HighResolutionStructuresof p-Aminobenzamidine-and ... · culated using the structure of Banner et...

High Resolution Structures of p-Aminobenzamidine- andBenzamidine-VIIa/Soluble Tissue FactorUNPREDICTED CONFORMATION OF THE 192–193 PEPTIDE BOND AND MAPPINGOF Ca2�, Mg2�, Na�, AND Zn2� SITES IN FACTOR VIIa*□S

Received for publication, September 12, 2005, and in revised form, June 1, 2006 Published, JBC Papers in Press, June 6, 2006, DOI 10.1074/jbc.M509971200

S. Paul Bajaj‡1, Amy E. Schmidt‡2, Sayeh Agah‡, Madhu S. Bajaj§, and Kaillathe Padmanabhan¶

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

Factor VIIa (FVIIa) consists of a�-carboxyglutamic acid (Gla)domain, two epidermal growth factor-like domains, and a pro-tease domain. FVIIa binds seven Ca2� ions in the Gla, one in theEGF1, and one in the protease domain.However, blood containsboth Ca2� and Mg2�, and the Ca2� sites in FVIIa that could bespecifically occupied by Mg2� are unknown. Furthermore,FVIIa contains a Na� and two Zn2� sites, but ligands for thesecations are undefined.Weobtainedp-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containingCa2�, Mg2�, Na�, and Zn2�. The crystal diffracted to 1.8 A res-olution, and the final structure has an R-factor of 19.8%. In thisstructure, the Gla domain has four Ca2� and three boundMg2�.The EGF1 domain contains one Ca2� site, and the proteasedomain contains oneCa2�, oneNa�, and twoZn2� sites. 45Ca2�

binding in the presence/absence ofMg2� to FVIIa, Gla-domain-less FVIIa, and prothrombin fragment 1 supports the crystaldata. Furthermore, unlike in other serine proteases, the amideNof Gly193 in FVIIa points away from the oxyanion hole in thisstructure. Importantly, the oxyanion hole is also absent in thebenzamidine-FVIIa/sTF structure at 1.87 A resolution. How-ever, 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 bya flip of the 192–193 peptide bond in FVIIa. Thus, it is the sub-strate and not theTF binding that induces oxyanion hole forma-tion and functional active site geometry in FVIIa. Absence ofoxyanion hole is unusual and has biologic implications for FVIIamacromolecular substrate specificity and catalysis.

Human factor VII (FVII)3 is a vitamin K-dependent traceplasma protein that is synthesized by hepatocytes and secretedinto the blood as a single chain molecule ofMr �50,000 (1, 2).Gene structure, amino acid sequence, and the modular struc-ture of FVII reveal that the protein is organized into severaldiscrete domains (3, 4). FVII consists of an N-terminal �-car-boxyglutamic acid domain (Gla), a short hydrophobic segment,two epidermal growth factor (EGF)-like domains, and a C-ter-minal serine protease module, which consists of two �-barrelsubdomains (3–5). Several coagulation enzymes, includingFIXa, FXa, and FVIIa, have been shown to activate FVII (2, 5–9).In each case, the activation of FVII involves the cleavage of asingle peptide bond between Arg152 and Ile153, located in theconnecting region between the EGF2 and the proteasedomains. This results in the formation of a two-chain FVIIamolecule 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 heavychain of FVIIa contains the catalytic triad, namely His57,Asp102, and Ser195, with Asp189 (chymotrypsin numbering) atthe 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). HumanTF consists of the following three regions: anN-terminal extra-cellular region of 219 residues, a transmembrane region of 23residues, and a C-terminal cystoplasmic region of 19 residues.The extracellular region of TF consists of two fibronectin typeIII repeats. The crystal structure of the extracellular domainpair termed soluble TF (sTF, residues 1–219) is known (12–15).The two fibronectin type III domains are connected end-to-endat an angle of 120° (12–15). Similar to full-length TF, sTF bindsFVIIa with high affinity and potentiates its enzymatic activity(16–18).Recent evidence indicates that theTF pathway (or the extrin-

sic pathway) plays a primary role in initiating blood coagulation

* This work was supported in part by National Institutes of Health GrantsHL-70369 and HL-36365. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

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

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

1 To whom correspondence should be addressed: UCLA/Orthopaedic Hospi-tal, 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: [email protected].

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

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 con-taining residues 1–243; PL, phospholipid; PEG, polyethylene glycol; BPTI,bovine pancreatic trypsin inhibitor; Ch�, choline; CHAPS, 3-[(3-cholamidopro-pyl)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.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 34, pp. 24873–24888, August 25, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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during normal hemostasis as well as during many pathologicsituations, including arteriosclerosis and septicemia (5). Thispathway begins by exposure of blood to TF in the extravascularspace at an injury site and formation of the complex betweenTFand plasma factor FVII/VIIa. The FVIIa/TF complex then acti-vates FIX, FX, and FVII. The FXa thus formed with FVa, Ca2�,and phospholipid (PL) then activates prothrombin to throm-bin, which cleaves fibrinogen to fibrin, which polymerizes toform 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� usingchloromethyl ketone-inhibited active site FVIIa and two frag-ments of sTF obtained by subtilisin treatment. The structurerevealed that part of the helix composed of residues 30–40 oftheGla domainmakes van derWaals contactswith sTF, and theEGF1 domain makes extensive contacts with sTF. The EGF2and the protease domain also make contacts with sTF, whichappears to be a complex interface region. Consistent with bio-chemical data (20–22), FVIIa in the crystal structure has nineCa2� ions bound, seven in the Gla domain, one in the EGF1domain, 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 inFVIIa have not been identified. Furthermore, it has been pro-posed that FVIIa contains a Na� site (27), but the site has notbeen defined in any of the crystal structures (19, 28–33). Nota-bly, FVIIa also contains two Zn2� sites in its protease domain(34). Occupancy of the Zn2� sites inhibits FVIIa catalytic activ-ity in the absence but poorly in the presence of Ca2�. Thisobservation led to the hypothesis that Zn2� binds to the prote-ase domain Ca2� loop and attenuates the activity of FVIIa (34,35). As is the case for Na�, the Zn2� sites in FVIIa are notdefined.Here we report a 1.8 Å crystal structure of p-aminobenzami-

dine-FVIIa/sTF (pAB-VIIa/sTF), solved from crystals grown inthe presence of Ca2�, Mg2�, Na�, and Zn2�. The Gla domainhas four Ca2� and three Mg2� sites, and the protease domainhas two Zn2� sites unique to FVIIa. The Na� site is structurallysimilar to the Na� site in FXa (36, 37), APC (38, 39), and theproposed site in FIXa (40) but not to that in thrombin (37, 41).Moreover, the main chain NH of 193 (chymotrypsin number-ing) in FVIIa points away from the oxyanion hole in the pAB-VIIa/sTF and benzamidine-VIIa/sTF structures. This is anuncharacteristic feature of serine proteases (10), and inclusionof pAB (42, 43) or benzamidine (44–46) at the S1 site isexpected not to disrupt the geometry of the oxyanion hole.Interestingly, soaking benzamidine-VIIa/sTF crystals withD-Phe-Pro-Arg-chloromethyl ketone (D-FPR-ck) resulted inbenzamidine displacement, D-FPR incorporation, and induc-tion of the oxyanion hole. From these data, we infer that TFbinding restructures the activation domains in FVIIa (47), but itdoes not induce formation of the oxyanion hole. Instead, it isthe binding of the substrate/inhibitor at the active site thatinduces oxyanion hole formation required for catalysis. Theimportance of this mechanism in attaining unique selectivity ofthe molecular substrates to initiate physiologic coagulation byFVIIa/TF is discussed.

MATERIALS AND METHODS

Reagents—Magnesium chloride, calcium chloride, and PEG4000 were purchased from Hampton Research. 45CaCl2 wasobtained from ICN Biochemicals. Phosphatidylcholine andphosphatidylserine, Trizma (Tris base), choline chloride(ChCl), zinc chloride, benzamidine, p-aminobenzamidine(pAB), and all other chemicals of the highest grade availablewere obtained from Sigma. D-FPR-ck was obtained fromCalbiochem.Proteins—Human FVII was expressed using pMon3360b

expression vector in BHK/VP16 cells as described by Hippen-meyer and Highkin (48) and Zhong et al. (49). The FVII waspurified using a Ca2�-dependent monoclonal antibody andFPLC Mono Q column chromatography (49). Gla analysis wasperformed by Commonwealth Biotechnologies, Inc. (Rich-mond, VA), using alkaline hydrolysis followed by high pressureliquid chromatography analysis. The amount of Gla was quan-titated based upon Asp and Asn present per mol of FVII. Puri-fied FVII contained nine Gla residues per mol. FVIIa wasobtained using FXa-Sepharose as described previously, and theresin was removed by gentle centrifugation (2, 49). The purifiedprotein was concentrated to �20 mg/ml and stored at �80 °Cuntil used. sTF (residues 1–219) was obtained from E. G.Tuddenham (Imperial College, London) and from TomGirardMonsanto/Pharmacia. sTF was concentrated to �10 mg/mland stored at �80 °C. Both proteins were �98% pure as judgedby SDS-gel electrophoresis (50). Human FX was purchasedfrom ERL (South Bend, IN). Recombinant human TF contain-ing the transmembrane domain (residues 1–243) was a giftfrom Genentech (San Francisco, CA).Preparation of Relipidated (r)TF—Phospholipid (PL) vesicles

(75% phosphatidylcholine, 25% phosphatidylserine) were pre-pared by a slight modification of the method of Husten et al.(51). Human TF was diluted to 30 �g/ml in 50 mM Tris-HCl,150 mM NaCl (TBS), pH 7.5, containing 10 mM CHAPS andmixed with an equal volume of 2 mM PL vesicles. The mixturewas incubated for 2 h at 37 °C. The rTFwas extensively dialyzedat 4 °C against TBS, pH 7.5, to remove CHAPS. The functionalTF concentration was determined as described previously(52) using FX as a substrate. Approximately 60% of TF (cal-culated from the starting concentration) was available on theoutside of the vesicles assuming a stoichiometry of 1:1between FVIIa and TF.Crystallization—The pAB-VIIa/sTF or the benzamidine-

VIIa/sTF complex was crystallized using the sitting drop orthe hanging drop vapor diffusion method. Specifically, theprotein drop contained 4 mg/ml VIIa/sTF complex, 20 mM

Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl2 � 20 �M

ZnCl2, and 10 mM pAB or 10 mM benzamidine, whereas thereservoir solution contained 16–22% PEG 4000, 100 mM

MgCl2, and 20 mM BisTris, pH 6.5. Drops were prepared bymixing 2 �l of protein solution with 2 �l of reservoir solutionat 20 °C. Crystals appeared within 7 days and were allowed togrow up to 14–20 days before being flash-frozen withoutadditional cryoprotectant.For soaking experiments, 50 �l of 100 mM Tris-HCl, pH 7.5,

200 mM NaCl, 20 �M ZnCl2, and 10 mM CaCl2 was mixed with

Induction of Oxyanion Hole in FVIIa by Substrate/Inhibitor

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50 �l of the reservoir solution (22% PEG 4000, 100 mM MgCl2,pH6.5). The abovemixturewas allowed to equilibratewith 1mlof the reservoir solution for 1week. Five-�l volume of D-FPR-ckwas added to the drop to achieve a final concentration of 250�M inhibitor. Two benzamidine-VIIa/sTF crystals grown in thepresence of 20 �M ZnCl2 were then soaked in the drop for 48 h.During soaking, the drop was allowed to equilibrate with thereservoir solution. The crystals were flash-frozen without addi-tional cryoprotectant.X-ray Data Collection—The pAB-VIIa/sTF data set was col-

lected at beamline X12C of the National Synchrotron LightSource in Upton, NY, using a Brandeis 4 CCD detector. Onecrystal containing 20 �M ZnCl2 diffracted to a resolution of 1.8Å, the details of which are presented here. Three different datasets were collected for the benzamidine-VIIa/sTF crystals. Onecrystal had 20 �M Zn2�, and the other two did not contain anyadded Zn2�. One of the crystals without added Zn2� diffractedto 1.87 Å, which is presented here. This data set was collectedat the European Synchrotron Radiation Facility beam 14-2 at0.93 �. The other two benzamidine crystals, one with addi-tional ZnCl2 and one without additional ZnCl2, each dif-fracted 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 collectedat beamline X12C of the National Synchrotron Light Source.All crystals belonged to the orthorhombic space groupP212121 with one molecule of the pAB-VIIa/sTF or benzami-dine-VIIa/sTF or D-FPR-VIIa/sTF complex in the asymmet-ric unit. The data were processed and scaled using the pro-grams DENZO and SCALEPACK (53).Structure Determination and Refinement—First, the struc-

ture of pAB-VIIa/sTF was determined. Initial phases were cal-culated using the structure of Banner et al. (19) (PDB code1DAN), as a starting model. This was followed by severalrounds of positional, B-factor, and simulated annealing proto-cols using the program XPLOR (54). Ten percent of the datawas kept out of refinement for cross-validation (55). Duringinitial refinement stages, it becameobvious that theGla domainis tilted �6° toward the TF2 domain, and that the � loop has aslightly different conformation in comparison to the Ca2� onlystructure (19). The residues in theGla domain were fitted usingthe program O (56). The Rcryst dropped to 24.1 with Rfree of30.2. Based upon previous information (57, 58) as well as thedistances and the positions of the Gla residues, Mg2� ions wereplaced at the 1-, 4-, and 7-position (59, Tulinsky numbering),4and Ca2� ions were placed at the 2-, 3-, 5-, and 6-positionsbased upon previous structures of Gla domains (19, 59). Thepositions of thesemetals were refined and subsequently solventmolecules surrounding these metals as well as other metal ions(EGF1 and protease domain Ca2�, Na�, and Zn2�) were addedto the model. At this point, it was also noted that the peptidebond between Lys341H (192)5 and Gly342H (193) must be

flipped to agree with the electron density maps. The structurewas put through refinement, and the resulting Rcryst was 22.4,and Rfree was 28.3. At this stage, additional solvent moleculeswere added in steps, and the structure was refined. The finalRcryst was 19.8, and Rfree was 25.9, and the refinement statisticsare given in Table 1. The coordinates are deposited into theRCSB Protein Data Bank with accession code 2A2Q.Initial phases for the benzamidine-VIIa/sTF crystal were cal-

culated using pAB-VIIa/sTF as a starting model; however, pABwas removed from the structure, and the Lys341H (192)–Gly342H (193) peptide bond was flipped such that a standardoxyanion hole conformation was attained prior to use for phas-ing. The refinement protocol was the same as described abovefor the pAB-VIIa/sTF crystal. Similar to the pAB-VIIa/sTF, itwas again noted that in all three structures of benzamidine-VIIa/sTF, the peptide bondbetweenLys341H (192) andGly342H(193) needed to be in the nonstandard oxyanion hole confor-mation in order to agree with the electron density maps. Therefinement statistics for the benzamidine-VIIa/sTF crystal thatdiffracted to 1.87 Å are given in Table 1. The coordinates aredeposited into theRCSBProteinData Bankwith accession code2AER.The refinement protocol for the D-FPR-VIIa/sTF crystal was

similar to those described for the benzamidine-VIIa/sTF crys-tals. During refinement, it was noted that soaking of benzami-dine-VIIa/sTF with D-FPR resulted in the D-FPR-VIIa/sTFstructure having the Lys341H (192)–Gly342H (193) peptidebond in standard oxyanion hole conformation. The refinementstatistics for D-FPR-VIIa/sTF are also given in Table 1. Thecoordinates are deposited into the RCSB Protein Data Bankwith accession code 2FIR.Ca2� Binding to Various Fragments of FVIIa or Prothrombin

Fragment 1—Calcium binding was determined by the tech-nique of equilibrium dialysis using 45Ca2� as a probe. The spe-cific procedure has been described earlier (20). Gla-domainlessFVIIa and human prothrombin fragment 1 were obtained asdescribed previously (20, 60). Prior to Ca2� binding studies,full-length FVIIa and Gla-domainless FVIIa were inactivatedusing dansyl-Glu-Gly-Arg-chloromethyl ketone as describedpreviously (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 proteinranged from 20 to 40 �M. The buffer used was TBS, pH 7.5.Activation of FX by FVIIa/sTF or FVIIa/rTF—FX was acti-

vated by either FVIIa/sTF or FVIIa/rTF in TBS, pH 7.5, con-taining 0.1% PEG 8000 using three different Ca2�/Mg2� con-ditions as follows: 1) 1.1mMCa2�, 0mMMg2�; 2) 1.1mMCa2�,0.6 mM Mg2�; and 3) 1.7 mM Ca2�, 0 mM Mg2�. Each reactioncontained 10 nM FX and either 1 nM FVIIa, 10 nM sTF, or 1 nMFVIIa, 10 pM rTF, 50 �M PL. FVIIa was incubated with sTF or

4 The metals in the Gla domain of pAB-VIIa are numbered 1–7 according to thesystem of Tulinsky and co-workers (59) who first described the structure ofthe 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 usedpreviously for D-FFR-VIIa (19) and BPTI-VIIa (30), the residue number is fol-lowed by the chain identifier in boldface. Thus, the light chain residues arefollowed by L, heavy chain residues by H, and the TF residues by T. Theheavy chain of FVIIa represents the serine protease domain and the stand-ard chymotrypsin numbering is given in square brackets after the sequen-tial FVIIa numbering. The insertions in the protease domain are followed bythe letter A, B, C, etc. Water molecules are labeled with a prefix S for solvent.




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rTF for 5 min at 25 °C to allow for complex formation. FX wasthen added, and reactions were carried out at 25 °C. At varioustimes, 95-�l aliquots were removed and added to tubes con-taining 5 �l of 0.5 M EDTA, pH 8.0 (final EDTA concentrationof 20 mM), to stop the reaction. From this mixture, 90 �l wasremoved and placed in awell on an Immulon 4HBX flat bottom96-well microtiter plate (Dynex Technologies), and 10 �l ofbenzoyl-Ile-Glu-Gly-Arg-p-nitroaniline was added to yield afinal concentration of 250 �M. The p-nitroaniline formed wasmeasured (�A405/min) for up to 30min.The FXa generatedwascalculated from a standard curve constructed using FXaobtained 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). sTFwas immobilized on a car-boxymethyl dextran (CM5) flow cell (752 response units).Bovine serum albumin was immobilized on a separate flow cellas a control for nonspecific binding. FVIIa (2.8–22.3 nM) wasinjected across the flow cell in 50 mM Tris-HCl, pH 7.4, con-taining 5 mM CaCl2 and either 185 mM ChCl or 185 mM NaCl(flow rate 10 �l min�1). Six-minute association and 10-mindissociation times were used. Dissociation was monitored afterreturn to buffer flow, and the chip surface was regenerated byinjecting 0.1 M EDTA followed by equilibration with 50 mMTris-HCl, pH 7.4, containing 5 mM CaCl2 and either 185 mMChCl or 185 mM NaCl. Data were corrected for nonspecificbinding by subtracting the signal for binding to bovine serumalbumin. Data were analyzed with BIAevaluation 3.1 software

(Biacore, Piscataway, NJ), and curve fitting was done with theassumption of one-to-one binding.

RESULTS

General Aspects of the pAB-VIIa/sTF, Benzamidine-VIIa/sTF, and D-FPR-VIIa/sTF Structures—Although overall struc-tures of pAB-VIIa/sTF, benzamidine-VIIa/sTF, and D-FPR-VIIa/sTF in the presence of Ca2�/Mg2� are similar to theD-FFR-VIIa/sTF structure (19), there is significant new infor-mation afforded by our structures. 1) The conformation of theGla domain � loop is different in our structures. 2) In the newstructures, two Zn2� sites and one putative Na� site are iden-tified in the protease domain. 3) Three Ca2� sites in the Gladomain at positions 1, 4, and 7 were replaced by Mg2� in thenew structures. Binding and kinetic data are presented thatshow that under physiologic conditions these three sites areoccupied byMg2� and play an important role in biologic coag-ulation. However, the EGF1 and protease domain Ca2� siteswere not displaced by Mg2�. 4) Importantly, as compared withthe D-FFR-VIIa (19), the oxyanion hole in the pAB-VIIa or ben-zamidine-VIIa structure is not preformed. This is in contrast toknown structures of serine proteases with pAB (42, 43) or ben-zamidine (44–46). However, soaking benzamidine-VIIa crys-tals with D-FPR-ck resulted in the formation of the oxyanionhole in the D-FPR-VIIa. This finding has important implicationsas to FVIIa substrate specificity. Each of these points is elabo-rated below primarily using the pAB-VIIa/sTF structure.

TABLE 1Data collection and refinement statistics

pAB-VIIa/sTF Benzamidine-VIIa/sTF D-FPR-VIIa/sTFData collectionSpace group P212121 P212121 P212121Unit cell dimensionsa (Å) 69.72 69.90 70.02b (Å) 81.00 81.20 81.00c (Å) 126.12 125.90 126.33

Beamline/generator X12-C ESRF/ID 14-2 X12CResolution (Å) 90.0-1.8 90.0-1.87 40.0-2.0Wavelength (Å) 1.10 0.931 1.10Molecules per asymmetric unit 1 1 1Measured reflections 216,363 481,210 788,105Unique reflections 60,130 59,109 41,633Redundancy 3.6 8.1 18.9Overall 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/�(I)a 7.7 (3.4) 8.1 (3.3) 11.5 (3.5)

Refinement statisticsResolution (Å) 8.0-1.8 10-1.87 8.0-2.0No. of atoms/residuesProtein 554 588 587Metal 12 12 12Water 721 715 626

Rcryst (%)c 19.8 19.1 20.7Rfree (%)c 25.9 25.6 28.1Root mean square deviationsBond length (Å) 0.010 0.011 0.012Bond angle (°) 1.71 1.70 1.70

Ramachandran plot (%)Favored 87.0 88.8 86.6Allowed 11.9 10.6 12.4Generously allowed 0.9 0.4 0.8Disallowedd 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 was omitted as a test set.d In all structures Lys32L is in the disallowed region in the Ramachandran plot. This residue is also in the disallowed region in the Banner structure (19).




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A ribbon diagram of the pAB-VIIa/sTF structure is pre-sented in Fig. 1 (left panel).When theC� carbons of all residuesexcept the first 46 residues of the light chain (i.e. Gla domain)were used for superpositioning of the Ca2� structure (19), theroot mean square deviation was 0.7 Å. However, the Gladomain in the present Ca2�/Mg2� structure is tilted �6°toward the C-terminal TF2 domain of sTF. This tilt results incloser and tighter hydrophobic interactions involving Gladomain residues Leu13L, Phe31L, Arg36L, Leu39L, and Phe40Lwith C-terminal TF residues Tyr156T, Trp158T, Cys186T,Val207T, and Cys209T. An expanded view of these interactionsis detailed in Fig. 1 (right panel). All of these residues are wellordered in the pAB-VIIa/sTF structure, and the van der Waalscontacts are considerably shorter as compared with the Ca2�

structure (19). For example, the distances between some of theresidues in this region in the present structure and the 1DANstructure that differ by�0.5 Å are as follows: 3.86 versus 4.33 Å

between Phe31L C�1 and Trp158TCz2; 4.18 Å versus 4.91 Å betweenArg36L C� and Trp158T Cz2; 4.75 Åversus 6.64 Å between Leu39L C1and Tyr156T CD2; and 4.14 Å versus4.65 Å between Leu39L C� andTrp158T CH2. The more intimatehydrophobic interface was alsoobserved in the FVIIa/sTF structure(PDB code 1Z6J) determined from acrystal grown in the presence ofMg2� and citrate. This ismost likelybecause of the binding of Mg2� toGla25 and Gla29 (see below) in theCa2�/Mg2� structures.Gla Domain—The conformation

of the � loop between the Ca2�/Mg2� structure (present structure)and the Ca2� structure (19) was alsoobserved to be different. Here, resi-dues 12L–46L of the Gla domainwere used to superimpose the twostructures. As shown in Fig. 2A, themain chain atoms and the side chainpositions of the three hydrophobicresidues (Phe4L, Leu5L, and Leu8L)as well as those of Gla6 and Gla7,differ in the two structures. Thepositions of the Ca2�/Mg2� ions intheGla domain of the present struc-ture and the positions of the Ca2�

ions in the Banner structure (19) arealso depicted in Fig. 2A. The hydro-phobic residues as well as Gla6 andGla7 are well ordered in the presentstructure. In the present structure(Fig. 2B), the side chain NH2 ofArg9Lmakes anH-bondwithO�1 ofGla6. The amide N of Phe4L makesan H-bond with O�3 of Gla7, andNH2 of Arg15L makes an H-bond

with the carbonyl O of Pro10L (not shown). In the Ca2� struc-ture (19), the NH2 groups of both Arg9L and Arg15L makeH-bonds with Gla6. An additional H-bond (amide N of Ala1Land Gla26) and the coordination of the carbonyl O of Ala1L toCa5 are common to both structures. Thus, there are sufficientinteractions that allow for well ordered folding of the � loop inthe present structure.The coordination of each Ca2� and Mg2� ion in the Ca2�/

Mg2� structure to the Gla residues and water molecules isshown in Fig. 2B. All Ca2�, Mg2�, and water molecules are wellordered, and the coordinations are listed in Table 2. Mg2� atposition 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 addi-tionalwatermolecules coordinated toMg1 in the present struc-ture. Furthermore, the position and coordination of Mg1 issimilar to that observed for the Gla domain of FIX in the pres-

FIGURE 1. View of the pAB-VIIa/sTF complex. Ribbon representation of the pAB-VIIa/sTF with metal ions isshown. The protease domain of FVIIa is in blue; the EGF2 domain is in cyan; the EGF1 domain is in red; and theGla domain is in yellow. The four Ca2� in the Gla domain, one (Ca8) in the EGF1 domain, and one (Ca9) in theprotease domain are shown as black spheres. The three Mg2� in the Gla domain are shown as cyan spheres. Mg1is in the back of the structure and is shown by an arrow. The Na� (putative) in the protease domain is shown asa lavender sphere, and the two Zn2� (Zn1 and Zn2) are shown as yellow spheres. The N-terminal domain of sTFis shown in black (TF1) and the C-terminal domain is shown in green (TF2). The FVIIa/sTF structure of Banner etal. (19) (PDB code 1DAN) in the presence of Ca2� is superimposed on the present Ca2�/Mg2� structure and isshown in magenta. The C� atoms of the Gla domain residues 1– 46 were excluded in superpositioning. Fororientation, 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 interac-tions between sTF and the FVIIa Gla domain. The �-helices are presented as ribbon cylinders and �-strands asthick arrows. FVIIa is in yellow, and sTF is in magenta. Residues Leu13L, Phe31L, Arg36L, Leu39L, and Phe40L ofFVIIa that interact with residues Tyr156T, Trp158T, Cys186T, Val207T, and Cys209T of sTF are labeled without thechain identifier. Overall, these residues make shorter contacts than that seen in the Banner structure (19).




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ence of Ca2�/Mg2� (57) and of FXain the presence of Mg2� only (58).Ca2� at position 2 coordinates to

Gla26, Gla29, and to a water mole-cule as in the structure of Banner etal. (19). However, coordination toGla7 in the D-FFR-VIIa structure(19) is replaced by coordination totwo water molecules (S508 andS602) that occupy the Gla7 positionin the present structure. Ca2� atposition 3 coordinates to Gla16,Gla26, and Gla29 as well as to threewater molecules one of which,namely S602, occupies the sameposition as Gla7 O�2 in the D-FFR-VIIa structure (19).Mg2� at position 4 coordinates to

Gla16 and Gla26, as is the Ca2� inthe Banner structure (19). Instead ofdirect coordination of Gla6 andGla7 to Ca2� in the D-FFR-VIIastructure, Gla7 coordinates to Mg4via two water molecules (S209 andS363). Furthermore, S209 coordi-nates to O1 of Gln2L as comparedwith its direct coordination withCa4 in the structure of Banner et al.(19). Again, the distances for theMg4 coordination are shorter(Table 2), and its position is com-patible with that of FXa structure inthe presence of Mg2� (58).Ca2� at position 5 coordinates to

Gla16, Gla20, and to the carbonyl Oof Ala-1L similar to that in theD-FFR-VIIa structure (19). How-ever, instead of coordination toGla6, two water molecules are pres-ent in the present structure, one(S630) of which coordinates toGla7.Ca2� at position 6 coordinates toGla6, Gla20, and to a water mole-cule (S630), which is connected toGla7. In the D-FFR-VIIa structure,coordination to only Gla20 isobserved.Mg2� at position 7 coordinates to

Gla14 and Gla19, as is the case forCa2� at this position in the D-FFR-VIIa structure (19). The coordina-tion distances are short and arecompatible with Mg2� and notCa2� (Table 2). This position andcoordination of Mg7 is consistentwith a previous report of FVIIa/sTFin Mg2�/Ca2�/citrate (PDB code1Z6J). However, the reported coor-

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 forsuperpositioning were residues 13L– 46L. The Ca2�/Mg2� structure is in blue and the Ca2� structure is inmagenta. Phe4, Leu5, Gla6, Gla7, and Leu8 are depicted for both structures. Ca2� (blue) and Mg2� (cyan) ions forthe present structure as well as Ca2� ions (magenta) for the PDB code 1DAN (19) are shown as spheres. Nrepresents the N terminus of the Gla domain of FVIIa. B, coordination of the Ca2� and Mg2� ions in the Gladomain of the Ca2�/Mg2� structure. Electron density (2Fobs � Fcalc) contoured at 1� of all nine Gla residues aswell as that of Ca2� (magenta spheres), Mg2� (cyan spheres), and coordinating water molecules (red spheres) isshown. Coordination of Ca5 to O of Ala1L and H-bonds between Gla6 and the NH2 side chain of Arg9L, Gla7, andN of Phe4L, and a water molecule with O�1 of Gln2L are indicated with dashed arrows. Black dashed lines depictall other coordinations and H-bonds. A stereo figure is given in the supplemental material as Fig. 1S.




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dination distances in the 1Z6J structure are long for Mg2�, andno 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 Gla7seen in the Banner structure and coordinate with Ca2 andCa3. Furthermore, Gla7 occupies the position of Gla6 in theBanner structure and substitutes for it directly in coordinat-ing 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 theBanner structure.Ca2� Binding to FVIIa, Gla-domainless FVIIa, and Pro-

thrombin Fragment 1—The goals of these experiments were2-fold. The first goal was to determine the number of Ca2� sitesthat would be occupied by FVIIa and prothrombin fragment 1in the presence of plasma concentrations of 0.6 mM Mg2� (65,66). The second goalwas to determine theCa2� sites thatwould

be occupied by FVIIa under our crystallization conditions (5mMCa2� and 50mMMg2�). Prothrombin fragment 1 was usedas a substitute for the Gla domain of FVIIa because we wereunable to obtain large enough amounts of the Gla domain ofFVIIa that were suitable for direct Ca2� binding studies. Theresults of the Ca2� binding experiments are presented in Fig. 3and summarized in Table 3. In agreement with previous data(20, 67), full-length FVIIa bound nine, Gla-domainless FVIIabound two, and prothrombin fragment 1 bound seven Ca2�

ions in the absence of Mg2� (Fig. 3, A–C). The two Ca2�-bind-ing sites in the Gla-domainless FVIIa could not be displaced byMg2� (Fig. 3C). In full-length FVIIa and in prothrombin frag-ment 1, Mg2� displaced a maximum of three Ca2� sites underplasma (Fig. 3, A and B) or crystallographic concentrations ofMg2� (Fig. 3C). From these data, we conclude that in full-lengthFVIIa,Mg2�occupies three sites in theGla domain. Based uponthe Ca2�/Mg2� structure of FIX Gla domain (57) as well as the

TABLE 2Ca2�/Mg2� coordination in the FVIIa Gla domain and its comparison with the 1DAN Ca2� Gla structure

1PF2 numbering (Ref. 52)Coordination in present structure (2A2Q) Coordination in 1DAN

1DAN numbering (Ref. 19)Ligand Distance Ligand Distance

Å ÅMg-1 (B-factor 26) O�2 Gla25 1.77a O�1 Gla25 2.95

O�3 Gla25 2.10 O�3 Gla25 2.48O�1 Gla29 2.23 O�1 Gla29 2.44 Ca-9O�3 Gla29 1.92 O�3 Gla29 3.11S228 (H2O228 in 2A2Q) 1.78S630 2.44

Ca-2 (B-factor 56) O�3 Gla26 2.27 O�2 Gla26 2.38O�1 Gla29 2.92 O�3 Gla29 2.28O�2 Gla29 3.00 O�4 Gla29 2.56 Ca-3S508 (O�4 Gla7 position in 1DAN) 3.39 O�4 Gla7 2.35S602 (O�2 Gla7 position in 1DAN) 2.82 O�2 Gla7 2.39S694 2.02 H2O 241 2.65

H2O 242 2.50Ca-3 (B-factor 53) O�3 Gla16 3.49 O�3 Gla16 2.54

O�2 Gla29 2.47 O�4 Gla29 2.57O�3 Gla26 3.34 O�1 Gla26 2.46 Ca-4O�4 Gla26 2.82 O�1 Gla7 2.68S602 (O�2 Gla7 position in 1DAN) 2.17 O�2 Gla7 2.53S262 2.62 H2O 188 2.57S411 3.39 H2O 189 2.51

H2O 190 2.61Mg-4 (B-factor 20) O�1 Gla16 1.92 O�1 Gla16 2.27

O�3 Gla16 1.97 O�3 Gla16 2.38O�1 Gla26 1.97 O�1 Gla26 2.49 Ca-5O�4 Gla26 1.95 O�4 Gla26 2.64S209 (to O1 Asn2 and O�1/O�2 Gla7) 1.98 O1 Asn2 2.34S363 (to O�2 Gla7, not shown in Fig. 2B) 2.11 O�1 Gla7 2.36

O�1 Gla6 2.56Ca-5 (B-factor 51) Carbonyl O Ala1 2.97 Carbonyl O Ala1 2.37

O�1 Gla16 2.31 O�1 Gla16 2.49O�2 Gla16 2.43 O�2 Gla16 2.41 Ca-6O�3 Gla20 3.03 O�3 Gla20 2.71O�2 Gla7 3.25 O�4 Gla20 2.63S722 2.71 O�1 Gla6 2.61

O�4 Gla6 2.69H2O 186 2.51

Ca-6 (B-factor 53) O�3 Gla20 2.89 O�3 Gla20 2.48O�4 Gla20 2.21 O�1 Gla20 2.62 Ca-8O�3 Gla6 2.18S722 to O�2 Gla7 2.33

Mg-7 (B-factor 41) O�1 Gla14 1.93 O�2 Gla14 2.71O�4 Gla14 1.86 O�3 Gla14 2.39 Ca-7O�2 Gla19 1.88 O�2 Gla19 2.28O�4 Gla19 1.87 O�3 Gla19 2.70

H2O 243 2.79a The coordination distances for Mg2� positions in the present structure are compatible withMg2�-oxygen coordination distances of �2 Å in small molecules (61, 62) and notwith 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 1DANstructure at 2.0 Å resolution is from only calcium conditions.




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structure of FXa in the presence of Mg2� (58), these three siteswere identified at positions 1, 4, and 7. The Mg2� ions at thesesites in the present structure coordinate to their respectiveligands with distances compatible with Mg2� and not Ca2�

(Table 2).Role of Mg2� in Activation of FX by FVIIa/TF—We next

examined whether plasma concentrations of Mg2� couldpotentiate FVIIa/sTF and/or FVIIa/rTF activation of FX. Thesedata are presented in Fig. 4A for sTF and in Fig. 4B for rTF/PL.As depicted in Fig. 4A, 1 nM FVIIa/sTF activated FX (10 nM) at0.6 pM/min in the presence of 1.1 mM Ca2�, 1 pM/min in thepresence of 1.7 mM Ca2�, and 5 pM/min in the presence of 1.1mMCa2�, 0.6 mMMg2�. As depicted in Fig. 4B, 10 pM of FVIIa/rTF/PL activated FX (10 nM) at 101 pM/min in the presence of1.1 mM Ca2�, 210 pM/min in the presence of 1.7 mM Ca2�, and456 pM/min in the presence of 1.1 mM Ca2�, 0.6 mM Mg2�.Thus, in systems with and without PL, plasma concentrationsof Mg2� potentiate the activation of FX 5 to 8-fold at physio-logic concentrations of Ca2�. Thus, the three sites in the FVIIaGla domain that are predicted to be occupied by Mg2� (Fig. 3)in vivo are likely to play a significant role in FX activation (Fig. 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 Zhanget al. (30) except that the present structure has two water mol-ecules as comparedwith one in the structure of Zhang et al. (30)and none in the structure of Banner et al. (19). The distances ofligands coordinating toCa2� are�2.4 Å (Table 4) in agreementwith the biochemical data presented in Fig. 3C, indicating thatMg2� cannot displace the EGF1 domain Ca2� site. As shown inFig. 5A, the EGF1 domain Ca2� (Ca8) is coordinated to fiveprotein ligands composed of the carbonyl O of Gln64L, the car-bonyl O of Gly47L, the side chain carboxyl group of Asp46L, andboth carboxyl groups of Asp63L as well as to two water mole-cules, which are well ordered. All major hydrophobic and polarinteractions observed previously (19, 30) involving the EGF1and 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 bindtwo Zn2� ions (34). Zn2� has also been shown to inhibit theactivity of FVIIa and reduce its affinity for TF (34, 35). Basedupon mutagenesis and modeling studies, it was proposed thattheZn2� sites involveHis216H (76) andHis257H (117) (34). Thisinformation was utilized in locating the Zn2� sites in the pro-tease domain (Fig. 5B). Site 1 involves direct coordination toHis216H (76), side chain Glu220H (80), side chain Ser222H (82),and three well ordered water molecules. Site 2 involves directcoordination to His257H (117), the side chain of Lys161H (24),the carboxyl group of Asp219H (79), the carbonyl group ofGly209H (69), and two well ordered water molecules. The dis-tances and geometry for the two Zn2� sites are given in Table 4.Zn1 is connected to the protease domain Ca2� site via the car-boxylate side chain of Glu220H (80), whereas Zn2 is connectedto theCa2� site viawatermolecules. Except forGlu220H (80), allside chains involved in Zn1 and Zn2 coordination are unique toFVIIa (68) and are ideal for Zn2� coordination (see “Discus-sion”). Furthermore, refining the structure with water mole-

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 prothrombinfragment 1 was 0.8 mg/ml, and the concentration of calcium varied from 0.1to 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 thepresence of various concentrations of Mg2� at 5 mM constant Ca2�. Opencircles, 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.




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cules at these two sites gave B values (crystallographic temper-ature factors) of �12.0, indicating that these sites are occupiedby ions with number of electrons much higher than water.These observations further validate assigning Zn2� ions tothese sites.

The protease domainCa2�-binding site in the pAB-VIIa/sTFstructure is located between the two Zn2� sites and is similar tothat described previously (19, 30). Ca2� coordinates to the sidechains of Glu210H (70) and Glu220H (80), to the carbonyl oxy-gens of Asp212H (72) and Glu215H (75), as well as to two wellordered water molecules (Fig. 5B). Moreover, in agreementwith Zhang et al. (30), we were also unable to observe coordi-nation of Asp217H (77) to Ca2� via a water molecule as initiallyobserved by Banner et al. (19). However, Asp217H (77) links toZn2 site via a water molecule (Fig. 5B and Table 3).Putative Na� Site in the Protease Domain of FVIIa—We

recently observed that Na� increases the catalytic activity ofFVIIa in the absence of Ca2� and a mutant in which Phe374H

FIGURE 4. Effect of plasma concentration of Mg2� on FX activation byFVIIa/sTF and FVIIa/rTF. A, activation of FX by FVIIa/sTF. Each reaction con-tained 1 nM FVIIa, 10 nM sTF, and 10 nM FX in TBS, pH 7.5, containing 0.1% PEG8000 and different concentrations of Ca2� and Mg2�. At different times, thereaction was stopped by the addition of EDTA, and the FXa generated wasmeasured using the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitro-aniline. 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 �M PL and 10 nM FX in TBS,pH 7.5, containing 0.1% PEG 8000 and different concentrations of Ca2� andMg2�. The amount of FXa generated was measured as described in A. Opencircles, 1.1 mM Ca2�; open squares, 1.1 mM Ca2� � 0.6 mM Ca2�; closed circles,1.1 mM Ca2� � 0.6 mM Mg2�.

TABLE 3Binding of Ca2� to FVIIa and prothrombin fragment 1

Proteinmol of Ca2�/mol of proteina

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 � 0.2FVIIades1–38 1.8 � 0.2 1.7 � 0.2 NDb

Prothrombin fragment 1 6.8 � 0.3 3.8 � 0.2 3.9 � 0.2aAll 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 adivalentmetal-binding site in the EGF1domain aswell as in the protease domain only (20). Prothrombin fragment 1 has only theGla domainmetal-binding sites. This fragmentwas used to confirm the results obtained with FVIIa.

b ND indicates not determined.

TABLE 4The EGF1 domain Ca2� and the protease domain Ca2�, Na�, andZn2� coordination in the pAB-VIIa/sTF crystal structureThe * denotes ligands that are in the apical position in octahedral geometry. Theremaining ligands occupy the square planar positions. Residue numbering systemfor Ca8 is that of light chain of FVIIa and that of Ca9, and Zn1, Zn2 and Na in theprotease domain is that of chymotrypsin. S denotes solvent (water).

Ion Ligand DistanceÅ

Ca8 Asp46 OD2 2.33aEGF1 domain Gly47 O 2.32

Gln49 O�1 2.27Asp63 OD1 2.73Asp63 OD2 2.19Gln64 O 2.33S24 O 2.53S441 O 2.52

Ca9 Glu70 O�2 2.19protease domain Asp72 O 2.17

Glu75 O 2.01Glu80 O�2 2.01S26* O 2.01S58* O 2.20

Zn1a His76* ND1 3.20protease domain Glu80 O�1 2.88

Ser82* O� 3.69S374 O 3.20S477 O 2.52S530 O 2.42

Zn2 Lys24 Nz 2.57protease domain Gly69 O 2.94

Asp79 OD2 3.84His117 ND1 3.03S528* O 2.84S531* O 3.30

Na Tyr184 O 2.91protease domain Ser185 O 3.36

Thr221 O 2.30His224* O 3.30S144* O 2.60S357 O 3.05

a The site occupancy for each Zn2� site appears to be 1. This is based upon the factthat theB-factors dropped from the low40s to�12when the structurewas refinedwith water molecules only. For Na�, the site occupancy could not be determinedbecause when a water molecule was substituted for Na�, the B-factor droppedonly slightly from 22 to 19.




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(225) is mutated to Pro loses the Na� potentiation effects (69).However, in the presence of Ca2�, physiologic concentrationsof Na� have minimal effect on the activity of FVIIa (69, 70).Based upon homology, FVIIa might contain a Na� site at aposition similar to that proposed for FXa, FIXa, and APC (36–40). In previous FVIIa structures, a water molecule has beenplaced at the proposed Na� site (19, 29–31). The location ofthis putative Na� site in FVIIa and its ligands are shown inFig. 6A and Table 4. As is the case with FXa, FIXa, and APC,Na� in FVIIa coordinates to the carbonyl oxygens ofTyr332H (184), Ser333H (185), Thr370H (221), and His373H(224) as well as to two water molecules, one of which is linkedto 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 islinked to the FVa-binding site (71, 72). In the pAB-FVIIa/sTFstructure, we observed that Phe374H (225), a key determinant ofthe Na�-binding site, is located in a hydrophobic cavity con-sisting of residues Pro303H (161), Leu305H (163), Thr307H (165),Cys310H (168), 311LeuH (169), Gln313H [170A], Ile323H (176),and Cys329H (182). This hydrophobic core extends toMet306H(164), Gln308H (166), and Asp309H (167) that are part of theTF-binding helix (Fig. 6B). In this context, the protease domainof FVIIa hasmany hydrophobic and polar interactions with TF.These interactions are not shown in the figure but are as fol-lows: Met306H (164) interacts with Arg74T, Phe76T, Pro92T,and Tyr94T. The amide N of Thr307H (165) makes an H-bond

with Glu91T; and NH1 and NH2 of Arg379H (230) makeH-bonds with Glu91T. Moreover, Gln308H (166) makes anH-bondwith the carbonylOofTyr94T. Furthermore, the amideN andO2 of Asp309H (167)make anH-bondwith the hydroxylof Tyr94T as well as with the N2 of Asn96T. The disruption ofthe Na� site when Phe374H (225) is changed to Pro could col-lapse this hydrophobic cavity and result in diminished interac-tions with TF. Notably, Phe374H (225) was shown to be neces-sary for optimal TF-mediated activation of the catalyticfunction of FVIIa (73).Weused surface plasmon resonance to experimentally deter-

mine the effect ofNa� on the binding of FVIIa to sTF.As shownin Fig. 7, in the presence of Na�, the kon for binding was 4.6 �0.6 105 M�1 s�1; koff was 2.4 � 0.2 10�3 s�1, and Kd was5.2 � 0.3 nM. The Kd value in the presence of Na� is similar tothat observed by Petrovan and Ruf (73). In the presence of Ch�,the kon was 1.8 � 0.3 105 M�1 s�1; koff was 2.5 � 0.2 10�3

s�1, andKdwas 13.9� 0.7 nM. Thus, Na� affects the binding ofFVIIa to sTF by �3-fold, which is primarily via an effect on kon.This may be due to stabilization of the interactions noted in thepreceding paragraph.Atypical Conformation of the Lys341H–Gly342H (192–193)

Peptide Bond in the pAB-VIIa/sTF Structure—During interme-diate stages of refinement, it was observed that the carbonyl Oof Lys341H (192), which normally points away from the oxyan-ion hole in serine proteases, was positioned in negative electrondensity (Fobs � Fcalc) contoured at�2.5� (Fig. 8A). At the same

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 EGF1domain. Electron density (2Fobs � Fcalc) contoured at 1� (gray) for Ca2� (magenta sphere) and two water molecules (red spheres) is shown. Electron densitycontoured 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. Electrondensity (2Fobs � Fcalc) contoured at 1� (gray) of Zn2� (cyan spheres), Ca2� (magenta sphere), and water molecules (red spheres) is shown. The electron densitycontoured 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 ligandsand 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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time, it was also noted that the side chain of Gln286H (143) waslocated in negative density as well. Moreover, there was signif-icant positive density (Fobs � Fcalc) contoured at 4� (Fig. 8A)

where the carbonyl O of Lys341H (192) could be moved by con-certed flipping of the Lys341H–Gly342H (192–193) peptidebond. Following this, it was then possible tomove the side chainof Gln286H (143) out of the negative density and into the posi-tive 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 negativeand positive density in the difference (Fobs � Fcalc) map validat-ing that the Lys341H–Gly342H (192–193) peptide bond confor-mation in pAB-VIIa/sTF is unconventional (Fig. 8B). Of note isthe observation that O� of Ser344H (195) makes an H-bondwith the amino group of pAB and with the carbonyl O ofLys341H (192). However, in the D-FFR-VIIa (19) and the BPTI-VIIa (30) structures, the carbonyl O of Lys341H (192) makes anH-bond with the amide N of Gln286H (143), whereas in thepAB-VIIa/sTF structure, the amide N of Gly342H (193) makesan H-bond with the carbonyl side chain of Gln286H (143).Structures of Benzamidine-VIIa/sTF and D-FPR-VIIa/sTF—

Both thebenzamidine-VIIa/sTF andDFPR-VIIa/sTF structuresare comparable with the pAB-VIIa/sTF structure with respectto the Gla domain � loop placement as well as Na�, Ca2�, andMg2� binding. However, the benzamidine-VIIa/sTF crystals,which were grown in the absence of added Zn2�, have watermolecules at the two Zn2� positions. In the benzamidine-VIIa/sTF structure, included in this report, the Zn2� sites were ini-tially refined with water molecules and had B-factors of 19 and22; however, when the water molecules were replaced withZn2�, the B-factors increased to 56 and 63. For consistency, theZn2� are left to occupy the Zn2� sites in the structure. It isrealized that in the benzamidine-VIIa/sTF structure included

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 involvescoordination 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) forNa� (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 shownin 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, hydrophobicenvironment surrounding Phe225. The �-helices are presented as ribbon cylinders and �-strands as thick arrows. FVIIa is in cyan, and sTF is in yellow. The hydrophobicresidues 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 areinvolved 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 bufferused 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 strengthconstant. sTF was immobilized on a CM5 chip by the amine coupling method.The chip was activated by mixing 400 mM N-ethyl-N-(3-dimethylamino-propyl)-carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide. Animmobilization level of 752 response units (RU) was attained for the boundprotein. Residual reactive groups on the chip surface were blocked using 1.0M 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 areshown as dashed lines, and Ch� curves are shown as solid lines. Magenta, 2.8nM 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-onebinding.




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here, the Zn2� only partially occupies these sites. In the othertwo benzamidine-VII/sTF structures and in the D-FPR-VIIa/sTF structures, which contained added 20 �M Zn2�, the B-fac-tors for the two Zn2� were in the low 40s, indicating morecomplete 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 nonstandard orientation such thattheGly342H (193) amideNpoints away from the oxyanion hole,and the carbonyl O of Lys341H (192) points into the oxyanionhole. This was noted in all three benzamidine-VIIa/sTF crystal

structures, two without the added Zn2�, including the one pre-sented here (Table 1 and Fig. 8C) and the one with added Zn2�.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) peptidebond to form the oxyanion hole. Refinement of the benzami-dine-VIIa/sTF crystal that has been soaked with D-FPR-ckrevealed benzamidine displacement, D-FPR incorporation, andinduction 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

FIGURE 8. Conformations of the Lys341H-Gly342H (192–193) peptide bond in pAB-VIIa, benzamidine-VIIa, and D-FFR-VIIa. A, negative electron densitysurrounding the carbonyl O of 192 at intermediate stages of pAB-VIIa refinement. The negative difference map (Fobs � Fcalc) contoured at �2.5� surroundingresidues 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 inblue. 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. The192–193 peptide bond was flipped 180°, and adjustment of the Gln143 side chain was made before further refinement. All maps were contoured at the samelevel 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. TheH-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 andMethods.” All maps were contoured at the same level as in A. Again note the absence of positive (blue) or negative electron density (red) encompassing thecarbonyl 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.




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of the oxyanion hole and makes an H-bond with the negativelycharged carbonyl oxygen (oxyanion) of the P1 Arg residue ofD-FPR (Fig. 8D). The formation of this H-bond is required forstabilization of the tetrahedral transition complex, an interme-diate formed during peptide bond cleavage. Cumulatively, ourdata imply that the enhancement of FVIIa catalytic activityupon TF binding does not entail correction of the impairedoxyanion hole to the standard conformation; however, activesite peptidyl inhibitor/substrate binding does induce the stand-ard oxyanion hole conformation.

DISCUSSION

The absence of an oxyanion hole in a crystal of FVIIa/sTF at2.2 Å with an amidinophenylurea-based inhibitor was firstreported in 2003 (74, 75). In this series of inhibitors, the nitro-gen at the para position of the benzamidine moiety and thecarbonyl group of Lys341H (192) make H-bonds with thehydroxyl 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 ofFVIIawith a sulfonamide inhibitor in the absence of TF. Similarto the FVIIa with the amidinophenylurea-based inhibitor (74),the nitrogen at the para position of the benzamidine moiety ofthe 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) observedthe same two H-bonds involving Ser344H (195), Lys341H (192),and the nitrogen at the para position of the benzamidinemoietyin FVIIa � sTF structures with a phenylglycine inhibitor withnonstandard oxyanion hole conformation. From the abovestudies it was inferred that the nonstandard conformation ofthe oxyanion hole in these structures is because of distinctiveproperties of the inhibitors.In the pAB-VIIa/sTF structure (Fig. 8B), the same two

H-bonds involving the p-amino group of the benzamidinemoi-ety and the carbonyl group of Lys341H (192) with the hydroxylof Ser344H (195) are observed. These observations indicate thatthe other segments of the respective inhibitors (32, 74–76) donot contribute to the nonstandard conformation of the oxyan-ion hole. The nonstandard oxyanion hole conformationsobserved in FVIIa with the above series of inhibitors as well aspAB may stem from the presence of nitrogen at the para posi-tion of the benzamidine moiety.To ascertain whether the presence of nitrogen at the para

position of the benzamidine moiety of the various inhibitors isthe cause of the nonstandard oxyanion hole conformation inFVIIa, we crystallized FVIIa/sTF with benzamidine. In all threebenzamidine-VIIa/sTF structures in the presence and absenceof added ZnCl2, we observed the nonstandard oxyanion holeconformation. This strongly indicates that the nonstandardoxyanion hole conformation in FVIIa is not related to theunusual properties of the FVIIa inhibitors used in previousstudies (32, 74–76). The structure obtained with benzamidineleads us to propose that the nonstandard oxyanion hole confor-mation is rather an inherent property of FVIIa.Soaking benzamidine-VIIa/sTF crystals with D-FPR-ck

resulted in benzamidine displacement and D-FPR incorpora-tion with resultant flip of the Lys341H–Gly342H (192–193) pep-

tide bond such that the oxyanion hole is fully formed (Fig. 8D).For this to occur the H-bond between the amide N of Gly342H(193) and side chain of Gln286H (143) must first be broken.Then, either concurrently or subsequently, the Lys341H–Gly342H (192–193) peptide bondmust be flippedwith resultantformation of three H-bonds, including one between the car-bonyl group of Lys341H (192) and the backbone N of Gln286H(143) and two between the oxyanion and the amide nitrogens ofGly342H (193) and Ser344H (195). The negative charge on thecarbonyl oxygen of the transition state intermediate shouldprovide enough energy to overcome the energetic barriers ofthe first two steps involving breaking the H-bond and flippingthe peptide bond. Subsequently, formation of the threeH-bonds results in stabilization of the tetrahedral intermediate,which is necessary for catalysis. The crystal soaking experi-ments strongly support that it is the substrate/inhibitor activesite occupancy with a developing oxyanion and not TF bindingthat induces formation of the oxyanion hole in FVIIa. The com-petent oxyanion hole observed in several FVIIa crystal struc-tures in the presence and absence of TF with transition stateanalogue inhibitors (19, 28–30, 77) must then be due to theabove 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 beenobserved for the toxin B (80, 81). In the case of toxin A, it is theproline at position 192 that causes the peptide bond betweenPro192 and Gly193 to flip �180° relative to that typically seen inserine proteases. Because both standard (competent) and non-standard (incompetent) conformations have been observed forthe toxin B (80, 81), it has been suggested that upon binding ofthe substrate (providing oxyanion), the peptide bond betweenPro192 and Gly193 in toxin A would flip 180° to provide compe-tent conformation for catalysis (78, 79). Interestingly, thrombinhas also been shown to have a reversed oxyanion hole in theabsence of Na� (82). Based upon these observations coupledwith our FVIIa/sTF soaking experiments and free energy sim-ulations, it appears that there is enough flexibility to adopt opti-mal oxyanion hole conformation upon substrate binding assuggested by Cavarelli et al. (79) and Bobofchak et al. (83).

FVIIa/TF recognizes its physiologic macromolecular sub-strates, FIX and FX, primarily via exosites distant from theactive site. In this regard, the Gla and/or EGF1 domains of FIXand FX have been implicated to be the primary determinants inbinding to the FVIIa/TF complex (49, 84–86, 88–92).Once thetrimolecular complex is formed, the cleavage site peptidesequence in FIX or FX approaches the active site cleft ofFVIIa/TF and induces formation of the oxyanion hole. Thismechanism provides discriminating specificity in activation ofFIX and FX by FVIIa/TF.One should note that in the reported structure of uninhibited

FVIIa, the Lys341H–Gly342H (192–193) peptide bond had nor-mal conformation (31). However, in this structure, the activesite of FVIIa was initially occupied by a sulfate ion in the oxya-nion hole similar to the carbonyl group of P1 residue. Thiscould reorient the Lys341H–Gly342H (192–193) peptide bondas seen in the D-FPR-VIIa (present paper), D-FFR-VIIa (19), andBPTI-VIIa (30) structures. Upon removal of the sulfate ion by




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soaking crystals under sulfate-free conditions, the Lys341H–Gly342H (192–193) peptide bond conformation may notchange because of its stabilization by the H-bond betweenLys341H (192) carbonyl group and Gln286H (143) main chain Nas 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 inFVIIa. This is supported by the metal-ligand coordination dis-tances, which are compatiblewithCa2�, despite the presence ofexcess Mg2�. The protease domain Ca2� site is similar to thatdescribed for trypsin, which also could not be replaced byMg2�

in the crystals (44). Moreover, the EGF1 domain of FXa wasdisordered when the crystals were grown in the presence ofMg2� (58), in agreementwith our data that FVIIa EGF1 domaincannot bind Mg2�. The significance of these two Ca2� sites isthat their occupancy is required for FVIIa binding to TF anddevelopment of its catalytic activity.The data presented in Fig. 3 demonstrate that under physio-

logic and crystallographic conditions, the FVIIa Gla domainbinds three Mg2� and four Ca2� ions. Three Mg2� ions werelocated based upon the structure of the Gla domain of FXa inthe presence ofMg2� (58) and the structure of FIX Gla domainin the presence of Ca2�/Mg2� (57). The four Ca2� ions werelocated based upon the Ca2� structures of prothrombin frag-ment 1 (59) and D-FFR-VIIa (19). Under plasma concentrationsof �1.1 mM free Ca2� and �0.6 mM free Mg2�, we predict thatthese three sites will be occupied by Mg2�. Interestingly, thesethree Mg2� sites are certainly the ones that could not be dis-placed by Ca2� in the Gla domain of prothrombin (Fig. 3) (93,94) or FVIIa (Fig. 3). In support of this, data have also beenpresented 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� sitesthat we have identified in the Gla domain of FVIIa are consist-ent with the biochemical and crystallographic data.The binding of the above Mg2� sites is expected to be coop-

erative in nature (58, 93–98), and filling of these sites wouldresult in an intermediate folding state of the Gla domain inwhich the � loop (residues L1–L12) of FVIIa is disordered asobserved in the case of FXa (58). Under in vivo conditions, it islikely thatMg2� and not Ca2� affords this intermediate confor-mational state (92, 94). Filling of the remaining four Ca2�-spe-cific sites to the intermediate state will promote proper foldingof the N-terminal residues into the � loop conformation forbinding to the PL surface. This two-state sequential model isconsistent with the earlier observations of Borowski et al. (98)and Wang et al. (58). Furthermore, it is possible that the Gladomain conformation seen in the present structure representsan advanced intermediate folding stage and that the Ca2� sitesidentified are only partially occupied.Evidence exists that Mg2� plays a role in physiologic coagu-

lation (23–26, 93, 99). It supports the activation of FIX by FXIaand activation of FX by FIXa at physiologic concentrations ofCa2� (23, 24, 99). It also supports the activation of prothrombinby FXa at very low concentrations of Ca2� (93). In this study, weprovide evidence that it supports the activation of FX byFVIIa/TF (Fig. 4). Thus, it appears thatMg2� and Ca2� work inconcert to promote coagulation in vivo.

Location (Fig. 6A) of the Na� site in FVIIa is based upon theprevious structures of other vitamin K-dependent proteins(36–40). Because sodium ions are particularly difficult to provefrom the electron density, we cannot make a definite statementat this time regarding theNa� site in FVIIa. Anomalous diffrac-tion data are needed to unequivocally establish the existence ofa Na� 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 ofNa� in blood clotting proteasesmay be toinfluence 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 involvesside chains of His, Asp, and Lys, two water molecules, and themain chain carbonyl group (Fig. 8). Both metal sites have octa-hedral coordination geometry (Table 4), and the implicatedside chains are excellent candidates for binding to zinc (100).Because the side chain of Glu220H (80) is involved in coordina-tion to Zn1, and the carboxyl group of Asp219H (79) and thecarbonyl group of Gly209H (69) are involved in coordination toZn2, binding of Ca2� to the 210H–220H loop [70–80 loop]could attenuate the binding of Zn2�. Biochemical data supportthis concept (34, 35). His211H (71), which was previously con-sidered a candidate for binding to Zn2�, is not unique to FVIIaand 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 forma-tion, �-granules would be released, which would increase thelocal Zn2� concentration. This increased local concentration ofZn2� is likely to inhibit FVIIa bound to TF. Thus, after theinitiation phase of clotting is achieved, Zn2� exerts a mecha-nism of control on FVIIa activity and regulates its ability toactivate its physiologic substrates FIX and FX. Understandingthe precise location of zinc sites could helpmutagenesis studiesin design of a better therapeutic FVIIamolecule, which is resist-ant to inhibition by zinc. Such data could also help understandhow Zn2� inhibits the activity of FVIIa.

CONCLUSION

In this study we have identified three Mg2� sites in the Gladomain of FVIIa. It is quite possible that Mg2� in vivo occupiesthese sites and promotes coagulation. Notably, under physio-logic conditions, the protease and EGF1 domain Ca2� sites willbe solely occupied by Ca2�. Furthermore, we have identifiedtwo Zn2� sites in the protease domain that were not definedpreviously. The ligands for the Zn2� sites are unique to FVIIaand are in agreement with the biochemical data. We also pro-pose a putative Na� site in FVIIa. Na� appears to promotewhereas Zn2� appears to down-regulate FVIIa activity. Atypi-cal conformation of the Lys341H–Gly342H (192–193) peptidebond in FVIIa/TF that results in the absence of an oxyanionhole characteristic of active serine proteases is unique to FVIIa.Because serine proteases play an important role in many phys-iologic processes, it is critical to understand how a given serineprotease recognizes its substrate among a milieu of other pro-teins. One determining factor is the flanking sequences (P4–P4residues) surrounding the peptidyl cleavage site in the substrates(87, 102, 103). Recently, a new concept has emerged in which




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exosites remote from the cleavage site recognition sequences playa dominant role in substrate specificity. In FVIIa/TF, binding toexosites of FIX and FX play a large role in determining specificity.Following the cofactor-enzyme-substrate complex assembly viathese exosites, the active site then approaches the cleavage site forproper catalysis. Such a mechanism introduces a higher level ofselectivity and specificity.

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

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PadmanabhanS. Paul Bajaj, Amy E. Schmidt, Sayeh Agah, Madhu S. Bajaj and Kaillathe

Zn2+ SITES IN FACTOR VIIaOF THE 192-193 PEPTIDE BOND AND MAPPING OF Ca2+, Mg2+, Na+, ANDBenzamidine-VIIa/Soluble Tissue Factor: UNPREDICTED CONFORMATION

-Aminobenzamidine- andpHigh Resolution Structures of

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