doi:10.1016/j.jmb.2007.05.042 J. Mol. Biol. (2007) 371, 774–786

Active and Exo-site Inhibition of Human Factor Xa:Structure of des-Gla Factor Xa Inhibited byNAP5, a Potent Nematode AnticoagulantProtein from Ancylostoma caninum

Jorge L. Rios-Steiner1, Mário T. Murakami2,3,4, Alexander Tulinsky1

and Raghuvir K. Arni2,3,4⁎

1Department of Chemistry,Michigan State University,East Lansing, MI 48824-1322,USA2Department of Physics,UNESP, São José do Rio Preto,15054-000, Brazil3Center for Applied Toxinology,CAT-CEPID, São Paulo,SP 05503-900, Brazil4Center for StructuralGenomics, UNESP, São José doRio Preto, 15054-000, Brazil

Present address: J. L. Rios-SteinerAbbreviations used: Gla, γ-carbox

tissue factor; fIXa, factor IXa; fVIIIa,proteins 5, 6 and c2; TAP, tick anticochymotrypsin-elastase inhibitor; desreferred to without confusion as fXain chymotrypsinogen numbering, Ilebovine pancreatic trypsin inhibitor;E-mail address of the correspondi

0022-2836/$ - see front matter © 2007 E

Hookworms are hematophagous nematodes capable of growth, develop-ment and subsistence in living host systems such as humans and othermammals. Approximately one billion, or one in six, people worldwide areinfected by hookworms causing gastrointestinal blood loss and irondeficiency anemia. The hematophagous hookworm Ancylostoma caninumproduces a family of small, disulfide-linked protein anticoagulants (75–84amino acid residues). One of these nematode anticoagulant proteins, NAP5,inhibits the amidolytic activity of factor Xa (fXa) with Ki=43 pM, and is themost potent natural fXa inhibitor identified thus far. The crystal structure ofNAP5 bound at the active site of γ-carboxyglutamic acid domainless factorXa (des-fXa) has been determined at 3.1 Å resolution, which indicates thatAsp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar tothat of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39of NAP5 additionally forms a hydrogen bond (2.5 Å) with His57 NE2 of thecatalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in thenative structure, resulting in an interaction that has not been observedbefore. Furthermore, the C-terminal extension of NAP5 surprisinglyinteracts with the fXa exosite of a symmetry-equivalent molecule forminga short intermolecular β-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, orthe exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does notinhibit fVIIa of the fVIIa/TF complex.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: nematode anticoagulant proteins; factor X/Xa; ixolaris; tissuefactor pathway inhibitor; active and exo-site binding

*Corresponding author

Introduction

Blood coagulation, a complex balanced event,requires the interaction of a series of proteins and

, Department of Chemistryglutamic acid; EGF, epidfactor VIIIa; fVa, factor Vagulant protein; fXIa, fac-fXa, fXa less its γ-carbox; fXa numbering, light cha16–Arg245; EGR-fXa, factoTFPI, tissue factor pathwang author: arni@ibilce.une

lsevier Ltd. All rights reserve

cofactors, in active and inactive states, to orchestrateand participate in selective and specific catalyticroles aimed at producing a thrombus clot. Humanblood coagulation factor X (fX), a vitamin K-depen-

y, University of Puerto Rico, Mayaguez, Puerto Rico.ermal growth factor; fXa, factor Xa; fVIIa, factor VIIa; TF,a; NAP5, NAP6 and NAPc2, nematode anticoagulanttor XIa; ATI, Ascaris trypsin inhibitor; C/E-1, Ascarisyglutamic acid (Gla) domain (Ala1–Tyr44), alternativelyin beginning with EGF2, Leu88–Arg139, catalytic domain,r Xa inhibited with GluGlyArgchloromethyl ketone; BPTI,y inhibitor.sp.br

d.

775Active- and exo-site inhibition of factor Xa

dent glycoprotein, synthesized in the liver, circulatesin the plasma as a two-chain protein linked by adisulfide bridge.1 The light chain (16.2 kDa, 139residues) is comprised of a γ-carboxyglutamic acid(Gla) domain containing 11 Gla residues,2 and twoepidermal growth factor (EGF)-like modules with aβ-hydroxyaspartic acid residue in the N-terminalEGF1 domain.3,4 The heavy chain (42.7 kDa, 303residues) with two N-linked carbohydrate attach-ment sites harbors the inactive catalytic serineprotease domain.5 fX is converted to its active fXaform by either the factor VIIa (fVIIa)/tissue factor(TF)/cellular surface/Ca2+ extrinsic pathway com-plex or by the fIXa/fVIIIa/cellular surface/Ca2+ X-ase intrinsic pathway complex and fXa alsoparticipates in the formation of the prothrombinasecomplex (prothrombin/fXa/fVa/cellular surface/Ca2+) that converts prothrombin to thrombin bylimited proteolysis.6 A number of enzymes thatparticipate in the blood coagulation cascade can beactivated by snake venom proteinases.7,8

Some organisms, such as leeches, ticks, certaintypes of bats and hookworms, have developed stra-tegic survival mechanisms that permit them to feedfrom a wide range of mammalian hosts, includingman.9–11 Human hookworm infection of the smallintestine dates back to the beginning of dog domes-tication (∼3500 BC), results in the loss of up to 0.2 mlof blood per day,12 and is a leading cause of anemia inthe tropics and subtropics, affecting nearly one bil-lion, or one in six, people worldwide,. Like other he-matophagous organisms, hookworms have evolvedhighly effective anticoagulant mechanisms andmole-cules to facilitate the acquisition of blood for theirsurvival. The hematophagous hookworm Ancylos-toma caninum produces a family of small disulfide-linked protein anticoagulants (75–84 amino acidresidues, Figure 1).10,11 One of these nematode antic-oagulant proteins, NAP5, cloned by Corvas Interna-tional and under development for the prevention andtreatment of disorders involving abnormal blood clotformation or thrombosis, inhibits the amidolyticactivity of fXa with Ki=43 pM.11,13 The only othernatural inhibitor of fXa with a comparable potency toNAP5 is the tick anticoagulant peptide (TAP) (Ki=59pM).8 A similar, highly homologous (90%)member ofthe family (NAP6) inhibits catalytic activity of fXawith Ki ∼1.0 nM. In testing NAP5 against 11 other

Figure 1. Alignment of amino acid sequences ofNAP5, NAP6 and NAPc2. Differences from NAP5 are inbold; disulfide bonds are shown as black lines; theconserved motif involved in exosite binding id boxed indark gray; non-conserved motifs involved in the exositebinding boxed are in light gray; the NAP5 sequence isnumbered consecutively.

serine proteases for potency and specificity with25 μMNAP5 showed only 50% inhibition of just one,fXIa (Ki=3.8 nM). The highest level of inhibitionamong the others (13–15%) was with kallikrein,activated protein C and tissue-type plasminogenactivator. NAP5 and NAP6 inhibit thrombin forma-tion by direct association at the catalytic site of fXa. Inthe case of NAP5, Arg40 is the specificity site P1residue, while phenylalanine is at this position inNAP6, which most likely accounts for the 20-folddecrease in potency of the latter, since the Phe40/Argmutant of NAP6 possesses the same relative activityas NAP5.11 NAPc2, another slightly larger (seven tonine residues) member of the A. caninum family ofanticoagulants differs from NAP5 and NAP6, since itonly partially inhibits the amidolytic activity of fXaand inhibits thrombin formation by binding to fXa atan exosite distinct and remote from the activecenter,14 with the resultant binary complex inactivat-ing the TF-fVIIa complex with Ki=35 pM.11

The non-hematophagous Ascaris family of nema-todes parallels that of Ancylostoma hookworms inproducing small proteins that inactivate chymotryp-sin, elastase, trypsin, pepsin and carboxypeptidase.15

The physiological roles of these inhibitory proteinsdiffer from those of Ancylostoma, in that they protectthe worms from proteolytic degradation by digestiveenzymes of the host through intestinal up-take,whereas the hematophagous hookworm inhibitorsshut down the coagulationmachinery in order to feedfreely on blood and subsist. The similarities betweenthe two notwithstanding, the sequences of theproteins from the two different species display littlehomology (b30% overall, 13% from the five disulfidesalone),11 although a high degree of sequentialcongruence exists among the different inhibitors ofeach species.11,15 An NMR structure determination ofAscaris trypsin inhibitor (ATI) has been described,16

as well as a crystallographic structure of the Ascarischymotrypsin-elastase (C/E-1) inhibitor bound toelastase.17 A unique feature of the C/E-1 elastasecomplex is the penetration of Arg217A of the enzymethrough a pore formed by the Cys17–Cys29 disulfideloop of the inhibitor.17Clinically used anticoagulants are based either on

coumarins,18 which interfere with the vitaminK-dependent proteins, or on heparins,19 which en-hance the inhibition of both thrombin and fXa byantithrombin III. Both these anticoagulants areclinically difficult to control, since they are highlyunspecific and often produce harmful side-effects.This has stimulated the study and search foralternative anticoagulants such as snake venomproteins that inhibit coagulation by binding to theGla domain of fXa,20 or highly specific exosite-binding peptide and protein inhibitors of bloodcoagulation factors such as fXa11 and fVIIa.21

We present here the crystal structure of recombi-nant NAP5 bound to the active site of des-fXa. Wedescribe the overall structure and the mode ofbinding assisted by an unexpected hydrogen bondformed between Ser39 from NAP5 and His57 of thecatalytic triad of fXa along with a regional (or local)

776 Active- and exo-site inhibition of factor Xa

subsidiary binding site. Surprisingly, the NAPc2-binding exosite14 of a symmetry-related fXa mole-cule in the NAP5-fXa crystal structure is also utilizedto bind NAP5 and involves the formation of anantiparallel β-strand between the C-terminal seg-ment of NAP5 with one of the β-strands of theseven-stranded β-barrel (β6) and part of theC-terminal helix of fXa. The fXa exosite interactionsof NAP5 and NAPc2 are compared, and models forthe binding of NAP6 and ixolaris, a two-Kunitzdomain fXa inhibitor from the salivary gland of thehard tick Ixodes scapularis,22 which is a vector forLymes disease, are discussed.

Results and Discussion

Structure of des-fXa

The C-terminal EGF2 module and the catalyticdomain of des-fXa are well defined in the final (2Fo–Fc) electrondensitymap ofNAP5-fXa toArg245 of thecatalytic domain and Arg139 of EGF2. However,several surface Glu, Lys and Arg side-chains havepartial or no density. In addition, the whole N-term-inal EGF1 module, and its leading pentapeptide, isflexibly disordered as in other des-fXa structures.23–25

Since the catalytic domain ends at Arg245, the fXa ofthe NAP5-fXa complex corresponds to the des-fXaβstructure5 lacking a C-terminal hexapeptide, whichwas probably lost by autolysis. Like other inhibitedfXa structures,24–26 there is no apparent cleavage inthe autolysis loop region of NAP5-fXa (His145–Thr153) as was observed in the native structure.23

Optimal superposition of the Cα positions of thecatalytic domain of fXa of NAP5-fXa on those ofnative fXa (PDB code 1HCG) results in a rmsd of0.50 Å for 216 of a total 233 (97%) Cα atoms (ninepositions deviating by more than 1.0 Å); the rmsd ofEGF2Cα positions is larger at 0.71 Å for 49 of 50 (98%)Cα atoms (five deviations greater than 1.0 Å). Thelarger deviation of the EGF2 domain is related to itshigher B-values (native fXa: bBN=27 Å for thecatalytic domain, bBN=37 Å2 for EGF2; NAP5-fXa,bBN=32 Å2 and 42 Å2, respectively).Specific monovalent cation effects in blood coagu-

lation were first reported for fXa,27 followed bythrombin,28 and activated protein C.29 The Na+

binding site in the structure of fXa is in the samerelative position as that in thrombin.30 An incom-plete octahedral Na+ site is present in the structure ofNAP5-fXa composed of carbonyl oxygen atoms fromTyr185, Asp185A, Ala221, Arg222 and Lys224 with aNa+ ion occupancy of 1.0 and B=16 Å2. The compo-sition of the site differs from that of native fXa, inlacking one water molecule and by Ala221O repla-cing a second water molecule. The catalytic domainof fXa also has a calcium ion-binding site31 in thevicinity of Arg70–Glu80, like fVIIa,32 fIXa,33 andtrypsin.34 However no calcium ion was found in thevicinity of the Ca2+-binding loop (no Ca2+ was pre-sent in the crystallization protocols and any residualCa2+ was essentially diluted and dialyzed out).

Structure of NAP5

The mean temperature factor of the NAP5 in-hibitor (45 Å2) is slightly greater than that of theEGF2 domain of fXa (42 Å2) but, like the latter, it isgenerally well defined in the final (2Fo–Fc) electrondensity maps, except for the first two N-terminalresidues, the C terminus (Val77) and some surfaceside-chains (Glu5, Glu27, Glu31, Lys19, Lys24, andArg65). The latter are characterized by breaks in thedensity but are sufficiently well defined to beincluded in the final model. The quality of the(2Fo–Fc) electron density of the whole NAP5-desfXastructure is exceptionally good ,considering theresolution (3.1 Å), most likely due to the highredundancy (9.1) of the diffraction measurements.The overall shape of NAP5 is that of a distorted

oval (axial lengths of 25 Å and 16 Å), wedge-shapeddiscoidwith theN andC termini located on the sameside of the molecule, physically separated by twoshort β-strands (β4, andβ5) and a 310-turn (Glu66–Cys69) but held together with the aid of a bivalentsalt-bridge (Glu10, Glu22, and Arg56) (Figure 2,Table 1). The C-terminal region is located across theoval diagonally opposite the reactive-site P1 residue(Arg40). An extended C terminus (∼29 Å) consistingof eight or nine residues emanates from the 310 turn(Figure 2). Although, as will be seen, this segmentbinds to fXa, it may be flexible and disordered in theunbound inhibitor state as described for NAPc2.The NAP5 molecule is stabilized by five disulfide

bridges, four short β-strands, two short helix-likestructures, a bivalent salt-bridge and numerous otherhydrogenbonds andburied hydrophobic interactions(Figure 2, Table 1). The four short β-strands that areapproximately perpendicular to each other in theNAP5 structure (β1, Glu10–Asp13; β3, Ala47–Cys50;β4, Tyr55–Asp57; β5, Asp62–Val64) are arranged inabout the same relative orientations as those of theATI and C/E-1 hookworm inhibitors (β-strandnumbering is that used byHuang et al.;17 β2 reportedin the latter does not appear to interact closelywithβ4and β5 in either inhibitor structure).16,17 Since the ATIand C/E-1 inhibitors contain only 62 and 63 residues,respectively, while NAP5 contains 77, and theirstructures basically differ from NAP5 because ofsmaller disulfide loop sizes, only the Cα coordinatesof 14 β-strand residues of NAP5 were superposed(Figure 2) on the corresponding coordinates of C/E-117 and resulted in rmsd=0.77 Å. About 50 of the 63C/E-1 residues fit the NAP5 fold reasonably well (5–35, 46–50, and 53–69), while the largest deviationoccurs in the overlap of the 36–46 loop, whichcontains the reactive-site position of the inhibitors(Arg40 for NAP5, Leu31 for C/E-1) (Figure 2). Threeof the five disulfide bridges located on the surfacecontaining the N and C-terminal residues (Cys6–Cys48, Cys50–Cys63, Cys25–Cys69) (Figure 2) super-pose well. The other two disulfide bridges differ inone of the cysteine positions. The latter (Cys37–Cys42) are in the 36–46 loop that does not superposeglobally between the two structures. One of thesedisulfides (Cys21–Cys37) bridges between two short

Figure 2. Stereoview of the superpositioning of C/E-1 on NAP5. A ribbon representation of NAP5 is in blue, theNAP5 disulfide bridges are numbered, reactive-site P1 residues are shown as yellow sticks (Arg40, NAP5; Leu31, C/E-1).

777Active- and exo-site inhibition of factor Xa

distorted helical regions unique to NAP5 (Gly16–Glu22, Asp34–Arg39) (Table 1). The C/E-1 inhibitoralso has a small turn around Pro42–Arg44 not presentin NAP5 and another one that corresponds to theC-terminal 310-turn of NAP5 (Figure 2).

Active site binding of NAP5

Incubation with fXa results in partial cleavagebetween the Arg40 and Gly41 of NAP5.11 In the

Table 1. Secondary structural and polar intramolecularinteractions of NAP5

A. Antiparallel β-strandsβ1 β3 d (Å) β4 β5 d (Å)Asn09 O Lys51 N 2.9 Tyr55 N Val64 O 3.0Trp11 N Val49 O 2.9 Tyr55 O Val64 N 2.9Tryp11 O Val49 N 3.1 Asp57 N Asp62 O 2.4Asp13 N Ala47 O 3.0 Asp57 O Asp62 N 3.2Asp13 O Ala47 N 3.1

B. Helices and turnsDistorted helices d (Å)

Gly16 O Lys19 N 2.6 gGln18 O Cys21 N 3.1 H1 ∼2.0 turnsLys19 O Glu22 N 3.0Asp34 O Cys37 N 2.9 gPro35 O Arg38 N 2.7 H2 ∼1.5 turnsIle36 O Ser39 N 3.1

310-TurnsGly07O Glu10N 3.1Asp13O Gly16N 3.0Lys51O Phe54N 3.4Glu66O Cys69N 3.4

C. Salt-bridgesGlu10 OE2 Arg56 NH1 3.5 g Salt-bridge-1Glu10 OE2 Arg56 NH2 2.4Glu22 OE1 Arg56 NE 2.8 g Salt-bridge-2Glu22 OE2 Arg NH2 3.1Glu32 OE1 Arg38 NH2 3.1 Salt-bridge-3

D. Additional hydrogen bondsd (Å) d (Å)

Gly07 N Glu10 OE1 2.7 Cys25 N Arg56 O 3.3Asp13 OD2 Gln18 N 3.0 Ser39 OG Gly41 N 2.8Cys15 O Leu44 N 2.8 Gly53 O Arg65 NH1 3.3Gln18 NE2 Cys37 O 2.7 Tyr55 OH Glu66 OE1 3.4Glu22 OE2 Cys50 N 3.2 Asp56 NH1 Gly61 O 3.2Ala23 O Arg56 N 3.0 Asp57 OD1 Val59 N 3.0Lys24 O Glu27 OE1 2.5 Asp57 OD2 Asp70 N 2.9

Ile75 O His76 ND1 2.5

NAP5-desfXa structure, the main chain of Arg38–Arg40 runs roughly antiparallel to Ser214–Gly216 offXa in a substrate-like binding manner, forming aβ-strand hydrogen bond between Arg38 O andGly216 N (3.0 Å), and possibly a longer one betweenArg40 N and Ser214 O (3.6 Å) (Figure 3). The side-chain of Arg40 occupies the S1 specificity site of fXa inan extended conformation, forming a single N–singleO hydrogen bonded salt-bridge through its guanidi-nium NH2 atom and one of the carboxylate oxygenatoms of Asp189 (OD1, 2.6 Å) (Figure 3). Thisinteraction differs from the twin N- twin O doublyhydrogen bonded salt-bridge usually encounteredbetween substrates and inhibitors at the S1 specificitysite of thrombin.35,36 Different types of salt-bridgeshave been observed with thrombin,35 but a survey ofintra-molecular arginine–carboxylate interactionsindicates that twin–twin contacts involving NE andNH1 (or NH2) of arginine and single N–single Ocontacts are much more prevalent.37 fXa displays asimilar variability in the S1 site binding with: (1) asingle O-twin amidino arrangement in the DX-9065acomplex;24 (2) a twin–twin geometry through anamidino group in the FX-2212- fXa inhibited struc-ture;26 and (3) through a lone hydrogen bond be-tween an aromatic tyrosyl hydroxyl group of TAPandAsp189OD2 inTAP–fXa.25 Although the locationof the hydrophobic TAP tyrosine residue in the S1 siteof fXa, that has arginine-like specificity, is somewhatunusual, an indole has been reported to bind in the S1site of thrombin.38

The carbonyl oxygen atom of Arg40 at the P1position is positioned in the oxyanion hole hydrogenbonding with Gly193 N (2.6 Å) and Ser195 N (2.8 Å).In addition, Ser195 OG is located over the plane ofthe Arg40 carboxyamide group directly bridgingArg40 C (2.8 Å) and Arg40N (3.1 Å) (Figure 3). Thus,the interaction of the electrophilic center of the P1residue of NAP5 at the catalytic site of fXa closelyresembles that of a substrate or inhibitor transitionstate intermediate, which has been observed in otherserine proteinase complexes.39–41

The Ser39, P2 residue, of NAP5 is somewhat un-usual because it is not generally encountered at thisposition in substrates or peptidic-like inhibitors ofserine proteases and it is the last residue of a shortdistorted helix containing a 310-turn of Pro35–Arg38

Figure 3. Stereoview of NAP5 (red) interacting with the active center and subsidiary binding region of the catalyticdomain of fXa (light gray). Ribbon representation with interacting side-chains as sticks: NAP5 atom colors are: carbon,white; nitrogen, blue; oxygen, red; fXa side-chains are in yellow; hydrogen bonds are shown as black broken lines.

778 Active- and exo-site inhibition of factor Xa

(H-2, Table 1) (Figure 3); Arg38 is at the P3 positionof the substrate. The turn of this distorted helix re-sults in the formation of a hydrogen bond betweenPro35 O (P5 residue) and Tyr99 OH (2.9 Å) es-sentially terminating NAP5 interactions in thisdirection. The hydroxyl group of Ser39 additionallyforms a hydrogen bond (2.75 Å) with His57 NE2 ofthe catalytic triad, replacing the hydrogen bond ofSer195 OG to the latter in the native structure(Figure 3).23 The latter distance is now 4.0 Å in theNAP5-des-fXa structure. In forming this hydrogenbond with NAP5, the imidazole group of His57maintains its position of the unbound structure andits native state hydrogen bond between His57 ND1and Asp102 OD1 (2.6 Å) (Figure 3). Both the formerinteractions have not been observed previouslybetween substrates or inhibitors of serine protei-nases. Other important hydrogen bonds of Ser39are: Ser39 N–Tyr99 OH (3.0 Å) and Ser39 O- Gln192NE2 (3.0 Å). The tyrosyl group of Tyr99 rotatesabout 90° around the CG-CZ direction with respectto its position in fXa,23 and its hydroxyl groupmoves 2.0 Å from its position in native fXa. TheSer39–Gln192 interaction is not unlike the Asn2 O–Gln192 OE1 (3.2 Å) interaction in TAP-fXa.Although the latter chain runs parallel withSer214–Glu217 of fXa, while the former is antipar-allel, Ser39 and Asn2 are the P2 residues. Theinteraction in both complexes is achieved by amovement of the Gln192 side-chain, which is largestin TAP-fXa. In TAP-fXa, Gln192 NE2 hydrogenbonds with Ala8 O of TAP,25 whereas its differentposition in NAP5-fXa leads to an intramolecularhydrogen bond with Arg143NH2 (3.2 Å). As will beseen next, both interactions involve the active centersubsidiary binding site of fXa. The methylenegroups of Arg38, located at the P3 position, aresurrounded on three sides by the tyrosyl group ofTyr99, the indole of Trp215 and the side-group ofIle36 of NAP5. The plane of the positively chargedguanidinium group of Arg38 stacks parallel with the

π-electron face of Phe174 of fXa (eight contactsb3.5Å)leading to a cation-π electron-mediated interaction,also called an ion-quadrupole attraction,42–44 whichappears to be unique and common to fXa andhas been observed at the S3 site of other fXacomplexes.24,25,36,45 The phenyl group of Phe174 isdisplaced slightly by the encroaching guanidiniumgroup of Arg38 of NAP5 compared to native fXa,while in TAP-Xa, the phenyl ring moves in theopposite direction. Since the main chain of TAPbinds in a retro-manner in the active center, itsArg3 is not positioned optimally for binding asArg38 in NAP5 (planes of guanidiniums differ by2.3 Å) but the interaction is nonetheless main-tained by the attraction (movement) of Phe174 inthe direction of the guanidinium of Arg3 of TAP.Two other aromatic residues in the vicinity (Tyr99,Trp215) have been suggested to assist in thecation recognition interaction by fXa.24,45

Subsidiary binding site of fXa

The P1′–P2′ residues of NAP5 (Gly41-Cys42),located on the C-terminal side of the scissile bond,are in close proximity to fXa. Since the NAP5 chainbranches at Cys42, Cys15–Asp53 and Leu43–Pro45are likely P3′–P5′ site residues (Figure 1). The Cys42residue is at the P2′ position of substrate and itsnitrogen atom forms a main chain hydrogen bondwith Phe41 O (3.3 Å) while the Asp14 residue makesa hydrogen bonded salt-bridge betweenAsp14OD1-Arg143 NH2 (2.4 Å) with another hydrogen bondfrom OD2 to Gln151 N (2.7 Å). This salt-bridge withArg143 is further shared with a hydrogen bond andthe negative charge between Asp13OD1 andArg143NH2 (3.4 Å). Since Cys15–Asp13 of NAP5 doublesback over the P1– P3 residues after branching atCys42, they are not positioned for substrate-likebinding (at the S3′–S5′ sites) but rather, bind at asubsidiary site assisting active site binding (Figure3), which positions also utilize most of the residues

779Active- and exo-site inhibition of factor Xa

and interacting atoms of the subsidiary binding siteof fXa observed in the TAP–fXa complex. A completelist of interactions of the subsidiary binding site aregiven in Table 2.25 Thus, the lengthy contiguousundecapeptide segment (Pro35–Asp13) of NAP5binds to and interacts with both the active andsubsidiary binding site regions of fXa. It is the uniquefolding conformations of NAP5 and TAP that lead tothe additional stabilization of the inhibitor–enzymecomplex beyond active site interactions throughhydrogen bonds and salt-bridge interactions withthis subsidiary binding region. Another subsidiarysite salt-bridge contact occurs between Asp52 OD1and Lys147NZ (3.0 Å) alongwith possibly a cation-πelectron-mediated interaction (∼2.7 Å) between Tyr3and the guanidinium moiety of Arg150 in the samevicinity (the density of the arginyl side-chain is welldefined only to NE). The remainder of the NAP5molecule extends outward from the surface and doesnot interact directly with fXa. However, the carbox-ylate oxygen atoms of Glu33 both hydrogen bondwith Ow821 (occupancy factor z=1.0, B=30 Å2),which is linked to Arg222 NH1 through Ow817(z=0.77, B=34 Å2) by two more hydrogen bonds,unlike TAP, the NAP5 structure extends away fromFXa in this region, hence the bridging watermolecules to Arg222 and the lack of more bindinginteractions; Arg222 is found also in the subsidiarybinding site of Tap-fXa. Thus, NAP5-fXa utilizes allbut two of the residues (Glu146 and Lys224) reportedin the subsidiary binding site of TAP-Xa. From Table1, it is clear that the subsidiary site of fXa is largerthan that of NAP5-fXa or TAP-fXa alone and isprincipally comprised of the so-called autolysis loopof fXa (Arg143–Gln151), found also in thrombin,46

along with at least the neighboring Arg222–Lys224segment. Proteolytic cleavages occur in this region innative fXa23 but, as here, not in active site inhibitedstructures of fXa.24–26 The binding of NAP5 and TAPin the autolysis loop region is in agreement with theprotection afforded by these inhibitors againstproteolytic cleavage of the Lys147- Gly148 bond bytrypsin. The amount of 13 kDa fXa fragmentgenerated by trypsin in 10 min of digestioncorresponds to 40% conversion, while it is only

Table 2. Polar interactions of the subsidiary bindingregion of the active site of fXa with NAP5 and TAP

NAP5 fXa d (Å) TAP fXa d (Å)

D13 OD1 R143 NH2 3.4 I60 O R143 NH2 2.7D14 OD1 R143 NH2 2.4 N57 O R143 NH2 2.7– – – Y49 OH E146 OE2 2.8D52 OD1 K147 NZ – Y49 OH E146 OE2 2.8Y03 OH R150 NE 2.7 – – –D14 OD2 Q151 N 2.7 – – –– – – A58 O Q192 NE2 3.4a

E33 OD1,OD2 R222 NH1 b D47 O R222 NH1 3.0– – – D47 OD1 R222 NE 2.8– – – D47 OD2 K224 NZ 2.5

a Different conformational changes in NAP5 and TAP (see thetext).

b Linked together by two hydrogen bonding water molecules(see the text).

15% for the same time in the presence of NAP5([fXa]= 11.3 μM, [NAP5]=13.6 μM, [trypsin] =182 nM). TAP inhibition protects fXa similarly. Thetotal surface buried between NAP5 and des-fXa inthe active and subsidiary binding site regions is954 Å2, which corresponds to about 16% of theNAP5surface and about 8.5% of fXa. Summarizing theNAP5 active site and subsidiary site interactionswith fXa: it forms four hydrogen bonded salt-bridgesand two cation-aromatic π electron-ion quadrupoleinteractions. This agrees well with the effect of NaClon the binding of NAP5 to immunocaptured FXa.The ED50 for blocking half of the NAP5 binding wasmeasured to be about 2 M NaCl. In contrast, NaCl(up to 1 M) had no effect on the binding of NAP5 toimmunocaptured EGR-fXa. Furthermore, it forms atotal of 16 hydrogen bonds, an antiparallel β-strandwith fXa, an enzyme–substrate transition stateintermediate complex, in addition to an undecapep-tide segment fitting closely in surface grooves of theactive site region (86 contacts b3.5 Å).

Comparison of NAP5-fXa with C/E-1 inhibitedelastase

The Cα coordinates of elastase of the C/E-1 elas-tase complexwere superposed on those of des-fXa ofthe NAP5-fXa complex with a rmsd=1.1 Å for 171 of227 (75%) Cα atoms (69 positions deviating by morethan 1.0 Å). The rotation matrix and translation vec-tor thus determined was then used to superimposethe C/E-1 elastase structure on that of NAP5-fXa.The C/E-1 inhibitor binds to elastase in a very

similar way to the manner in which NAP5 binds tofXa. The principal difference between the two in-hibitors is a rotation of the C/E-1 β-strand structurewith respect to that of NAP5, since the respectivereactive-site residues are in different spatial positionsin a global superposition (Figure 1). This results in theclose overlap of the P1 residues of both inhibitors(Arg40 of NAP5, Leu31 of C/E-1) in the specificitysites of their cognate enzymes. The catalytic sitebinding loop of NAP5 extends from Cys37 to Cys42while that of C/E-1 is from Cys29 to Cys33. Theadditional residue in the NAP5 stretch compared toC/E-1, which is N-terminal to the scissile bond is ab-sorbed by a 310-turn in NAP5 (Pro35–Arg38) notpresent in the C/E-1 inhibitor. Comparing the CA, C,N, O coordinates of the P3–P2′ residues of the re-active-site loops of NAP5 and C/E-1 gives rmsd=0.70 Å. Thus, although the global positioning of thereactive-site loop is different in the two inhibitors(Figure 1), its local superposition corresponds to thesame arrangement. This same canonical conforma-tion, also found in the bovine pancreatic trypsin in-hibitor (BPTI)–trypsin complex,39,47 is seen in anumber of other protein inhibitors of serineproteases.17

A unique feature of the C/E-1 elastase complex isthe penetration of the side-group ofArg217A througha pore in the inhibitor formed by the Cys17–Cys29loop, where Arg221A makes 14 interactions withresidues around the pore.17 Since fXa does not have

780 Active- and exo-site inhibition of factor Xa

the Arg217A insertion and the corresponding dis-ulfide loop is larger (Cys21–Cys37), a pore-penetra-tion interaction is not found in the NAP5-fXacomplex. The pore appears to be preformed and notthe result of a conformational change in C/E-1 due toArg217A because it is found also in the ATI structuredetermined by NMR16 and is present in NAP5-fXa,which, however, lacksArg217A. The samemost likelyapplies when C/E-1 binds to chymotrypsin (whichalso does not have Arg217A).

Location of the exosite on fXa

In the crystal structure of NAP5-fXa, about 20C-terminal residues (Asp57–His76) of the NAP5molecule bound to the active site of fXa interact in aspectacular, intricate andmost likely physiologicallyrelevant manner with the N-terminal seven-stranded β-barrel (β2, Gln30–Ile34; β3, Gly40–Leu47; β4, Tyr51–Thr54; β7, Ala104–Leu108; β6,Ala81–Lys90; β5, Lys65–Val68. β6 contributingtwice to the formation of the barrel with Ala81–Glu84, Glu86–Lys90 and the C-terminal α-helix of asymmetry-related fXa molecule (Figure 4). Theextended conformation of Glu71–His76 of NAP5runs antiparallel to His91–Val86 of fXa making fivemain chain β-strand hydrogen bonds with the latter(Table 3) and in this manner adds another β-strandto the fXa β-barrel (Figure 5(a) and (b)). Conversely,it is the electropositive polar side-groups of twoturns of the amphipathic-like C-terminal helix of fXa(Lys236–Lys243), which appear to be even moreimportant, that interact with a highly electronega-tive cluster of NAP5 (Asp57–Asp70), forming salt-bridges and hydrogen bonds (Figure 5(a); Table 3).This stretch of NAP5 is stabilized by two disulfidebridges, Cys25–Cys69 and Cys50–Cys63 and a short

β-strand (Asp62–Val64) (Figures 2 and 3), whereabout half (six) of the stretch are either Asp or Gluresidues (Figures 1 and 5(a)). The Arg240 side-chaininserts into a deep surface pocket of NAP5 ringed bythe negative charges from side-chains of Asp62 andGlu68 along with Gln71, with Asp57 located near itsbottom (Figure 5(a)). The arginyl residue is welldefined in the final electron density and makeshydrogen bonded salt-bridge contacts with Asp57and Asp62 (Table 3). Two other electropositive side-chains of fXa are in the region Lys236–Lys243making hydrogen bonded salt-bridges with Glu68and Asp62, respectively, of the negatively chargedpocket of NAP5. The total number of contacts b3.5 Åbetween NAP5 and the fXa exosite-like bindingregion is 52, of which four are salt-bridges of varyinggeometry and strength along with an impressivetotal of 18 hydrogen bonds. The buried surface inthis region is a surprising 802 Å2, which comparesvery favorably with the NAP5-active/subsidiarybinding site (954 Å). Thus, 30% of the NAP5 isburied in binding with two fXa molecules.With NAP5 linking fXa at two different sites,

infinite NAP5-fXa helical chains are formed parallelwith the 61 screw axis of the crystal. In particular,there are two such chains running through the unitcell in opposite directions as a double helix satisfy-ing crystallographic 2-fold rotation axes. The dia-meter of these antiparallel helical arrays is about80 Å. The remainder of the space of the unit cell,which includes that inside the double helix, is filledwith solvent giving a 73% (v/v) solvent fraction.

Comparison of NAP5, NAP6 and NAPc2

The NAP inhibitors possess the same disulfidepairing, display a high level of sequence identity,

Figure 4. NAP5 (white) inter-acting with the active site of a des-fXa (red) molecule and the bindingexosite of an adjacent fXa catalyticdomain (blue). The active site isshown in ribbon representation: thesubsidiary binding region of fXainteracting with NAP5 is in yellow;the exosite-binding regions of fXainteracting with the C-terminalregion of NAP5 are in light blue;EGF2 of fXa is in green. The reac-tive-site P1 residue (Arg40) ofNAP5is shown as a white stick structure.

Table 3. Exosite polar interactions of NAP5-des-fXa

NAP5 fXa d (Å)

Asp57 OD2 Arg240 NH1 3.4 Salt-bridge-1Asp57 OD2 Arg240 NH2 2.8 Salt-bridge-1Asp62 OD1 Arg240 NH2 2.6 Salt-bridge-2Asp62 OD1 Arg240 CZ 3.5 Salt-bridge-2Asp62 OD2 Arg240 NE 3.2 Salt-bridge-2Asp62 OD2 Lys243 NZ 2.9 Salt-bridge-3Glu68 OE1 Lys236 NZ 2.6 Salt-bridge-4Glu68 OE2 Lys236 NZ 3.0 Salt-bridge-4Glu68 O Arg240 NH1 2.4Gln71 NE2 Trp237 NE1 3.4Gln71 NE2 Trp237 O 3.1His72 N Lys90 O 3.0 β-StrandHis72 ND1 Asn92 ND2 2.7His72 O Lys90 N 2.9 β-StrandGlu73 OE2 Val88 O 3.3Ile74 N Val88 O 2.9 β-StrandIle74 O Val88 N 2.7 β-StrandHis76 N Gln86 O 3.5 β-StrandHis76 O Lys109 NZ 2.9

781Active- and exo-site inhibition of factor Xa

share similar β-strand structural elements and con-tain a C-terminal extension (8–14 amino acidresidues) (Figures 1 and 6).11 NAP5 and NAP6share 86% sequence identity, and the C-terminalextension is strictly conserved, whereas NAPc2shares only 45% sequence identity with NAP5 andhas a longer C-terminal extension (Figure 1). Thestructure of NAP5 bound to fXa has been comparedwith the crystallographic and NMR structures ofNAPc2, which indicates that three of the disulfidebridges of NAPc2, (like C/E-1), overlap well and arelocated on the surface. The remainder of the NAPc2structure, beyond this 10 Å×20 Å surface strip showsonly very little congruence (some possibly betweenCys21 and Cys25). The β-strands and the two turnsof α-helix are fairly well defined in all 18 differentNMR structures,48 and the NAPc2-fXa crystalstructure.14 The C-terminal region of NAPc2 is or-dered in the crystal structure but a dramatic confor-mational change occurs at the C terminus of NAPc2when bound to fXa compared to the relative orienta-tion of the C terminus observed in theNMR structureof NAPc2. This C-terminal conformation is quitesimilar to that observed in NAP5 on binding at thefXa exosite. Thus, it would seem that bothNAP5 andNAPc2 undergo similar conformational changesupon binding at the FX/fXa exosite. The principalinteractions in the fXa exosite are the formation of anintermolecular anti-parallel β-sheet between thesegments comprised of residues 86–93 of fXa andthe C terminus of NAPc2/NAP5 (a complete list ofcontacts is given in Table 3; and see Figure 5(b)).Analogous to the P1 reactive-site NAP5 residue

Arg40, NAPc2 contains arginine at position 44(Figure 1) that likely interacts with the active site offVIIa. However, NAP5 does not bind to fVIIa due tothe presence of arginine and serine at positions P3and P2 instead of leucine and valine as in NAPc2. Ingeneral, the reactive –P and –P' residues in NAPc2are predominantly hydrophobic, whereas in NAP5they are hydrophilic, with a large volume side-chainat position P3.

It is of significance that the loop containing theArg40 reactive-site P1 residue of NAP5 adopts a verydifferent conformation in NAPc2, so that the positionof the equivalent arginine in the latter is very dif-ferent. Thus, when NAPc2-fXa binds to TF-fVIIa, aconformational change most likely occurs in NAPc2(Figure 6) to a more canonical one,39–47 either onbinding to the fXa exosite,which is different from thatreported here for NAP5, or it would suggest when itbinds to the TF-fVIIa complex inhibiting the initiationof blood coagulation by the extrinsic pathway.

Ixolaris, a tissue factor pathway inhibitor(TFPI)-like protein with NAPc2-like properties

Ixolaris, a potent anticoagulant protein isolatedfrom the salivary gland of the hard tick I. scapularisconsists of two Kunitz-like domains linked by a shortpentapeptide sequence (Figure 7),22 is a vector forLymes disease and inhibits the extrinsic pathway,These Kunitz-like domains share moderate sequenceand structural similarities with TFPI, which consistsof three multivalent Kunitz-type domains with anacidicN terminus and a basic C-terminal extension. InTFPI, the first domain binds to the active site of fVIIa,the second binds to and inhibits fXa, and the thirddomain has been implicated in modulating theinteraction with lipoproteins. The 20 peptide linkerbetween the first and seconddomains of TFPI permitssimultaneous binding at the active sites of fVIIa andfXa, respectively. Ixolaris possesses a shorter linkerpeptide that contains two proline residues, bindsstoichiometrically either to fX or to fXa at the exositeand inhibits the fVIIa/TF complex in a mode similarto NAPc2. The first domain of ixolaris contains aglutamate residue instead of lysine or arginine atposition 36 (the sequence numbering is based onTFPI), which is not productive for binding to theactive site of fVIIa. Sequence alignments indicate thatthe C-terminal region of ixolaris18 shares significanthomology with the NAPc2 and NAP5 C-termini, thisregion of ixolaris was modeled on the basis of thecrystal structure of the fXa/NAPc2 complex (Figure7). Since the first domaindoesnot contain either lysineor arginine residues at the P1 position, the seconddomain likely plays a dual role in the inhibition of theextrinsic pathway. The P1 position in the seconddomain is occupied by arginine, which can interactwith Asp189 (S1 subsite), whereas the C terminusinteracts with the fX/fXa exosite. These featuressuggest that the second Kunitz domain and theextended C terminus of ixolaris likely play rolessimilar to that of the analogous domains of NAPc2 ininhibiting the extrinsic pathway.

Biological implications

The lack of selectivity of the coumarin andheparin-based compounds and the strategic positionof fXa in the blood coagulation cascade makes it anattractive target for the development of anticoagula-tion drugs. NAP5 is the most potent natural fXainhibitor characterized to date and is being tested

Figure 5. (a) A stereo view of NAP5 binding at the exosite of fXa. The backbone and side-chains are shown in ribbonand in stick/ball representations for NAP5 (Cα in yellow) and fXa (Cα in white), respectively. (b) Hydrogen bonds thatform the intermolecular β-strand between fXa (carbon in white) and NAP5 (carbon in yellow) superposed on NAPc2(carbon in green).

782 Active- and exo-site inhibition of factor Xa

clinically for the prevention and treatment ofthrombosis. The unique structural features of themode of fXa inhibition by NAP5 described hereshould provide a platform for the understanding anddevelopment of highly specific active site and exo-site inhibitor analogues.

Materials and Methods

Crystallization

Human des-fXa49 (39,800 kDa) was purchased fromHaematologic Technologies, Inc. as 1 ml frozen aliquots ata concentration of 2 mg/ml in 0.1 M Tris–HCl (pH 7.5),

buffer containing 150 mM NaCl. The recombinant NAP5(8–700 kDa) was a kind gift from Corvas International,Inc.50 A 0.54 mg sample of freeze-dried NAP5 wasdissolved in 50 μM 0.05 M Tris–HCl ( pH 7.5) and addedto a 1 ml aliquot of des-fXa solution to attain a 1.2:1 molarratio of NAP5 to des-fXa. The complex thus formed wasallowed to equilibrate overnight at 4 °C and subsequentlyconcentrated using a Centricon 10 kDa cut-off filter and thebuffer concentration was adjusted to obtain a stocksolution of 10 mg/ml of NAP5-des-fXa in 0.1 M Tris–HCl(pH 7.5), 40–50 mM NaCl. A 5.0 mg/ml solution of thecomplex was used to carry out an initial sparse factorialcrystal screening in a 1:1 (v/v) ratio with well solution(crystal screen I, Hampton Research) using the hanging-drop, vapour-diffussion crystallization method. Smallhexagonal rod-like crystals grew within a week with

Figure 6. A stereo view of thesuperposition of the average NMRstructure (white) and only the Cterminus of the crystal structure ofNAPc2-fXa (blue) on the NAP5structure (red). NAP5 disulfidebridges are shown in stick/balstructure and are numbered.

783Active- and exo-site inhibition of factor Xa

overall dimensions less than 0.1 mm from conditions 5, 8,13, 19, 21, 23, 33 and 36 and a NAP5-des-fXa concentrationof 2.5mg/ml.51,52 Of these, the condition that produced thelargest crystals was number 13 (30% (w/v) PEG400, 0.1 MTris–HCl (pH 7.5), 0.2M sodium citrate). Repetitivemacro-seeding in 8 μl hanging-drop experiments, reducing theprecipitant and the concentration of NAP5-des-fXa of thedrop, optimized macro-seeding growth at 0.75 mg/ml ofNAP5-des-fXa, 14% (w/v) PEG400, 0.1 M Tris–HCl (pH7.5), 0.1 M sodium citrate, 10–13 mM NaCl drop composi-tion suspended over well solution number 13. Bulky hex-agonal crystals with overall dimensions of up to 0.3 mmwere obtained within a period of two to three weeks.

X-ray data collection, processing and structuredetermination

X-rays were produced by a Rigaku RU200 rotatinganode generator equipped with Osmic confocal optics anddiffraction intensities were measured utilizing an RAXISIIc imaging plate detector from a flash-frozen crystal ofdimensions 0.25 mm×0.25 mm×0.30 mm. Autoindexingindicated that the crystals of the NAP5-des-fXa complexbelong to the space group P6122 (or enantiomorph) witha=b=137.2 Å, c=167.7 Å and Vm=4.6 Å3/Da (0.27 proteinfraction) for 12 molecules in the asymmetric unit,consistent with the low resolution of the diffraction data

Figure 7. A model of ixolaris(ribbon representation in red) binding with fVIIa (surface representation in white) and fXa (surfacerepresentation in lavender). Thereactive-site P1 residue Arg40 isshown in stick/ball structure withatom colors as in Figure 3.

l

that fell off very rapidly beyond 3.1 Å, and with theextremely fragile nature of the crystals. The raw intensitydata were integrated and scaled in point group 622 withthe programs DENZO and SCALEPACK.53 The statisticsof the intensity data processing are given in Table 4.The orientation and position of the catalytic and EGF2

domains of des-fXa were determined by molecularreplacement techniques with the program AMoRe,54

using the coordinates of the same modules of native des-fXa (PDB code 1HCG)23 stripped of solvent as a searchmodel. The Patterson rotation search within a sphericalradius of 25 Å using reflections in the (10.0–3.5) Å rangesatisfying |F|/σ (|F|)N1.0 resulted in one distinct peakwith a correlation coefficient of 0.13. The translation searchconducted in space group P6122 gave the most out-standing solution (correlation coefficient 0.69) and thecrystallographic R-factor was 33.7% (44.8% for P61). Rigidbody refinement increased the correlation to 0.72 anddecreased the R-factor to 31.3%.Examination of the (2Fo–Fc) and (Fo–Fc) difference

electron density maps revealed density corresponding tomuch of the NAP5molecule as well as density for residuesHis145–Thr153 of the autolysis loop of fXa, which aremissing from the native des-fXa structure.23 Both maps,however, indicated no electron density for the EGF1domain as in native des-fXa23 and other inhibited des-fXastructures.24,25 Positional and thermal parameter refine-

--

Table 4. Intensity data processing statistics of NAP5-des-fXa

Resolution (Å) 3.1No. reflections 155,990Total unique reflections 17,024No. I/σ(I)N1.0 14,878Average redundancy 9.1Coverage completeness (%) 96.9Inner rangea 89.6,Outer rangea 97.8Rmerge

Inner rangea 0.036Total rangea 0.098Outer rangea 0.51

I/σ(I)Inner rangea 40.3Total rangea 16.2Outer rangea 3.7a Inner, 35.0–6.7 Å; total, 35.0–3.1 Å; outer, 3.2–3.1 Å.

784 Active- and exo-site inhibition of factor Xa

ment was conducted using the program CNS.55 An initialsimulated annealing refinement of the des-fXa model withan average B-factor of 28 Å2 was carried out with the slow-cool procedure in which the temperature was decreasedfrom 2500 to 300 K in 25 intervals. In this and allsubsequent refinements, about 10% of the data set (1486reflections) was withheld for calculating the Rfree value.The slow-cool procedure calculation began at R=39.0%(Rfree=39.8%) and converged at R=31.3% (Rfree=36.2%).The corresponding difference maps showed: (1) Arg40 ofNAP5 located in the S1 specificity site of fXa forming asalt-bridge with Asp189 and continuous electron densityfor the Cys21–Cys42 disulfide loop of NAP5, (2) cleardensity corresponding to the autolysis loop of fXa notincluded in calculations; and (3) that a few residues ofEGF2 were misplaced and required refitting. Incorporat-ing all the preceding additions and changes, followed bypositional and individual isotropic B-factor refinement,resulted in a crystallographic residual of 28.5%(Rfree=33.7%).Thereafter, further manual fitting of NAP5 proceeded

using the program TURBO-FRODO56 (through omit mapswith a 3.0 Å radius cut-off, Ramachandran plot examina-tions, and comparisons with high-resolution proteinstructural fragments from the Protein Data Bank) followedby rounds of positional and individual B-factor refine-ment. A bulk solvent mask correction using (35.0–3.1) Ådata was introduced into calculations when most of theNAP5 structure was included and another simulatedannealing refinement was carried out. The bulk solventcorrection improved the (2Fo–Fc) maps, significantlyaiding tracing and map fitting of NAP5. The total numberof reflections used in the bulk solvent refinement was13,542, which satisfied I/σ(I)N1.5 in the 35.0–3.1 Åresolution range, while the corresponding number in thenon-bulk solvent refinement range (8.0–3.1) Å was 12,706.In the final stages, 163 water molecules (Occ.N0.5,bBN=35 Å2) were included to account for residual densityat geometrically plausible water positions (ΔR=–2.5%,while Rfree essentially remained the same). The final R-factor of the NAP5-des-fXa structure in the 8.0–3.1 Å rangewithout a bulk solvent correction is 16.6% (Rfree=26.9%).The final structure corresponds to rmsd values of 0.02 Åand 2.6° from ideal values in bond distances and bondangles. The Ramachandran plot of NAP5 had 40 residues(66.7%) in the most favored regions and 33.3% inadditionally allowed regions. Various PROCHECK

parameters57 compared well against expected values ofstructure determinations at 3.1 Å resolution.

Molecular modeling and quality analysis

The atomic coordinates of the bovine pancreatic trypsininhibitor mutant with an altered binding loop (PDBaccession code 1QLQ)58 and the second Kunitz domainof the tissue factor pathway inhibitor (PDB accession code1TFX)59 stripped of solvent and ligands were used toproduce a scaffold to obtain the structural model of thetwo Kunitz-type domains of ixolaris. Each domain wasmodeled individually and the structure of the NAPc2 C-terminal extension was used to model the correspondingregion of ixolaris. Homology-based molecular modelingwas carried out using a restraint-based modeling algo-rithm as implemented in the MODELLER program.60 Theoverall model was improved, enforcing proper stereo-chemistry using spatial restraints and CHARMM energyterms, followed by conjugate gradient simulation basedon the variable target function method.60 The loops wereoptimized using ModLoop61 based on the satisfaction ofspatial restraints, without relying on a database of knownprotein structures. The final model was submitted toexplicit solvent molecular dynamics simulations using theGROMACS 3.2 software package,62 and the GROMOS-96(43a1) force-field to check stability and consistency of thefinal model. The overall stereochemical quality of the finalmodel was assessed by the programs PROCHECK57 andVERIFY3D.63 The mode of interaction of ixolaris withfVIIa was modeled on the basis of the coordinates ofextracellular tissue factor complexed with fVIIa andinhibited by a BPTI mutant (PDB accession code 1FAK)64

and the exosite interaction with fXa was based on thestructure of the fXa/NAPc2 complex (PDB accession code2H9E).14

Protein Data Bank, accession code

The coordinates of NAP5-des-fXa have been depositedin the Brookhaven Protein Data Bank: accession code2P3F.

Acknowledgements

We thank George Vlasuk of Corvas Internationalfor providing us with NAP5. This research wassupported by NIH grants HL-25942 and HL-43229(AT) grants from FAPESP, CAT/CEPID, SMOLBNet,CNPq, PROBRAL/DAAD and CAPES (RKA). J.L.R.-S. was an NIH Underrepresented Minority Inves-tigator (HL43229). M.T.M. is the recipient of aFAPESP fellowship.

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Edited by R. Huber

(Received 20 March 2007; received in revised form 10 May 2007; accepted 15 May 2007)Available online 18 May 2007