HIGH RESULUTION 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 Bajaj‡§, Amy E. Schmidt‡, Sayeh Agah‡, Madhu S. Bajaj¶, and Kaillathe Padmanabhan¶

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

Running title: Induction of Oxyanion Hole in FVIIa by Substrate/Inhibitor Send correspondence to: S. Paul Bajaj, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery, Molecular Biology Institute, Box 951795, Rehab 22-53, Los Angeles, CA 90095-1795,Telephone: 310-825-5622/7603, Fax: 310-825-5972, E-mail: pbajaj@mednet.ucla.edu Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two EGF-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. Further, 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 1.8 Å resolution and the final structure has an R-factor of 19.8%. In this structure, 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 support the crystal data. Further, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87 Å resolution. However, soaking benzamidine-FVIIa/sTF crystals with D-Phe-Pro-Arg-chloromethylketone 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 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

Human factor VII (FVII)1 is a vitamin K-dependent trace plasma protein that is synthesized by hepatocytes and secreted into the blood as a single chain molecule of Mr ~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 γ-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 β-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 a 152-residue light chain and a 254-residue heavy chain held together by a single disulfide bridge. Characteristic of trypsin like

http://www.jbc.org/cgi/doi/10.1074/jbc.M509971200The latest version is at JBC Papers in Press. Published on June 6, 2006 as Manuscript M509971200

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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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 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 degrees (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 and coworkers first reported the 2.0 Å crystal structure of D-Phe-Phe-Arg-chloromethylketone (DFFR) inhibited FVIIa/sTF (19). The crystals were grown in the presence of Ca2+ using chloromethylketone-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 Mg

2+ plays an important role in physiologic

coagulation (23-26). However, Mg2+-sites in FVIIa have not been identified. Further, 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 1.8 Å crystal structure of p-aminobenzamidine-FVIIa/sTF (pAB-VIIa/sTF), solved from crystals grown in the presence of Ca

2+, Mg

2+, Na+, and Zn

2+. The Gla

domain has four Ca2+- and three Mg2+-sites, and the protease domain has two Zn2+-sites unique to FVIIa. 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 p-AB (42,43) or benzamidine (44-46) at the S1 site is expected not to disrupt the geometry of oxyanion hole. Interestingly, soaking benzamidine-VIIa/sTF crystals with D-Phe-Pro-Arg-chloromethylketone (DFPR-ck) resulted in benzamidine displacement, DFPR 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. Importance of this mechanism in attaining unique selectivity of the molecular substrates

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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. 45CaCl2 was obtained from ICN Biochemicals. Phosphatidylcholine and phosphatidylserine, trizma base, choline chloride (ChCl), zinc chloride, benzamidine, p-aminobenzamidine (pAB), and all other chemicals of the highest grade available were obtained from Sigma. DFPR-ck was obtained from Calbiochem, San Diego, CA.

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 MonoQ column chromatography (49). Gla analysis was performed by Commonwealth Biotechnologies, Inc. (Richmond, VA) using alkaline hydrolysis followed by HPLC 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 as well as from Tom Girard. 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 hr 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 previously described using FX as a substrate (52). 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, while the reservoir solution contained 16-22% PEG 4000, 100 mM MgCl2, and 20 mM bis-Tris, pH 6.5. Drops were prepared by mixing 2 µL protein solution with 2 µL 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 one week. Five-µL volume of DFPR-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 hrs. 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 beam-line X12C of the National Synchrotron Light Source (NSLS) 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

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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 (ESRF) beam 14-2 at 0.93 λ. 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 DFPR (subsequently referred to as DFPR-VIIa/sTF) diffracted to 2.0 Å. These three data sets were collected at beam-line 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 DFPR-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 were kept out of refinement for cross validation (55). During initial refinement stages, it became obvious that the Gla domain is tilted ~6° towards the TF2 domain and that the omega-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 Rcryst dropped to 24.1 with Rfree 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 (Ref. 59, Tulinsky numbering2) 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] 3 and Gly342H [193] must be flipped to agree with the electron density maps. The structure was put through refinement and the resulting Rcryst was 22.4 and Rfree was 28.3. At this stage, additional solvent molecules were added in steps and the structure was refined. The final Rcryst was 19.8 and Rfree was 25.9, and the refinement statistics are given in Table I. 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 Lys341H [192]-Gly342H [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 Lys341H [192] and Gly342H [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 I. The coordinates are deposited into the RCSB Protein Data Bank with accession code 2AER.

The refinement protocol for the DFPR-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 DFPR resulted in the DFPR-VIIa/sTF structure having the Lys341H [192]-Gly342H [193] peptide bond in standard oxyanion hole conformation. The refinement statistics for DFPR-VIIa/sTF are also given in Table I. 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 previously described (20,60). Prior to Ca2+-binding studies, full-length FVIIa and Gla-

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domainless FVIIa were inactivated using dansyl-Glu-Gly-Arg-chloromethylketone 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 µM 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: 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 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 S-2222 was added to yield a final concentration of 250 µM. The p-nitroaniline formed was measured (∆A405/min) for up to 30 minutes. 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 (SPR)—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 RU). Bovine serum albumin (BSA) was immobilized on a separate flow cell as a control for non-specific 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 min1). 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 non-specific binding by subtracting the signal for binding to BSA. 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 DFPR-VIIa/sTF structures—Although, overall structures of pAB-VIIa/sTF, benzamidine-VIIa/sTF, and DFPR-VIIa/sTF in the presence of Ca2+/Mg2+ are similar to the DFFR-VIIa/sTF structure (19), there is significant new information afforded by our structures. 1) The conformation of the Gla domain omega-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 position 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 to the DFFR-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 DFPRck resulted in the formation of the oxyanion hole in the DFPR-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 figure 1(left panel). When the Cα 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 rms∆ was 0.7 Å. However, the Gla domain in the present Ca2+/Mg2+ structure is tilted ~6° towards the carboxy terminal TF2 domain of

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sTF. This tilt results in closer and tighter hydrophobic interactions involving Gla domain residues Leu13L, Phe31L, Arg36L, Leu39L, and Phe40L with carboxy terminal TF residues Tyr156T, Trp158T, Cys186T, Val207T, and Cys209T. An expanded view of these interactions is detailed in figure 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 to 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 Å versus 4.63 Å between Phe31L Cε1 and Trp158T Cz2; 4.18 Å versus 4.91 Å between Arg36L Cγ and Trp158T Cz2; 4.75 Å versus 7.37 Å between Leu39L Cδ1 and Tyr156T CD2; and 4.14 Å versus 4.65 Å between Leu39L Cγ and Trp158T CH2. The more intimate hydrophobic interface was also observed in VIIa/sTF structure (pdb code 1Z6J) determined from a crystal grown in the presence of Mg2+ and citrate. This is most likely due to the binding of Mg2+ to Gla25 and Gla29 (see below) in the Ca2+/Mg2+ structures.

Gla Domain—The conformation of the omega-loop between the Ca2+/Mg2+ structure (present structure) and the Ca2+ 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 figure 2A, the main chain atoms and the side chain positions of the three hydrophobic residues (Phe4L, Leu5L, and Leu8L) as well as those of Gla6 and Gla7 differ in the two structures. The positions of the Ca2+/Mg2+ ions in the Gla domain of the present structure and the positions of the Ca2+ ions in the Banner structure (19) are also depicted in figure 2A. The hydrophobic residues as well as Gla6 and Gla7 are well ordered in the present structure. In the present structure (figure 2B), the side chain NH2 of Arg9L makes a H-bond with Oε1 of Gla6. The amide N of Phe4L makes a H-bond with Oε3 of Gla7, and NH2 of Arg15L makes a H-bond with the carbonyl O of Pro10L (not shown). In the Ca2+ structure (19), the NH2 groups of both Arg9L and Arg15L make H-bonds with

Gla6. An additional H-bond (amide N of Ala1L and Gla26) and the coordination of the carbonyl O of Ala1L to Ca5 are common to both structures. Thus, there are sufficient interactions that allow for well-ordered folding of the omega-loop in the present structure.

The coordination of each Ca2+ and Mg2+ ion in the Ca2+/Mg2+ structure to the Gla residues and water molecules is shown in figure 2B. All Ca2+, Mg2+, and water molecules are well ordered and the coordinations are listed in Table II. Mg2+ at position 1 has similar coordination to the Ca2+ in the DFFR-VIIa structure except that the distances are shorter (Table III), compatible with Mg2+ and not Ca2+. There are also two additional water molecules coordinated to Mg1 in the present structure. Further, 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).

Ca2+ 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 DFFR-VIIa structure (19) is replaced by coordination to two water molecules (S508 and S602) that occupy the Gla7 position in the present structure. Ca2+ 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 Oε2 in the DFFR-VIIa structure (19).

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 DFFR-VIIa structure, Gla 7 coordinates to Mg4 via two water molecules (S209 and S363). Further, S209 coordinates to Oδ1 of Gln2L as compared to its direct coordination with Ca4 in the structure of Banner et al (19). Again, the distances for the Mg4 coordination are shorter (Table III) 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 Ala1L similar to that in the DFFR-VIIa structure (19). However, instead of coordination to Gla6, two

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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 DFFR-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 DFFR-VIIa structure (19). The coordination distances are short and are compatible with Mg2+ and not Ca2+ (Table III). This position and coordination of Mg7 is consistent with that report and 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. Further, Gla7 occupies the position of Gla 6 in the Banner structure and substitutes for it directly in coordinating to Ca5 and via a water molecule (S363) to Mg4 (figure 2B). The side chains of Gla6 and Gla7 are well ordered (figures 2B) and provide additional coordination (Table II) not seen in the Banner structure.

Ca2+ Binding to FVIIa, Gla-domainless FVIIa, and Prothrombin Fragment 1—The goals of these experiments were two-fold. One, 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). Two, 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 is suitable for direct Ca2+ binding studies. The results of the Ca2+ binding experiments are presented in figure 3 and summarized in Table IV. 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+

(figure 3A-C). The two Ca2+ binding sites in the Gla-domainless FVIIa could not be displaced by Mg2+ (figure 3C). In full-length FVIIa and in prothrombin fragment 1, Mg2+ displaced a maximum of three Ca2+-sites under plasma (figures 3A and 3B) or crystallographic concentrations of Mg2+ (figure 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 III). Role of Mg2+ in Activation of FX by FVIIa/TF— We next examined whether plasma concentrations of Mg2+ could potentiate FVIIa/sTF and/or FVIIa/rTF activation of FX. These data are presented in figure 4A for sTF and in figure 4B for rTF/PL. As depicted in figure 4A, 1 nM of FVIIa/sTF activated FX (10 nM) at 0.6 pM/min in the presence of 1.1 mM Ca2+, 1 pM/min in the presence of 1.7 mM Ca2+, and 5 pM/min in the presence of 1.1 mM Ca2+/0.6 mM Mg2+. As depicted in figure 4B, 10 pM of FVIIa/rTF/PL activated FX (10 nM) at 101 pM/min in the presence of 1.1 mM Ca2+, 210 pM/min in the presence of 1.7 mM Ca2+, and 456 pM/min in the presence of 1.1 mM Ca2+/0.6 mM Mg2+. Thus, in systems with and without PL, plasma concentrations of Mg2+ potentate the activation of FX 5 to 8-fold at physiologic concentrations of Ca2+. Thus, the three sites in the FVIIa Gla domain that are predicted to be occupied by Mg2+ (figure 3) in vivo are likely to play a significant role in FX activation (figure 4) during physiologic coagulation (25,26).

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 to 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 ~2.5 Å in agreement with the biochemical data presented in figure 3C, indicating that Mg2+ cannot displace the

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EGF1 domain Ca2+-site. As shown in figure 5A, the EGF1 domain Ca2+ (Ca8) is coordinated to five protein ligands comprised 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.

Zn2+-sites and the Ca2+-site in the Protease Domain of FVIIa—The protease domain of FVIIa has been shown to bind two Zn2+ ions (34). Zn2+ 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 Zn2+-sites involve His216H [76] and His257H [117] (34). This information was utilized in locating the Zn2+-sites in the protease domain (figure 5B). Site 1 involves direct coordination to His216H [76], side chain Glu220H [80], side chain Ser222H [82] and three well-ordered water molecules. Site 2 involves direct coordination to His257H [117], the side chain of Lys161H [24], the carboxyl group of Asp219H [79], the carbonyl group of Gly209H [69], and two well-ordered water molecules. The distances and geometry for the two Zn2+-sites is given in Table III. Zn1 is connected to the protease domain Ca2+-site via the carboxylate side chain of Glu220H [80] whereas Zn2 is connected to the Ca2+-site via water molecules. Except for Glu220H [80], all side chains involved in Zn1 and Zn2 coordination are unique to FVIIa (68) and are ideal for Zn2+ coordination (see Discussion). Furthermore, refining the structure with water molecules at these two sites gave B values (crystallographic temperature factors) of ~12.0, indicating that these sites are occupied by ions with number of electrons much higher that water. These observations further validate assigning Zn2+ ions to these sites.

The protease domain Ca2+ binding site in the pAB-VIIa/sTF structure is located between the two Zn2+-sites and is similar to that described previously (19,30). Ca2+

coordinates to the side chains of Glu210H [70] and Glu220H [80], to the carbonyl oxygens of Asp212H [72] and Glu215H [75], as well as to two well-ordered water molecules (figure 5B). The coordination distances are compatible with Ca2+ (63,64) and not with Mg2+. This is consistent with the biochemical data in which the protease domain Ca2+ binding site could not be displaced by Mg2+ (figure 3C). Moreover, in agreement with Zhang et al. (30), we were also unable to observe coordination of Asp217H [77] to Ca2+ via a water molecule as initially observed by Banner et al (19). However, Asp217H [77] links to Zn2 site via a water molecule (figure 5B).

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 figure 6A. 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]. In previous studies it has been proposed that the Na+-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 Phe374H [225], a key determinant of the Na+ binding site, is located in a hydrophobic cavity consisting of residues Pro303H [161], Leu305H [163], Thr307H [165], Cys310H [168], 311LeuH [169], Gln313H [170A], Ile323H [176] and Cys329H [182]. This hydrophobic core extends to Met306H [164], Gln308H [166], and Asp309H [167] that are part of the TF binding helix (figure 6B). In this context, the protease domain of FVIIa has

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many hydrophobic and polar interactions with TF. These interactions are not shown in the figure but are as follows: Met306H [164] interacts with Arg74T, Phe76T, Pro92T, and Tyr94T. The amide N of Thr307H [165] makes a H-bond with Glu91T; and NH1 and NH2 of Arg379H [230] make H-bonds with Glu91T. Moreover, Gln308H [166] makes a H-bond with the carbonyl O of Tyr94T. Further, the amide N and Oδ2 of Asp309H [167] makes a H-bond with the hydroxyl of Tyr94T as well as with the Nδ2 of Asn96T. The disruption of the Na+-site when Phe374H [225] is changed to Pro could collapse this hydrophobic cavity and result in diminished interactions with TF. Notably, Phe374H [225] was shown to be necessary for optimal TF-mediated activation of FVIIa’s catalytic function (73).

We used SPR to experimentally determine the effect of Na+ on the binding of FVIIa to sTF. As shown in figure 7, in the presence of Na+, the kon for binding was 4.6 ± 0.6 x 105 M-1s-1, koff was 2.4 ± 0.2 x 10-3 s-1, and Kd was 5.2 ± 0.3 nM. The Kd value in the presence of Na+ is similar to that observed by Petrovan and Ruf (73). In the presence of Ch+, the kon was 1.8 ± 0.3 x 105 M-1s-1, koff was 2.5 ± 0.2 x 10-3 s-1, and Kd 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 kon. This may be due to stabilization of the interactions noted above 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 (Fobs – Fcalc) contoured at –2.5σ (figure 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 (Fobs – Fcalc) contoured at 4σ (figure 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 a H-bond with the amide N of Gly342H [193]. Subsequent cycles of refinement eliminated both the negative and positive density in the difference (Fobs – Fcalc) map validating that the Lys341H-Gly342H [192-193] peptide bond conformation in pAB-VIIa/sTF is unconventional (figure 8B). Of note is the observation that Oγ of Ser344H [195] makes a H-bond with the amino group of pAB and with the carbonyl O of Lys341H [192]. However, in the DFFR-VIIa (19) and the BPTI-VIIa (30) structures, the carbonyl O of Lys341H [192] makes a H-bond with the amide N of Gln286H [143] whereas in the pAB-VIIa/sTF structure, the amide N of Gly342H [193] makes a H-bond with the carbonyl side chain of Gln286H [143].

Structures of benzamidine-VIIa/sTF and DFPR-VIIa/sTF —Both the benzamidine-VIIa/sTF and DFPR-VIIa/sTF structures are comparable to the pAB-VIIa/sTF structure with respect to the Gla domain omega-loop placement as well as Na+, Ca2+ and Mg2+ binding. However, the benzamidine-VIIa/sTF crystals, which were grown in the absence of added Zn2+ have water molecules at the two Zn2+ positions. In the benzamidine-VIIa/sTF structure, included in this report, the Zn2+-sites were initially refined with water molecules and had B-factors of 19 and 22; however, when the water molecules were replaced with Zn2+, the B-factors increased to 56 and 63. For consistency, the Zn2+ are left to occupy the Zn2+-sites in the structure. It is realized that in the benzamidine-VIIa/sTF structure included here, the Zn2+ only partially occupies these sites. In the other two benzamidine-VII/sTF structures and in the DFPR-VIIa/sTF structures, which contained added 20 µM Zn2+, the B-factors for the two Zn2+ were in the low 40’s indicating more complete Zn2+ occupancy.

As was the case with the pAB-VIIa/sTF structure, the Lys341H [192]-Gly342H [193] peptide bond in benzamidine-VIIa/sTF structures was in non-standard

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orientation such that the Gly342H [193] amide N points away from the oxyanion hole and the carbonyl O of Lys341H [192] points into the oxyanion hole. This was noted in all three benzamidine-VIIa/sTF crystal structures, two without the added Zn2+ including the one presented here (Table I and figure 8C) and the one with added Zn2+.

Next, we examined whether soaking of benzamidine-VIIa/sTF crystals with an active site peptide inhibitor, DFPR-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 DFPR-ck revealed benzamidine displacement, DFPR incorporation, and induction of the oxyanion hole by a flip of the Lys341H-Gly342H [192-193] peptide bond in FVIIa (figure 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 DFPR (figure 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 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 coworkers (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. 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 (76). From the above studies it was inferred that the nonstandard conformation of the oxyanion hole in these structures is due to distinctive properties of the inhibitors. In the pAB-VIIa/sTF structure (figure 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 DFPR-ck resulted in benzamidine displacement and DFPR incorporation with resultant flip of the Lys341H-Gly342H [192-193] peptide bond such that the oxyanion hole is fully formed (figure 8D). For this to occur

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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 ~180º relative to that typically seen in serine proteases. Since 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 Pro192 and Gly193 in toxin A would flip 180º to provide competent conformation for catalysis (78,79). 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. (82). Interestingly, thrombin has also been shown to have a reversed oxyanion hole in the absence of Na+ (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 (84-92). 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 DFPR-VIIa (present paper), DFFR-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 DFPR-VIIa and DFFR-VIIa (19).

Concerning metal binding studies, Mg2+ cannot displace EGF1 domain Ca2+ site or the protease domain Ca2+-site 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

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binding to TF and development of its catalytic activity.

The data presented in figure 3 demonstrate that under physiologic and crystallographic conditions, 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 DFFR-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 (figure 3; 93, 94) or FVIIa (figure 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 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 omega-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 in to the omega-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).

Evidence exits 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

report, we provide evidence that it supports the activation of FX by FVIIa/TF (figure 4). Thus, it appears that Mg2+ and Ca2+ work in concert to promote coagulation in vivo.

Location (figure 6A) of the Na+ site in FVIIa is based upon the previous structures of other vitamin K-dependent proteins (36-40). Since 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 a Na+ site in FVIIa. Nonetheless, as in FIXa (40) and FXa (72), Na+ increases the affinity of FVIIa for its cofactor TF (figure 7). Thus, one function of Na+ in blood clotting proteases may be to influence cofactor binding.

As shown in figure 8, Zn1 site involves side chains of His, Glu, and Ser, and three water molecules. Zn2 site involves side chains of His, Asp, and Lys, two water molecules, and the main chain carbonyl group (figure 8). Both metal sites have octahedral coordination geometry (Table III) and the implicated side chains are excellent candidates for binding to zinc (100). Since 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+ (figure 8).

Platelets store large amounts of Zn2+ in their cytoplasm and α-granules at concentrations 30-60 times that of the plasma (101). Notably, upon platelet activation at the site of clot formation, α-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

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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.

Concluding Remarks—In this paper, we have identified three Mg2+-sites in the Gla domain of FVIIa. It is quite possible that Mg2+ in vivo occupies these sites and promote coagulation. Notably, under physiologic conditions, the protease and EGF1 domain Ca2+-sites will be solely occupied by Ca2+. Further, we have identified two Zn2+-sites in the protease domain that were not previously defined. The ligands for Zn 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. Since serine proteases play an important role in many physiologic processes, it is critical to understand how a given serine protease recognizes its substrate amongst a milieu of other proteins. One determining factor is the flanking sequences (P4-P4' residues) surrounding the peptidyl cleavage site in the substrates (102-104). 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.

ACKNOWLEDGEMENTS We thank Dr. Dulio Cascio (UCLA X-ray Crystallography Core Facility) for his 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 ESRF. We also thank Vivian Wang for her assistance with the Biacore experiments. SPB thanks Bob Sweet and his staff for their help in data collection during the Rapidata 2003 course at the National Synchrotron Light Source.

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FOOTNOTES * This work was supported by NIH Grants HL-70369 and HL-36365. AES is supported, in part, by a fellowship from the American Association of University Women Educational Foundation. The costs of Publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 to indicate this fact. § To whom correspondence should be addressed: S. Paul Bajaj, Protein Science Laboratory, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, Rehab 22-53, Box 951795, Los Angeles, CA 90095; Telephone: 310-825-5622; Fax: 310-825-5972; E-mail: pbajaj@mednet.ucla.edu 1. The abbreviations used are: pAB, p-aminobenzamidine; Gla, γ-carboxyglutamic acid; EGF, epidermal growth factor; FVII, factor VII; 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; BSA, bovine serum albumin; PL, phospholipid; PEG, polyethylene glycol; SPR, surface plasmon resonance; TBS, 50 mM Tris-HCl, 150 mM NaCl; DFFR-VIIa, D-Phe-Phe-Arg-chloromethylketone inhibited FVIIa; DFPRck, D-Phe-Pro-Arg-chloromethylketone; DFPR-VIIa, D-Phe-Pro-Arg-chloromethylketone inhibited FVIIa; BPTI, bovine pancreatic trypsin inhibitor; S-2222, benzoyl-Ile-Glu-Gly-Arg-p-nitroaniline; Ch+, choline. 2. The metals in the Gla domain of pAB-VIIa are numbered 1-7 according to the system of Tulinsky and coworkers who first described the structure of the Gla domain of prothrombin (59). The EGF1 domain Ca2+ is numbered 8 and the protease domain Ca2+ is numbered 9. 3. 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 DFFR-VIIa (19) and BPTI-VIIa (30), the residue number is followed by the chain identifier in bold. 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.

FIGURE LEGENDS

Figure 1. View of the pAB-VIIa/sTF complex. Ribbon representation of the pAB-VIIa /sTF with metal ions. The protease domain of FVIIa is in blue, the EGF2 domain 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 Cα 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 α-helices are presented as ribbon cylinders and β-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,

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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). Figure 2. Conformation of the omega-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 Cα 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-terminal 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 1σ 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 N of Phe4L, and a water molecule with Oε1 of Gln2L are indicated with stripped arrows. Black dashed lines depict all other coordinations and H-bonds. 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 mM 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 mM to 5 mM. 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+ at 5 mM 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 Ca2+ bound/mol protein. 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 S-2222. 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+. 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 1σ (grey) for Ca2+ (magenta sphere) and two water molecules (red spheres) is shown. Electron density contoured at 5σ 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 that of 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 1σ (grey) of Zn2+ (cyan spheres), Ca2+ (magenta sphere), and water molecules (red spheres) is shown. The electron density contoured at 3σ 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.

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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 1σ (grey) for Na+ (cyan sphere) and water molecules (red spheres). The electron density contoured at 2.5σ 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 α-helices are presented as ribbon cylinders and β-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. 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 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 nM, 3.7 nM, 5.5 nM, 7.5 nM, 9.3 nM, 11.1 nM, 14.9 nM, and 22.3 nM) were used. Only three are shown. Na+ curves are shown in 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 BIAevaluation 3.1 software and curve fitting was done assuming one-to-one binding. Figure 8. Conformations of the Lys341H-Gly342H [192-193] peptide bond in pAB-VIIa, benz-VIIa and DFFR-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 –2.5σ surrounding residues 192-195 and Gln-143 is shown in red. The positive difference map (Fobs – Fcalc) contoured at 4σ surrounding residues 192-195 and Gln143 is shown in blue. The 2Fobs – Fcalc map contoured at 1σ is shown in grey. B, Nonstandard conformation of the 192-193 peptide bond in pAB-VIIa after final refinement. The 192-193 peptide bond was flipped 180º 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 Oγ of Ser195 and amino group of pAB and C=O of 192, and between 193N and side chain C=O of Q143 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 DFPR-VIIa/sTF structure. The benzamidine-VIIa/sTF crystals were soaked with DFFR-ck as described in the Materials and Methods section. 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, B, and C, H-bonds are depicted by black dashed lines. Chymotrypsin numbering is used in the figure.

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TABLE I Data collection and refinement statistics

pAB-VIIa/sTF Benz-VIIa/sTF DFPR-VIIa/sTF

Data collection

Space Group P212121 P212121 P212121 Unit cell dimensions: a (Å) 69.72 69.90 70.02 b (Å) 81.00 81.20 81.00 c (Å) 126.12 125.90 126.33 Beamline/generator X12-C ESRF/ID 14-2 X12C Resolution (Å) 90.0-1.8 90.0-1.87 40.0-2.0 Wavelength (Å) 1.10 0.931 1.10 Molecules per asymmetric unit

1 1 1

Measured reflections 216,363 481,210 788,105 Unique reflections 60,130 59,109 41,633 Redundancy 3.6 8.1 18.9 Overall completeness (%)a 90.0 (65) 98 (89) 84 (63) Rmerge (%)a,b 5.7 (20.9) 5.0 (29.1) 7.1 (28.5) I/σ(I) a 7.7 (3.4) 8.1 (3.3) 11.5 (3.5)

Refinement statistics

Resolution (Å) 8.0-1.8 10-1.87 8.0-2.0 Number of atoms/residues Protein 554 588 587 Metal 12 12 12 Water 721 715 626 Rcryst (%)c 19.8 19.1 20.7 Rfree (%)c 25.9 25.6 28.1 r.m.s. deviations Bond length (Å) 0.010 0.011 0.012 Bond angle (º) 1.71 1.70 1.70 Ramachandran plot (%) Favored 87.0 88.8 86.6 Allowed 11.9 10.6 12.4 Generously allowed 0.9 0.4 0.8 Disallowedd 0.2 0.2 0.2 a Numbers in parentheses represent data in the highest resolution shell, 1.80-1.86 Å. bRmerge(I) = ∑hkl((∑i⏐Ihkl,i – [Ihkl]⏐)/ ∑iIhkl,i). c Rcryst = ∑hkl⏐⏐Fobs⏐- ⏐Fcalc⏐⏐/ ∑ hkl⏐Fobs⏐. Rfree was computed identically, except that 10% of the reflections were omitted as a test set. d In all structures Lys32L is in the disallowed region in the Ramachandaran plot. This residue is also in the disallowed region in the Banner structure (19).

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TABLE II. Ca2+/Mg2+ coordination in the FVIIa Gla domain and its comparison with the 1DAN Ca2+ Gla Structure

1PF2 Numbering (Reference 52)

Coordination in Present Structure (2A2Q) Ligand Distance (Å)

Coordination in 1DAN Ligand Distance (Å)

1DAN Numbering (Reference 19)

Oε2 Gla25 1.77a Oε1 Gla25 2.95 Oε3 Gla25 2.10 Oε3 Gla25 2.48 Mg-1 (B-factor 26) Oε1 Gla29 2.23 Oε1 Gla29 2.44 Ca-9

Oε3 Gla29 1.92 Oε3 Gla29 3.11 S228 (H2O228 in 2A2Q) 1.78

S722 2.44

Oε3 Gla26 2.27 Oε2 Gla26 2.38 Oε1 Gla29 2.92 Oε3 Gla29 2.28

Ca-2 (B-factor 56) Oε2 Gla29 3.00 Oε4 Gla29 2.56 Ca-3 S508 (Oε4 Gla7 position in 1DAN) 3.39

S602 (Oε2 Gla7 position in 1DAN) 2.82 S694 2.02

Oε4 Gla7 2.35 Oε2 Gla7 2.39 H2O 241 2.65

H2O 242 2.50

Oε3 Gla16 3.39 Oε3 Gla16 2.54 Oε2 Gla29 2.47 Oε4 Gla29 2.57

Ca-3 (B-factor 53) Oε3 Gla26 3.34 Oε1 Gla26 2.46 Ca-4 Oε4 Gla26 2.82 Oε1 Gla7 2.68 S602 (Oε2 Gla7 position in 1DAN) 2.17

S262 2.62 Oε2 Gla7 2.53 H2O 188 2.57

S411 3.39 H2O 189 2.51 H2O 190 2.61 Oε1 Gla16 1.92 Oε1 Gla16 2.27 Oε3 Gla16 1.97 Oε3 Gla16 2.38

Mg-4 (B-factor 20) Oε1 Gla26 1.97 Oε1 Gla26 2.49 Ca-5 Oε4 Gla26 1.95 Oε4 Gla26 2.64 S209 (to Oδ1 Asn2 and Oε1 /Oε2 Gla7) 1.98

S363 (to Oε2 Gla7, not shown in fig. 2B) 2.11 Oδ1 Asn2 2.34 Oε1 Gla7 2.36

Oε1 Gla6 2.56 Carbonyl O Ala1 2.97 Carbonyl O Ala1 2.37 Oε1 Gla16 2.31 Oε1 Gla16 2.49

Ca-5 (B-factor 51) Oε2 Gla16 2.43 Oε2 Gla16 2.41 Ca-6 Oε3 Gla20 2.87 Oε3 Gla20 2.71 Oε2 Gla7 3.25 Oε4 Gla20 2.63 S630 2.71 Oε1 Gla6 2.61 Oε4 Gla6 2.69 H2O 186 2.51 Oε3 Gla20 2.89 Oε3 Gla20 2.48

Ca-6 (B-factor 53) Oε4 Gla20 2.21 Oε1 Gla20 2.62 Ca-8 Oε3 Gla6 2.18 S630 to Oε2 Gla7 2.33

Oε1 Gla14 1.93 Oε2 Gla14 2.71

Mg-7 (B-factor 41) Oε4 Gla14 1.86 Oε3 Gla14 2.39 Ca-7 Oε2 Gla19 1.88 Oε2 Gla19 2.28 Oε4 Gla19 1.87 Oε3 Gla19 2.70

H2O 243 2.79

a The coordination distances for Mg2+ positions in the present structure are compatible with Mg2+-oxygen coordination distances of ~2 Å in small molecules (61,62) and not with Ca2+-oxygen average distances of ~2.4 Å (63,64). 2A2Q structure at 1.8 Å resolution is from crystals grown from calcium/magnesium conditions whereas 1DAN structure at 2.0 Å resolution is from only calcium conditions.

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Table III: The EGF1 domain Ca2+, and the protease domain Ca2+, Na+ and Zn2+ Coordination in the pAB-VIIa/sTF crystal Structure.

Ion Ligand Distance (Å)

Ca8

EGF1 Domain

Asp46 OD2 Gly47 O Gln49 Oε1 Asp63 OD1

Asp63 OD2 Gln64 O S24 O S441 O

2.33a

2.32 2.27 2.73 2.19 2.33 2.53 2.52

Ca9 Protease Domain

Glu70 Oε2 Asp72 O Glu75 O Glu80 Oε2 S26* O S58* O

2.19 2.17 2.01 2.01 2.01 2.20

Zn1a

Protease Domain

His76* ND1

Glu80 Oε1 Ser82* Oγ S374 O S477 O S530 O

3.20 2.88 3.38 3.20 2.52 2.42

Zn2 Protease Domain

Lys24 Nz Gly69 O Asp79 OD2 His117 ND1 S528* O S531* O

2.57 2.94 3.84 3.03 2.84 3.30

Na

Protease Domain

Tyr184 O Ser185 O Thr221 O His224* O S144* O S357 O

2.91 3.36 2.30 3.30 2.60 3.05

*Denotes ligands that are in the apical position in the octahedral geomtry. 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).

a The site occupancy for each Zn2+ site appears to be 1. This is based upon the fact that the B-factors dropped from the low 40s to ~12 when the structure was refined with water molecules only. For Na+, the site occupancy could not be determined because when a water molecule was substituted for Na+, the B-factor dropped only slightly from 22 to 19.

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TABLE IV

Binding of Ca2+ to FVIIa and Prothrombin Fragment 1 Mol Ca2+/Mol Proteina Protein 5 mM Ca2+ 5 mM Ca2+/50 mM Mg2+ 1.1 mM Ca2+/ 0.6 mM Mg2+

FVIIa 8.7 ± 0.3 5.8 ± 0.2 6.0 ± 0.2 FVIIades1-38 1.8 ± 0.2 1.7 ± 0.2 NDb

Prothrombin fragment 1 6.8 ± 0.3 3.8 ± 0.2 3.9 ± 0.2 a All measurements were made in triplicate. The FVIIades1-38 represents FVIIa in which residues 1-38 of

the Gla domain have been cleaved off; this molecule should have a divalent metal binding site in the EGF1 domain as well as in the protease domain only.20 Prothrombin fragment 1 has only the Gla domain metal binding sites. This fragment was used to confirm the results obtained with FVIIa.

b ND = not determined

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S. Paul Bajaj, Amy E. Schmidt, Sayeh Agah, Madhu S. Bajaj and Kaillathe Padmanabhan sites in factor VIIa2+ and Zn+, Na2+, Mg2+Ca

tissue factor: Unpredicted conformation of the 192-193 peptide bond and mapping of High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble

published online June 6, 2006J. Biol. Chem. 

  10.1074/jbc.M509971200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2006/06/20/M509971200.DC1

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