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J. Biol. Chem., Vol. 281, Issue 34, 24873-24888, August 25, 2006

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

UNPREDICTED CONFORMATION OF THE 192-193 PEPTIDE BOND AND MAPPING OF Ca²⁺, Mg²⁺, Na⁺, AND Zn²⁺ SITES IN FACTOR VIIa^*

S. Paul Bajaj¹, Amy E. Schmidt², Sayeh Agah, Madhu S. Bajaj, and Kaillathe Padmanabhan^¶

From the Protein Science Laboratory, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, and the Department of Medicine-Pulmonary Division, UCLA, Los Angeles, California 90095 and the ^¶Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Received for publication, September 12, 2005 , and in revised form, June 1, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Factor VIIa (FVIIa) consists of a -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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
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 50,000 (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 (3-5). Several coagulation enzymes, including FIXa, FXa, and FVIIa, have been shown to activate FVII (2, 5-9). In each case, the activation of FVII involves the cleavage of a single peptide bond between Arg¹⁵² and Ile¹⁵³, 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 254-residue 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 His⁵⁷, Asp¹⁰², and Ser¹⁹⁵, with Asp¹⁸⁹ (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 (12-15). The two fibronectin type III domains are connected end-to-end at an angle of 120° (12-15). 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, Ca²⁺, 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 Ca²⁺ 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 Ca²⁺ 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 Mg²⁺ plays an important role in physiologic coagulation (23-26). However, Mg²⁺ sites in FVIIa have not been identified. Furthermore, it has been proposed that FVIIa contains a Na⁺ site (27), but the site has not been defined in any of the crystal structures (19, 28-33). Notably, FVIIa also contains two Zn²⁺ sites in its protease domain (34). Occupancy of the Zn²⁺ sites inhibits FVIIa catalytic activity in the absence but poorly in the presence of Ca²⁺. This observation led to the hypothesis that Zn²⁺ binds to the protease domain Ca²⁺ loop and attenuates the activity of FVIIa (34, 35). As is the case for Na⁺, the Zn²⁺ 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 Ca²⁺,Mg²⁺,Na⁺, and Zn²⁺. The Gla domain has four Ca²⁺ and three Mg²⁺ sites, and the protease domain has two Zn²⁺ sites unique to FVIIa. The Na⁺ site is structurally similar to the Na⁺ 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Reagents—Magnesium chloride, calcium chloride, and PEG 4000 were purchased from Hampton Research. ⁴⁵CaCl₂ 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 Ca²⁺-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 20 mg/ml and stored at -80 °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 10 mg/ml and stored at -80 °C. Both proteins were 98% 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 µg/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 CaCl₂ ± 20 µM ZnCl₂, and 10 mM pAB or 10 mM benzamidine, whereas the reservoir solution contained 16-22% PEG 4000, 100 mM MgCl₂, and 20 mM BisTris, pH 6.5. Drops were prepared by mixing 2 µl of protein solution with 2 µl 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 µl of 100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 20 µM ZnCl₂, and 10 mM CaCl₂ was mixed with 50 µl of the reservoir solution (22% PEG 4000, 100 mM MgCl₂, pH 6.5). The above mixture was allowed to equilibrate with 1 ml of the reservoir solution for 1 week. Five-µl volume of D-FPR-ck was added to the drop to achieve a final concentration of 250 µM inhibitor. Two benzamidine-VIIa/sTF crystals grown in the presence of 20 µM ZnCl₂ 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 µM ZnCl₂ 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 µM Zn²⁺, and the other two did not contain any added Zn²⁺. One of the crystals without added Zn²⁺ 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 ZnCl₂ and one without additional ZnCl₂, each diffracted to 2.2 Å, and the crystal (which contained ZnCl₂) 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 P2₁2₁2₁ 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 [PDB] ), 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 6° toward the TF2 domain, and that the loop has a slightly different conformation in comparison to the Ca²⁺ 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, Mg²⁺ ions were placed at the 1-, 4-, and 7-position (59, Tulinsky numbering),⁴ and Ca²⁺ 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 Ca²⁺, Na⁺, and Zn²⁺) were added to the model. At this point, it was also noted that the peptide bond between Lys³⁴¹H (192)⁵ and Gly³⁴²H (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.

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 Lys³⁴¹H (192)-Gly³⁴²H (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.

Ca²⁺ Binding to Various Fragments of FVIIa or Prothrombin Fragment 1—Calcium binding was determined by the technique of equilibrium dialysis using ⁴⁵Ca²⁺ 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 Ca²⁺ 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 Gla-domainless FVIIa (20) were used. The concentration of protein ranged from 20 to 40 µM. 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 Ca²⁺/Mg²⁺ conditions as follows: 1) 1.1 mM Ca²⁺, 0 mM Mg²⁺; 2) 1.1 mM Ca²⁺, 0.6 mM Mg²⁺; and 3) 1.7 mM Ca²⁺, 0 mM Mg²⁺. Each reaction contained 10 nM FX and either 1 nM FVIIa, 10 nM sTF, or 1 nM FVIIa, 10 pM rTF, 50 µM PL. FVIIa was incubated with sTF or 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-µl aliquots were removed and added to tubes containing 5 µl of 0.5 M EDTA, pH 8.0 (final EDTA concentration of 20 mM), to stop the reaction. From this mixture, 90 µl was removed and placed in a well on an Immulon 4 HBX flat bottom 96-well microtiter plate (Dynex Technologies), and 10 µl of benzoyl-Ile-Glu-Gly-Arg-p-nitroaniline was added to yield a final concentration of 250 µM. The p-nitroaniline formed was measured (A₄₀₅/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 CaCl₂ and either 185 mM ChCl or 185 mM NaCl (flow rate 10 µl 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 CaCl₂ 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
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 Ca²⁺/Mg²⁺ 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 Zn²⁺ sites and one putative Na⁺ site are identified in the protease domain. 3) Three Ca²⁺ sites in the Gla domain at positions 1, 4, and 7 were replaced by Mg²⁺ in the new structures. Binding and kinetic data are presented that show that under physiologic conditions these three sites are occupied by Mg²⁺ and play an important role in biologic coagulation. However, the EGF1 and protease domain Ca²⁺ sites were not displaced by Mg²⁺. 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.

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 Ca²⁺ structure (19), the root mean square deviation was 0.7 Å. However, the Gla domain in the present Ca²⁺/Mg²⁺ structure is tilted 6° toward the C-terminal TF2 domain of sTF. This tilt results in closer and tighter hydrophobic interactions involving Gla domain residues Leu¹³L, Phe³¹L, Arg³⁶L, Leu³⁹L, and Phe⁴⁰L with C-terminal TF residues Tyr¹⁵⁶T, Trp¹⁵⁸T, Cys¹⁸⁶T, Val²⁰⁷T, and Cys²⁰⁹T. 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 Ca²⁺ 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 >0.5 Å are as follows: 3.86 versus 4.33 Å between Phe³¹L C₁ and Trp¹⁵⁸T Cz₂; 4.18 Å versus 4.91 Å between Arg³⁶L C and Trp¹⁵⁸T Cz₂; 4.75 Å versus 6.64 Å between Leu³⁹L C₁ and Tyr¹⁵⁶T C_D2; and 4.14 Å versus 4.65 Å between Leu³⁹L C and Trp¹⁵⁸T 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 Mg²⁺ and citrate. This is most likely because of the binding of Mg²⁺ to Gla25 and Gla29 (see below) in the Ca²⁺/Mg²⁺ structures.

View larger version (59K): [in this window] [in a new window]   FIGURE 1. View of the pAB-VIIa/sTF complex. Ribbon representation of the pAB-VIIa/sTF with metal ions is shown. The protease domain of FVIIa is in blue; the EGF2 domain is in cyan; the EGF1 domain is in red; and the Gla domain is in yellow. The four Ca2+ in the Gla domain, one (Ca8) in the EGF1 domain, and one (Ca9) in the protease domain are shown as black spheres. The three Mg2+ in the Gla domain are shown as cyan spheres. Mg1 is in the back of the structure and is shown by an arrow. The Na+ (putative) in the protease domain is shown as a lavender sphere, and the two Zn2+ (Zn1 and Zn2) are shown as yellow spheres. The N-terminal domain of sTF is shown in black (TF1) and the C-terminal domain is shown in green (TF2). The FVIIa/sTF structure of Banner et al. (19) (PDB code 1DAN) in the presence of Ca2+ is superimposed on the present Ca2+/Mg2+ structure and is shown in magenta. The C atoms of the Gla domain residues 1-46 were excluded in superpositioning. For orientation, Ser195 (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 Leu13L, Phe31L, Arg36L, Leu39L, and Phe40L of FVIIa that interact with residues Tyr156T, Trp158T, Cys186T, Val207T, and Cys209T of sTF are labeled without the chain identifier. Overall, these residues make shorter contacts than that seen in the Banner structure (19).

The coordination of each Ca²⁺ and Mg²⁺ ion in the Ca²⁺/Mg²⁺ structure to the Gla residues and water molecules is shown in Fig. 2B. All Ca²⁺, Mg²⁺, and water molecules are well ordered, and the coordinations are listed in Table 2. Mg²⁺ at position 1 has similar coordination to the Ca²⁺ in the D-FFR-VIIa structure except that the distances are shorter (Table 2), compatible with Mg²⁺ and not Ca²⁺. 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 presence of Ca²⁺/Mg²⁺ (57) and of FXa in the presence of Mg²⁺ only (58).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Conformation of the loop and positions of the Ca2+ and Mg2+ ions in the FVIIa Gla domain. A, superpositioning of the Gla domain in the presence of Ca2+/Mg2+ versus Ca2+ (19). The C atoms used for superpositioning were residues 13L-46L. The Ca2+/Mg2+ structure is in blue and the Ca2+ structure is in magenta. Phe4, Leu5, Gla6, Gla7, and Leu8 are depicted for both structures. Ca2+ (blue) and Mg2+ (cyan) ions for the present structure as well as Ca2+ ions (magenta) for the PDB code 1DAN (19) are shown as spheres. N represents the N terminus of the Gla domain of FVIIa. B, coordination of the Ca2+ and Mg2+ ions in the Gla domain of the Ca2+/Mg2+ structure. Electron density (2Fobs - Fcalc) contoured at 1 of all nine Gla residues as well as that of Ca2+ (magenta spheres), Mg2+ (cyan spheres), and coordinating water molecules (red spheres)is shown. Coordination of Ca5 to O of Ala1L and H-bonds between Gla6 and the NH2 side chain of Arg9L, Gla7, and NofPhe4L, and a water molecule with O1 of Gln2L are indicated with dashed arrows. Black dashed lines depict all other coordinations and H-bonds. A stereo figure is given in the supplemental material as Fig. 1S.

Mg²⁺ at position 4 coordinates to Gla16 and Gla26, as is the Ca²⁺ in the Banner structure (19). Instead of direct coordination of Gla6 and Gla7 to Ca²⁺ in the D-FFR-VIIa structure, Gla7 coordinates to Mg4 via two water molecules (S209 and S363). Furthermore, S209 coordinates to O₁ 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 Mg²⁺ (58).

Ca²⁺ 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. Ca²⁺ 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.

Mg²⁺ at position 7 coordinates to Gla14 and Gla19, as is the case for Ca²⁺ at this position in the D-FFR-VIIa structure (19). The coordination distances are short and are compatible with Mg²⁺ and not Ca²⁺ (Table 2). This position and coordination of Mg7 is consistent with a previous report of FVIIa/sTF in Mg²⁺/Ca²⁺/citrate (PDB code 1Z6J). However, the reported coordination distances in the 1Z6J structure are long for Mg²⁺, and no other metals in the Gla domain were defined.

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.

Ca²⁺ 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 Ca²⁺ sites that would be occupied by FVIIa and prothrombin fragment 1 in the presence of plasma concentrations of 0.6 mM Mg²⁺ (65, 66). The second goal was to determine the Ca²⁺ sites that would be occupied by FVIIa under our crystallization conditions (5 mM Ca²⁺ and 50 mM Mg²⁺). 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 Ca²⁺ binding studies. The results of the Ca²⁺ 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 Ca²⁺ ions in the absence of Mg²⁺ (Fig. 3, A-C). The two Ca²⁺-binding sites in the Gla-domainless FVIIa could not be displaced by Mg²⁺ (Fig. 3C). In full-length FVIIa and in prothrombin fragment 1, Mg²⁺ displaced a maximum of three Ca²⁺ sites under plasma (Fig. 3, A and B) or crystallographic concentrations of Mg²⁺ (Fig. 3C). From these data, we conclude that in full-length FVIIa, Mg²⁺ occupies three sites in the Gla domain. Based upon the Ca²⁺/Mg²⁺ structure of FIX Gla domain (57) as well as the structure of FXa in the presence of Mg²⁺ (58), these three sites were identified at positions 1, 4, and 7. The Mg²⁺ ions at these sites in the present structure coordinate to their respective ligands with distances compatible with Mg²⁺ and not Ca²⁺ (Table 2).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. 45Ca2+ binding measurements by equilibrium dialysis. A, Ca2+ binding data for full-length FVIIa. The concentration of FVIIa was 1.5 mg/ml, and the concentration of calcium varied from 0.1 to 5 mM. Open circles, absence of Mg2+; closed circles, presence of 0.6 mM Mg2+ in buffer. B, Ca2+ binding data for prothrombin fragment 1. The concentration of prothrombin fragment 1 was 0.8 mg/ml, and the concentration of calcium varied from 0.1 to5mM. Open circles, absence of Mg2+; closed circles, presence of 0.6 mM Mg2+ in buffer. C, displacement of Ca2+ by Mg2+.Ca2+ sites were determined in the presence of various concentrations of Mg2+ at5mM constant Ca2+. Open circles, full-length FVIIa; closed circles, prothrombin fragment 1; open squares, Gla domainless FVIIa. Each point is an average of triplicate measurements, and r represents mol of Ca2+ bound/mol of protein.

EGF1 and EGF2 Domains—The Ca²⁺-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 Ca²⁺ are 2.4 Å (Table 4) in agreement with the biochemical data presented in Fig. 3C, indicating that Mg²⁺ cannot displace the EGF1 domain Ca²⁺ site. As shown in Fig. 5A, the EGF1 domain Ca²⁺ (Ca8) is coordinated to five protein ligands composed of the carbonyl O of Gln⁶⁴L, the carbonyl O of Gly⁴⁷L, the side chain carboxyl group of Asp⁴⁶L, and both carboxyl groups of Asp⁶³L 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.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Effect of plasma concentration of Mg2+ on FX activation by FVIIa/sTF and FVIIa/rTF. A, activation of FX by FVIIa/sTF. Each reaction contained 1 nM FVIIa, 10 nM sTF, and 10 nM FX in TBS, pH 7.5, containing 0.1% PEG 8000 and different concentrations of Ca2+ and Mg2+. At different times, the reaction was stopped by the addition of EDTA, and the FXa generated was measured using the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroaniline. Open circles, 1.1 mM Ca2+; open squares, 1.1 mM Ca2+ + 0.6 mM Ca2+; closed circles, 1.1 mM Ca2+ + 0.6 mM Mg2+. B, activation of FX by FVIIa/rTF. Each reaction contained 1 nM FVIIa, 10 pM rTF, 50 µM PL and 10 nM FX in TBS, pH 7.5, containing 0.1% PEG 8000 and different concentrations of Ca2+ and Mg2+. The amount of FXa generated was measured as described in A. Open circles, 1.1 mM Ca2+; open squares, 1.1 mM Ca2+ + 0.6 mM Ca2+; closed circles, 1.1 mM Ca2+ + 0.6 mM Mg2+.

Putative Na⁺ Site in the Protease Domain of FVIIa—We recently observed that Na⁺ increases the catalytic activity of FVIIa in the absence of Ca²⁺ and a mutant in which Phe³⁷⁴H (225) is mutated to Pro loses the Na⁺ potentiation effects (69). However, in the presence of Ca²⁺, physiologic concentrations of Na⁺ have minimal effect on the activity of FVIIa (69, 70). Based upon homology, FVIIa might contain a Na⁺ 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⁺ site (19, 29-31). The location of this putative Na⁺ site in FVIIa and its ligands are shown in Fig. 6A and Table 4. As is the case with FXa, FIXa, and APC, Na⁺ in FVIIa coordinates to the carbonyl oxygens of Tyr³³²H (184), Ser³³³H (185), Thr³⁷⁰H (221), and His³⁷³H (224) as well as to two water molecules, one of which is linked to Asp³³⁸H (189).

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Ca2+ site in the EGF1 domain and location of the Zn2+ sites and their linkage to the protease domain Ca2+ site in FVIIa. A, Ca2+ site in the EGF1 domain. Electron density (2Fobs - Fcalc) contoured at 1 (gray) for Ca2+ (magenta sphere) and two water molecules (red spheres) is shown. Electron density contoured at 5 for Ca2+ is shown in blue. Note that in the presence of Ca2+, Mg2+ 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 Zn2+ sites and their linkage to the protease domain Ca2+ site. Electron density (2Fobs - Fcalc) contoured at 1 (gray) of Zn2+ (cyan spheres), Ca2+ (magenta sphere), and water molecules (red spheres) is shown. The electron density contoured at 3 for Zn2+ and Ca2+ ions is shown in blue. Note the linkage between the Zn2+ sites and the Ca2+ 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.

We used surface plasmon resonance to experimentally determine the effect of Na⁺ on the binding of FVIIa to sTF. As shown in Fig. 7, in the presence of Na⁺, the k_on for binding was 4.6 ± 0.6 x 10⁵ M^-1 s^-1; k_off was 2.4 ± 0.2 x 10^-3 s^-1, and K_d was 5.2 ± 0.3 nM. The K_d value in the presence of Na⁺ is similar to that observed by Petrovan and Ruf (73). In the presence of Ch⁺, the k_on was 1.8 ± 0.3 x 10⁵ M^-1 s^-1; k_off was 2.5 ± 0.2 x 10^-3 s^-1, and K_d was 13.9 ± 0.7 nM. Thus, Na⁺ affects the binding of FVIIa to sTF by 3-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 Lys³⁴¹H-Gly³⁴²H (192-193) Peptide Bond in the pAB-VIIa/sTF Structure—During intermediate stages of refinement, it was observed that the carbonyl O of Lys³⁴¹H (192), which normally points away from the oxyanion hole in serine proteases, was positioned in negative electron density (F_obs - F_calc) contoured at -2.5 (Fig. 8A). At the same time, it was also noted that the side chain of Gln²⁸⁶H (143) was located in negative density as well. Moreover, there was significant positive density (F_obs - F_calc) contoured at 4 (Fig. 8A) where the carbonyl O of Lys³⁴¹H (192) could be moved by concerted flipping of the Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond. Following this, it was then possible to move the side chain of Gln²⁸⁶H (143) out of the negative density and into the positive density where the carbonyl side chain of Gln²⁸⁶H (143) could make an H-bond with the amide N of Gly³⁴²H (193). Subsequent cycles of refinement eliminated both the negative and positive density in the difference (F_obs - F_calc) map validating that the Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond conformation in pAB-VIIa/sTF is unconventional (Fig. 8B). Of note is the observation that O of Ser³⁴⁴H (195) makes an H-bond with the amino group of pAB and with the carbonyl O of Lys³⁴¹H (192). However, in the D-FFR-VIIa (19) and the BPTI-VIIa (30) structures, the carbonyl O of Lys³⁴¹H (192) makes an H-bond with the amide N of Gln²⁸⁶H (143), whereas in the pAB-VIIa/sTF structure, the amide N of Gly³⁴²H (193) makes an H-bond with the carbonyl side chain of Gln²⁸⁶H (143).

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Putative Na+ site in the protease domain of FVIIa and the environment surrounding Phe225. A, Na+ site coordination. The Na+ site in FVIIa involves coordination from four carbonyl groups (184, 185, 221, and 224) from the protein and two water molecules. Electron density (2Fobs - Fcalc) is contoured at 1 (gray) for Na+ (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+ is shown in blue. Note that the Na+ site is linked through water molecules to the carboxylate of Asp189. The salt bridge between Asp189 and pAB is also shown. B, hydrophobic environment surrounding Phe225. 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 Phe225 and lead to Met164, Gln166, and Asp167 that interact with TF are depicted and colored by atom type. Residues whose C=O groups are involved in coordinating to Na+ are also shown. Na+, magenta sphere; water, red spheres. Residue numbering is that of chymotrypsin.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Effect of Na+ on the interaction of FVIIa with sTF. The buffer used was 50 mM Tris-HCl, pH 7.5, containing 5 mM Ca2+ and either 185 mM Na+ or 185 mM Ch+.Ch+ was used as an inert ion to keep the ionic strength constant. sTF was immobilized on a CM5 chip by the amine coupling method. The chip was activated by mixing 400 mM N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide. An immobilization level of 752 response units (RU) was attained for the bound protein. Residual reactive groups on the chip surface were blocked using 1.0 M ethanolamine-HCl, pH 8.5. Eight concentrations of FVIIa (2.8, 3.7, 5.5, 7.5, 9.3, 11.1, 14.9, and 22.3 nM) were used. Only three are shown. Na+ curves are shown as dashed lines, and Ch+ curves are shown as solid lines. Magenta, 2.8 nM FVIIa; blue, 5.5 nM FVIIa; black, 9.3 nM FVIIa. Data were analyzed with BIAe-valuation 3.1 software, and curve fitting was done assuming one-to-one binding.

View larger version (70K): [in this window] [in a new window]   FIGURE 8. Conformations of the Lys341H-Gly342H (192-193) peptide bond in pAB-VIIa, benzamidine-VIIa, and D-FFR-VIIa. A, negative electron density surrounding the carbonyl O of 192 at intermediate stages of pAB-VIIa refinement. The negative difference map (Fobs - Fcalc) contoured at -2.5 surrounding residues 192-195 and Gln143 is shown in red. The positive difference map (Fobs - Fcalc) contoured at 4 surrounding residues 192-195 and Gln143 is shown in blue. The 2Fobs - Fcalc map contoured at 1 is shown in gray. B, nonstandard conformation of the 192-193 peptide bond in pAB-VIIa after final refinement. The 192-193 peptide bond was flipped 180°, and adjustment of the Gln143 side chain was made before further refinement. All maps were contoured at the same level as in A. Note that no positive density (blue) or negative density (red) was observed surrounding the 192-193 peptide bond or side chain of Gln143. The H-bonds between the O of Ser195 and amino group of pAB and C=O of 192 and between 193N and side chain C=O of Gln143 are depicted by black dashed lines. C, final electron density map surrounding the 192-193 peptide bond in benzamidine-VIIa. All maps were contoured at the same level as in A. As in A and B, H-bonds are depicted by black dashed lines. Note the absence of positive (blue) or negative electron density (red) encompassing the carbonyl O of Lys341H (192). D, fully formed oxyanion hole in D-FPR-VIIa/sTF structure. The benzamidine-VIIa/sTF crystals were soaked with D-FPR-ck as described under "Materials and Methods." All maps were contoured at the same level as in A. Again note the absence of positive (blue) or negative electron density (red) encompassing the carbonyl O of Lys341H (192). The inhibitor is completely defined by the electron density, and the 192-193 peptide bond is in the standard orientation. As in A-C, H-bonds are depicted by black dashed lines. Chymotrypsin numbering is used in the figure.

Next, we examined whether soaking of benzamidine-VIIa/sTF crystals with an active site peptide inhibitor, D-FPR-ck, could restructure the Lys³⁴¹H-Gly³⁴²H (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 Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond in FVIIa (Fig. 8D). Now, the Gly³⁴²H (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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
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 nitrogen at the para position of the benzamidine moiety and the carbonyl group of Lys³⁴¹H (192) make H-bonds with the hydroxyl of Ser³⁴⁴H (195) (74, 75). Olivero and co-workers (32) also observed a nonstandard conformation of the Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond in the crystal structure of FVIIa with a sulfonamide inhibitor in the absence of TF. Similar to the FVIIa with the amidinophenylurea-based inhibitor (74), the nitrogen at the para position of the benzamidine moiety of the sulfonamide inhibitor and the carbonyl group of Lys³⁴¹H (192) make H-bonds with the hydroxyl group of Ser³⁴⁴H (195) in this structure (32). Furthermore, Zbinden et al. (76) observed the same two H-bonds involving Ser³⁴⁴H (195), Lys³⁴¹H (192), and the nitrogen at the para position of the benzamidine moiety in FVIIa ± sTF structures with a phenylglycine inhibitor with nonstandard oxyanion hole conformation. From the above studies it was inferred that the nonstandard conformation of the oxyanion hole in these structures is because of distinctive properties of the inhibitors.

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 Lys³⁴¹H (192) with the hydroxyl of Ser³⁴⁴H (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 ZnCl₂, 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 Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond such that the oxyanion hole is fully formed (Fig. 8D). For this to occur the H-bond between the amide N of Gly³⁴²H (193) and side chain of Gln²⁸⁶H (143) must first be broken. Then, either concurrently or subsequently, the Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond must be flipped with resultant formation of three H-bonds, including one between the carbonyl group of Lys³⁴¹H (192) and the backbone N of Gln²⁸⁶H (143) and two between the oxyanion and the amide nitrogens of Gly³⁴²H (193) and Ser³⁴⁴H (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 Pro¹⁹² and Gly¹⁹³ to flip 180° relative to that typically seen in serine proteases. Because both standard (competent) and non-standard (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 Pro¹⁹² and Gly¹⁹³ 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⁺ (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 Lys³⁴¹H-Gly³⁴²H (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 Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond as seen in the D-FPR-VIIa (present paper), D-FFR-VIIa (19), and BPTI-VIIa (30) structures. Upon removal of the sulfate ion by soaking crystals under sulfate-free conditions, the Lys³⁴¹H-Gly³⁴²H (192-193) peptide bond conformation may not change because of its stabilization by the H-bond between Lys³⁴¹H (192) carbonyl group and Gln²⁸⁶H (143) main chain N as seen in D-FPR-VIIa and D-FFR-VIIa (19).

Concerning metal binding studies, Mg²⁺ cannot displace Ca²⁺ from the EGF1 domain or protease domain Ca²⁺ sites in FVIIa. This is supported by the metal-ligand coordination distances, which are compatible with Ca²⁺, despite the presence of excess Mg²⁺. The protease domain Ca²⁺ site is similar to that described for trypsin, which also could not be replaced by Mg²⁺ in the crystals (44). Moreover, the EGF1 domain of FXa was disordered when the crystals were grown in the presence of Mg²⁺ (58), in agreement with our data that FVIIa EGF1 domain cannot bind Mg²⁺. The significance of these two Ca²⁺ 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 Mg²⁺ and four Ca²⁺ ions. Three Mg²⁺ ions were located based upon the structure of the Gla domain of FXa in the presence of Mg²⁺ (58) and the structure of FIX Gla domain in the presence of Ca²⁺/Mg²⁺ (57). The four Ca²⁺ ions were located based upon the Ca²⁺ structures of prothrombin fragment 1 (59) and D-FFR-VIIa (19). Under plasma concentrations of 1.1 mM free Ca²⁺ and 0.6 mM free Mg²⁺, we predict that these three sites will be occupied by Mg²⁺. Interestingly, these three Mg²⁺ sites are certainly the ones that could not be displaced by Ca²⁺ 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 Mg²⁺-binding sites in the Gla domain of prothrombin to which Ca²⁺ has difficult access (95). Thus, location of Mg²⁺ and Ca²⁺ sites that we have identified in the Gla domain of FVIIa are consistent with the biochemical and crystallographic data.

The binding of the above Mg²⁺ sites is expected to be cooperative in nature (58, 93-98), 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 Mg²⁺ and not Ca²⁺ affords this intermediate conformational state (92, 94). Filling of the remaining four Ca²⁺-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 Ca²⁺ sites identified are only partially occupied.

Evidence exists that Mg²⁺ 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 Ca²⁺ (23, 24, 99). It also supports the activation of prothrombin by FXa at very low concentrations of Ca²⁺ (93). In this study, we provide evidence that it supports the activation of FX by FVIIa/TF (Fig. 4). Thus, it appears that Mg²⁺ and Ca²⁺ work in concert to promote coagulation in vivo.

Location (Fig. 6A) of the Na⁺ 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⁺ site in FVIIa. Anomalous diffraction data are needed to unequivocally establish the existence of aNa⁺ site in FVIIa. Nonetheless, as in FIXa (40) and FXa (72), Na⁺ increases the affinity of FVIIa for its cofactor TF (Fig. 7). Thus, one function of Na⁺ 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 Glu²²⁰H (80) is involved in coordination to Zn1, and the carboxyl group of Asp²¹⁹H (79) and the carbonyl group of Gly²⁰⁹H (69) are involved in coordination to Zn2, binding of Ca²⁺ to the 210H-220H loop [70-80 loop] could attenuate the binding of Zn²⁺. Biochemical data support this concept (34, 35). His²¹¹H (71), which was previously considered a candidate for binding to Zn²⁺, is not unique to FVIIa and does not appear to be involved in binding to Zn²⁺ (Fig. 8).

Platelets store large amounts of Zn²⁺ 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 Zn²⁺ concentration. This increased local concentration of Zn²⁺ is likely to inhibit FVIIa bound to TF. Thus, after the initiation phase of clotting is achieved, Zn²⁺ 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 Zn²⁺ inhibits the activity of FVIIa.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
In this study we have identified three Mg²⁺ sites in the Gla domain of FVIIa. It is quite possible that Mg²⁺ in vivo occupies these sites and promotes coagulation. Notably, under physiologic conditions, the protease and EGF1 domain Ca²⁺ sites will be solely occupied by Ca²⁺. Furthermore, we have identified two Zn²⁺ sites in the protease domain that were not defined previously. The ligands for the Zn²⁺ sites are unique to FVIIa and are in agreement with the biochemical data. We also propose a putative Na⁺ site in FVIIa. Na⁺ appears to promote whereas Zn²⁺ appears to down-regulate FVIIa activity. Atypical conformation of the Lys³⁴¹H-Gly³⁴²H (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-P4' 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.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2A2Q, 2AER, and 2FIR) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Grants HL-70369 and HL-36365. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

² Supported in part by a fellowship from the American Association of University Women Educational Foundation.

¹ To whom correspondence should be addressed: UCLA/Orthopaedic Hospital, Dept. of Orthopaedic Surgery, Molecular Biology Institute, Box 951795, Rehab 22-53, Los Angeles, CA 90095-1795. Tel.: 310-825-5622; Fax: 310-825-5972; E-mail: pbajaj{at}mednet.ucla.edu.

³ The abbreviations used are: FVII, factor VII; pAB, p-aminobenzamidine; Gla, -carboxyglutamic acid; EGF, epidermal growth factor; FVIIa, factor VIIa; FXa, factor Xa; FIXa, factor IXa; APC, activated protein C; TF, full-length tissue factor; sTF, soluble tissue factor containing 1-219 residues; rTF, relipidated TF containing residues 1-243; PL, phospholipid; PEG, polyethylene glycol; BPTI, bovine pancreatic trypsin inhibitor; Ch⁺, choline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PDB, Protein Data Bank; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; ck, chloromethyl ketone.

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 Ca²⁺ is numbered 8, and the protease domain Ca²⁺ 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.

	ACKNOWLEDGMENTS

 
We thank Dr. Dulio Cascio (UCLA X-ray Crystallography Core Facility) for help with the crystal structure analysis. We are grateful to A. Liesum, J. Dumas, D. Prevost, and Dr. H. Schreuder of Sanofi-Aventis for one of the benzamidine-VIIa/sTF x-ray data collection at the European Synchrotron Radiation Facility. We also thank Vivian Wang for assistance with the Biacore experiments. S. P. B. thanks Bob Sweet and staff for their help in data collection during the Rapidata 2003 course at the National Synchrotron Light Source.

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