Zn 2ϩ are left to occupy the Zn 2ϩ sites in the structure. It is realized that in the benzamidine-VIIa/sTF structure included FIGURE 6. Putative Na ؉ site in the protease domain of FVIIa and the environment surrounding Phe 225 . A, Na ϩ site coordination. The Na ϩ site in FVIIa involves coordination from four carbonyl groups (184, 185, 221, and 224) from the protein and two water molecules. Electron density (2F obs Ϫ F calc ) is contoured at 1 (gray) for Na ϩ (cyan sphere) and water molecules (red spheres) with the exception of S144 which is contoured at 0.7. The electron density contoured at 2.5 for Na ϩ is shown in blue. Note that the Na ϩ site is linked through water molecules to the carboxylate of Asp 189 . The salt bridge between Asp 189 and pAB is also shown. B, hydrophobic environment surrounding Phe 225 . The ␣-helices are presented as ribbon cylinders and ␤-strands as thick arrows. FVIIa is in cyan, and sTF is in yellow. The hydrophobic residues that surround Phe 225 and lead to Met 164 , Gln 166 , and Asp 167 that interact with TF are depicted and colored by atom type. Residues whose CϭO groups are involved in coordinating to Na ϩ are also shown. Na ϩ , magenta sphere; water, red spheres. Residue numbering is that of chymotrypsin. here, the Zn 2ϩ only partially occupies these sites. In the other two benzamidine-VII/sTF structures and in the D-FPR-VIIa/ sTF structures, which contained added 20 M Zn 2ϩ , the B-factors for the two Zn 2ϩ were in the low 40s, indicating more complete Zn 2ϩ occupancy.
As was the case with the pAB-VIIa/sTF structure, the Lys 341 H (192)-Gly 342 H (193) peptide bond in benzamidine-VIIa/sTF structures was in nonstandard orientation such that the Gly 342 H (193) amide N points away from the oxyanion hole, and the carbonyl O of Lys 341 H (192) points into the oxyanion hole. This was noted in all three benzamidine-VIIa/sTF crystal structures, two without the added Zn 2ϩ , including the one presented here (Table 1 and Fig. 8C) and the one with added Zn 2ϩ .
Next, we examined whether soaking of benzamidine-VIIa/ sTF crystals with an active site peptide inhibitor, D-FPR-ck, could restructure the Lys 341 H-Gly 342 H (192)(193) peptide bond to form the oxyanion hole. Refinement of the benzamidine-VIIa/sTF crystal that has been soaked with D-FPR-ck revealed benzamidine displacement, D-FPR incorporation, and induction of the oxyanion hole by a flip of the Lys 341 H-Gly 342 H (192)(193) peptide bond in FVIIa (Fig. 8D). Now, the Gly 342 H (193)-amide nitrogen of FVIIa is favorably situated to be a part of the oxyanion hole and makes an H-bond with the negatively charged carbonyl oxygen (oxyanion) of the P1 Arg residue of D-FPR (Fig. 8D). The formation of this H-bond is required for stabilization of the tetrahedral transition complex, an intermediate formed during peptide bond cleavage. Cumulatively, our data imply that the enhancement of FVIIa catalytic activity upon TF binding does not entail correction of the impaired oxyanion hole to the standard conformation; however, active site peptidyl inhibitor/substrate binding does induce the standard oxyanion hole conformation.

DISCUSSION
The absence of an oxyanion hole in a crystal of FVIIa/sTF at 2.2 Å with an amidinophenylurea-based inhibitor was first reported in 2003 (74,75). In this series of inhibitors, the nitro- In the pAB-VIIa/sTF structure (Fig. 8B), the same two H-bonds involving the p-amino group of the benzamidine moiety and the carbonyl group of Lys 341 H (192) with the hydroxyl of Ser 344 H (195) are observed. These observations indicate that the other segments of the respective inhibitors (32, 74 -76) do not contribute to the nonstandard conformation of the oxyanion hole. The nonstandard oxyanion hole conformations observed in FVIIa with the above series of inhibitors as well as pAB may stem from the presence of nitrogen at the para position of the benzamidine moiety.
To ascertain whether the presence of nitrogen at the para position of the benzamidine moiety of the various inhibitors is the cause of the nonstandard oxyanion hole conformation in FVIIa, we crystallized FVIIa/sTF with benzamidine. In all three benzamidine-VIIa/sTF structures in the presence and absence of added ZnCl 2 , we observed the nonstandard oxyanion hole conformation. This strongly indicates that the nonstandard oxyanion hole conformation in FVIIa is not related to the unusual properties of the FVIIa inhibitors used in previous studies (32, 74 -76). The structure obtained with benzamidine leads us to propose that the nonstandard oxyanion hole conformation is rather an inherent property of FVIIa.
Soaking benzamidine-VIIa/sTF crystals with D-FPR-ck resulted in benzamidine displacement and D-FPR incorporation with resultant flip of the Lys 341 H- Gly 342 H (192-193) pep-tide bond such that the oxyanion hole is fully formed (Fig. 8D). For this to occur the H-bond between the amide N of Gly 342 H (193) and side chain of Gln 286 H (143) must first be broken. Then, either concurrently or subsequently, the Lys 341 H-Gly 342 H (192)(193) peptide bond must be flipped with resultant formation of three H-bonds, including one between the carbonyl group of Lys 341 H (192) and the backbone N of Gln 286 H (143) and two between the oxyanion and the amide nitrogens of Gly 342 H (193) and Ser 344 H (195). The negative charge on the carbonyl oxygen of the transition state intermediate should provide enough energy to overcome the energetic barriers of the first two steps involving breaking the H-bond and flipping the peptide bond. Subsequently, formation of the three H-bonds results in stabilization of the tetrahedral intermediate, which is necessary for catalysis. The crystal soaking experiments strongly support that it is the substrate/inhibitor active site occupancy with a developing oxyanion and not TF binding that induces formation of the oxyanion hole in FVIIa. The competent oxyanion hole observed in several FVIIa crystal structures in the presence and absence of TF with transition state analogue inhibitors (19, 28 -30, 77) must then be due to the above conformational alterations in solution.
The nonstandard oxyanion hole conformation has also been observed for Staphylococcus aureus exfoliative toxin A (78,79), and both nonstandard and standard conformations have been observed for the toxin B (80,81). In the case of toxin A, it is the proline at position 192 that causes the peptide bond between Pro 192 and Gly 193 to flip ϳ180°relative to that typically seen in serine proteases. Because both standard (competent) and nonstandard (incompetent) conformations have been observed for the toxin B (80,81), it has been suggested that upon binding of the substrate (providing oxyanion), the peptide bond between Pro 192 and Gly 193 in toxin A would flip 180°to provide competent conformation for catalysis (78,79). Interestingly, thrombin has also been shown to have a reversed oxyanion hole in the absence of Na ϩ (82). Based upon these observations coupled with our FVIIa/sTF soaking experiments and free energy simulations, it appears that there is enough flexibility to adopt optimal oxyanion hole conformation upon substrate binding as suggested by Cavarelli et al. (79) and Bobofchak et al. (83).
FVIIa/TF recognizes its physiologic macromolecular substrates, FIX and FX, primarily via exosites distant from the active site. In this regard, the Gla and/or EGF1 domains of FIX and FX have been implicated to be the primary determinants in binding to the FVIIa/TF complex (49, 84 -86, 88 -92). Once the trimolecular complex is formed, the cleavage site peptide sequence in FIX or FX approaches the active site cleft of FVIIa/TF and induces formation of the oxyanion hole. This mechanism provides discriminating specificity in activation of FIX and FX by FVIIa/TF.
One should note that in the reported structure of uninhibited FVIIa, the Lys 341 H-Gly 342 H (192-193) peptide bond had normal conformation (31). However, in this structure, the active site of FVIIa was initially occupied by a sulfate ion in the oxyanion hole similar to the carbonyl group of P1 residue. This could reorient the Lys 341 H-Gly 342 H (192-193) peptide bond as seen in the D-FPR-VIIa (present paper), D-FFR-VIIa (19), and BPTI-VIIa (30) (19).
Concerning metal binding studies, Mg 2ϩ cannot displace Ca 2ϩ from the EGF1 domain or protease domain Ca 2ϩ sites in FVIIa. This is supported by the metal-ligand coordination distances, which are compatible with Ca 2ϩ , despite the presence of excess Mg 2ϩ . The protease domain Ca 2ϩ site is similar to that described for trypsin, which also could not be replaced by Mg 2ϩ in the crystals (44). Moreover, the EGF1 domain of FXa was disordered when the crystals were grown in the presence of Mg 2ϩ (58), in agreement with our data that FVIIa EGF1 domain cannot bind Mg 2ϩ . The significance of these two Ca 2ϩ sites is that their occupancy is required for FVIIa binding to TF and development of its catalytic activity.
The data presented in Fig. 3 demonstrate that under physiologic and crystallographic conditions, the FVIIa Gla domain binds three Mg 2ϩ and four Ca 2ϩ ions. Three Mg 2ϩ ions were located based upon the structure of the Gla domain of FXa in the presence of Mg 2ϩ (58) and the structure of FIX Gla domain in the presence of Ca 2ϩ /Mg 2ϩ (57). The four Ca 2ϩ ions were located based upon the Ca 2ϩ structures of prothrombin fragment 1 (59) and D-FFR-VIIa (19). Under plasma concentrations of ϳ1.1 mM free Ca 2ϩ and ϳ0.6 mM free Mg 2ϩ , we predict that these three sites will be occupied by Mg 2ϩ . Interestingly, these three Mg 2ϩ sites are certainly the ones that could not be displaced by Ca 2ϩ in the Gla domain of prothrombin (Fig. 3) (93,94) or FVIIa (Fig. 3). In support of this, data have also been presented that demonstrate the existence of specific Mg 2ϩbinding sites in the Gla domain of prothrombin to which Ca 2ϩ has difficult access (95). Thus, location of Mg 2ϩ and Ca 2ϩ sites that we have identified in the Gla domain of FVIIa are consistent with the biochemical and crystallographic data.
The binding of the above Mg 2ϩ sites is expected to be cooperative in nature (58,(93)(94)(95)(96)(97)(98), and filling of these sites would result in an intermediate folding state of the Gla domain in which the loop (residues L1-L12) of FVIIa is disordered as observed in the case of FXa (58). Under in vivo conditions, it is likely that Mg 2ϩ and not Ca 2ϩ affords this intermediate conformational state (92,94). Filling of the remaining four Ca 2ϩ -specific sites to the intermediate state will promote proper folding of the N-terminal residues into the loop conformation for binding to the PL surface. This two-state sequential model is consistent with the earlier observations of Borowski et al. (98) and Wang et al. (58). Furthermore, it is possible that the Gla domain conformation seen in the present structure represents an advanced intermediate folding stage and that the Ca 2ϩ sites identified are only partially occupied.
Evidence exists that Mg 2ϩ plays a role in physiologic coagulation (23-26, 93, 99). It supports the activation of FIX by FXIa and activation of FX by FIXa at physiologic concentrations of Ca 2ϩ (23,24,99). It also supports the activation of prothrombin by FXa at very low concentrations of Ca 2ϩ (93). In this study, we provide evidence that it supports the activation of FX by FVIIa/TF (Fig. 4). Thus, it appears that Mg 2ϩ and Ca 2ϩ work in concert to promote coagulation in vivo. Location (Fig. 6A) of the Na ϩ site in FVIIa is based upon the previous structures of other vitamin K-dependent proteins (36 -40). Because sodium ions are particularly difficult to prove from the electron density, we cannot make a definite statement at this time regarding the Na ϩ site in FVIIa. Anomalous diffraction data are needed to unequivocally establish the existence of a Na ϩ site in FVIIa. Nonetheless, as in FIXa (40) and FXa (72), Na ϩ increases the affinity of FVIIa for its cofactor TF (Fig. 7). Thus, one function of Na ϩ in blood clotting proteases may be to influence cofactor binding.
As shown in Fig. 8, the Zn1 site involves side chains of His, Glu, and Ser and three water molecules. The Zn2 site involves side chains of His, Asp, and Lys, two water molecules, and the main chain carbonyl group (Fig. 8). Both metal sites have octahedral coordination geometry (Table 4), and the implicated side chains are excellent candidates for binding to zinc (100). Because the side chain of Glu 220 H (80) is involved in coordination to Zn1, and the carboxyl group of Asp 219 H (79) and the carbonyl group of Gly 209 H (69) are involved in coordination to Zn2, binding of Ca 2ϩ to the 210H-220H loop [70 -80 loop] could attenuate the binding of Zn 2ϩ . Biochemical data support this concept (34,35). His 211 H (71), which was previously considered a candidate for binding to Zn 2ϩ , is not unique to FVIIa and does not appear to be involved in binding to Zn 2ϩ (Fig. 8).
Platelets store large amounts of Zn 2ϩ in their cytoplasm and ␣-granules at concentrations 30 -60 times that of the plasma (101). Notably, upon platelet activation at the site of clot formation, ␣-granules would be released, which would increase the local Zn 2ϩ concentration. This increased local concentration of Zn 2ϩ is likely to inhibit FVIIa bound to TF. Thus, after the initiation phase of clotting is achieved, Zn 2ϩ exerts a mechanism of control on FVIIa activity and regulates its ability to activate its physiologic substrates FIX and FX. Understanding the precise location of zinc sites could help mutagenesis studies in design of a better therapeutic FVIIa molecule, which is resistant to inhibition by zinc. Such data could also help understand how Zn 2ϩ inhibits the activity of FVIIa.

CONCLUSION
In this study we have identified three Mg 2ϩ sites in the Gla domain of FVIIa. It is quite possible that Mg 2ϩ in vivo occupies these sites and promotes coagulation. Notably, under physiologic conditions, the protease and EGF1 domain Ca 2ϩ sites will be solely occupied by Ca 2ϩ . Furthermore, we have identified two Zn 2ϩ sites in the protease domain that were not defined previously. The ligands for the Zn 2ϩ sites are unique to FVIIa and are in agreement with the biochemical data. We also propose a putative Na ϩ site in FVIIa. Na ϩ appears to promote whereas Zn 2ϩ appears to down-regulate FVIIa activity. Atypical conformation of the Lys 341 H-Gly 342 H (192-193) peptide bond in FVIIa/TF that results in the absence of an oxyanion hole characteristic of active serine proteases is unique to FVIIa. Because serine proteases play an important role in many physiologic processes, it is critical to understand how a given serine protease recognizes its substrate among a milieu of other proteins. One determining factor is the flanking sequences (P4 -P4Ј residues) surrounding the peptidyl cleavage site in the substrates (87, 102, 103). Recently, a new concept has emerged in which exosites remote from the cleavage site recognition sequences play a dominant role in substrate specificity. In FVIIa/TF, binding to exosites of FIX and FX play a large role in determining specificity. Following the cofactor-enzyme-substrate complex assembly via these exosites, the active site then approaches the cleavage site for proper catalysis. Such a mechanism introduces a higher level of selectivity and specificity.