Proc. Natl. Acad. Sci. USA
`Vol. 95, pp. 6630–6635, June 1998
`Biochemistry
`
`Structural basis for chemical inhibition of human blood
`coagulation factor Xa
`KENJI KAMATA*†, HIROSHI KAWAMOTO†, TERUKI HONMA†, TOSHIHARU IWAMA†, AND SUNG-HOU KIM*‡
`*Department of Chemistry and Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720-5230; and †Tsukuba Research Institute,
`Banyu Pharmaceutical Co., Ltd., Okubo 3 Tsukuba 300–33, Japan
`
`Contributed by Sung-Hou Kim, March 18, 1998
`
`Factor Xa, the converting enzyme of pro-
`ABSTRACT
`thrombin to thrombin, has emerged as an alternative (to
`thrombin) target for drug discovery for thromboembolic
`diseases. An inhibitor has been synthesized and the crystal
`structure of the complex between Des[1–44] factor Xa and the
`inhibitor has been determined by crystallographic methods in
`two different crystal forms to 2.3- and 2.4-Å resolution. The
`racemic mixture of inhibitor FX-2212, (2RS)-(3*-amidino-3-
`biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor
`Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212
`(FX-2212a) was found to bind to the active site of factor Xa in
`both crystal forms. The biphenylamidine of FX-2212a occu-
`pies the S1-pocket, and the pyridine ring makes hydrophobic
`interactions with the factor Xa aryl-binding site. Several water
`molecules meditate inhibitor binding to residues in the active
`site. In contrast to the earlier crystal structures of factor Xa,
`such as those of apo-Des[1–45] factor Xa and Des[1–44]
`factor Xa in complex with a naphthyl inhibitor DX-9065a, two
`epidermal growth factor-like domains of factor Xa are well
`ordered in both our crystal forms as well as the region between
`the two domains, which recently was found to be the binding
`site of the effector cell protease receptor-1. This structure
`provides a basis for designing next generation inhibitors of
`factor Xa.
`
`Thromboembolic disease is caused by the improper function-
`ing of the blood coagulation process. Blood clots are formed
`by a zymogen activation cascade of serine proteases, and the
`last protease of the cascade, thrombin, converts fibrinogen to
`fibrin, which cross-links to form blood clots (for a review, see
`ref. 1). To find antithrombotic drugs, many inhibitors of
`thrombin have been developed (2–5). But factor Xa, which is
`also essential for both the intrinsic and extrinsic pathways of
`the coagulation process, is thought to be a better target of
`antithrombotic drugs because many thrombin inhibitors have
`been shown to increase the risk of abnormal bleeding (6–8).
`Factor X is secreted into the blood as the zymogen form of
`the serine protease and is converted to an active form, factor
`Xa, by the factor VIIaytissue factor complex (in the extrinsic
`pathway) or by the factor IXayfactor VIIIa complex (in the
`intrinsic pathway) (1). Both complexes remove the activation
`peptide of factor X by limited proteolytic cleavage to form
`mature factor Xa. Factor Xa leads to blood clot formation by
`converting prothrombin to thrombin. In the presence of Ca21
`ions, factor Xa forms prothrombinase with factor Va on the
`phospholipid membrane of the activated platelets.
`Furthermore, the binding of factor Xa to effector cell
`protease receptor-1 (EPR-1) participates in the activation of
`lymphocytes (9, 10) and arterial smooth muscle cells (11).
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked ‘‘advertisement’’ in
`accordance with 18 U.S.C. §1734 solely to indicate this fact.
`© 1998 by The National Academy of Sciences 0027-8424y98y956630-6$2.00y0
`PNAS is available online at http:yywww.pnas.org.
`
`Recent research also suggests that EPR-1 is required for the
`prothrombinase formation on the platelet membranes (12).
`Factor Xa consists of a light chain and a heavy chain linked
`by a single disulfide bond. The light chain contains the
`N-terminal Gla domain and two epidermal growth factor
`(EGF)-like domains. The Gla domain contains 11 g-carboxy-
`glutamic acid residues and mediates binding to the negatively
`charged phospholipid membrane in the presence of Ca21 ions.
`The role of the EGF domains are not clear yet, but recent
`research suggests that the region between the two EGF
`domains is the binding site of EPR-1 (13, 14). The heavy chain
`contains a trypsin-like serine protease domain. This domain
`organization is very similar to those of other blood coagulation
`enzymes such as factor VIIa, factor IXa, and protein C (1).
`The crystal structure of human factor Xa has been deter-
`mined in apo form (15) and as a complex with the inhibitor
`DX-9065a, (2S)-{4-[1-acetimidoyl-(3S)-pyrrolidinyl]oxyphe-
`nyl}-3-(7-amidino-2-naphthyl)propionic acid (Fig. 1; ref. 16).
`But in both structures, the first EGF domain and the region
`between the two EGF domains were disordered. In contrast,
`these regions are well ordered in our crystal structure of the
`complex between Des[1–44] factor Xa and FX-2212a (Fig. 1),
`a new inhibitor with an IC50 of 272 nM and an apparent Ki of
`131 nM (measured for the racemic mixture of FX-2212). This
`inhibitor was synthesized as an initial lead for structure-based
`inhibitor design for factor Xa. Our structure reveals the details
`of the binding mode of the S-isomer of FX-2212, FX-2212a, as
`well as the structures of two, hitherto unknown regions: the
`first EGF domain and the binding site of EPR-1.
`
`METHODS
`Human factor Xa b-form was purchased from Haematologic
`Technologies, (Burlington, VT) and converted to Des[1–44]
`factor Xa by chymotrypsin digestion (17, 18), thus removing
`the Gla domain. FX-2212 was synthesized as outlined in Fig.
`2. The inhibitor was confirmed by 1H-NMR spectra on a
`Varian VXR 300 (300 MHz) spectrometer and high resolution
`mass spectra on a JEOL JMS-SX102A spectrometer. FX-2212
`is one of the strongest inhibitors among ’200 initial lead
`compounds we have synthesized. Inhibition assays of factor Xa
`and thrombin were measured by using S-2765 (Chromogenix,
`Molndal, Sweden) as a substrate in a solution of 20 mM Hepes
`(pH 7.4), 150 mM NaCl, and 2 mM CaCl2 at various inhibitor
`concentrations. IC50 was determined at 0.2 mM S-2765. To
`
`Abbreviations: FX-2212, (2RS)-(39-amidino-3-biphenylyl)-5-(4-
`pyridylamino)pentanoic acid; FX-2212a, (2S)-(39-amidino-3-
`biphenylyl)-5-(4-pyridylamino)pentanoic acid; DX-9065a, (2S)-{4-[1-
`acetimidoyl-(3S)-pyrrolidinyl]oxyphenyl}-3-(7-amidino-2-naphthyl)
`propionic acid; EPR-1, effector cell protease receptor-1; EGF, epi-
`dermal growth factor; Gla, g-carboxyglutamic acid.
`Data deposition: The atomic coordinates have been deposited in the
`Protein Data Bank, Biology Department, Brookhaven National Lab-
`oratory, Upton, NY 11973 (references 1XKA and 1XKB).
`‡To whom reprint requests should be addressed. e-mail: shkim@
`lbl.gov.
`
`6630
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`MYLAN - EXHIBIT 1014
`
`

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`Biochemistry: Kamata et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`6631
`
`search were .9 times and 2.5 times for the form 1 crystal and
`18 times and 9 times for the form 2 crystal, respectively. After
`rigid body refinement, the R-factors were 38.9% and 42.5% for
`the form 1 and 2 crystals, respectively. After building the first
`EGF domain model and a few refinement cycles, the electron
`density for the inhibitor was found in a difference density map
`(see Fig. 5a). Model building, electron density map calculation,
`and model refinement were performed with the X-PLOR (23–
`25) and the O (26) programs. The crystal structure in the form
`1 crystal was refined to an R value of 20.6% (R free 5 29.4%),
`and that in the form 2 crystal was refined to an R value of
`19.6% (R free 5 28.7%) with good stereochemistry (Table 1).
`
`RESULTS
`Overall Structure of Factor Xa. The crystal structure of
`Des[1–44] factor Xa (Fig. 3) has an elongated shape similar to
`those of protein C (27), factor VIIa (28), and factor IXa (29)
`except for the Gla domain, which was removed. Although the
`first EGF domain was disordered in both structures of apo (15)
`and DX-9065a-bound factor Xa (16), electron density for this
`domain was unambiguous in both forms 1 and 2 crystals. The
`structures of the second EGF domain and the catalytic domain
`have the same structure as those of the apo factor Xa structure
`(18) for the most part. Between our structures and the apo
`factor Xa structure, rms differences in the Capositions of the
`catalytic domains average 0.41 Å for the form 1 crystal and 0.43
`Å for the form 2 crystal. The small structural differences were
`caused by the inhibitor FX-2212 and calcium binding, as well
`as by the presence of an autolysis loop in our structures. The
`autolysis loop [Arg-143–Arg-154 using the chymotrypsin num-
`bering system (15)], which was cleaved off of the apo factor Xa
`crystals (15), clearly was ordered in both form 1 and 2 crystals.
`The catalytic domain has one bound calcium in both crystal
`forms. The presence of the autolysis loop and the calcium
`binding induced only small structural changes.
`The folding of the first EGF domain is similar to that of
`factor IXa (29) and bovine factor X (30–32). The domain has
`one calcium binding site as in factor VII and factor IX. Among
`three molecules in our two crystal forms, we found only one
`poorly ordered Ca21 ion in this site despite the presence of 4
`mM CaCl2 in the crystallization solutions. The lack of the Gla
`domain might have destabilized the structure of the calcium
`binding site and decreased the affinity for calcium. The ligands
`for Ca21 are O« of Gln-L49, Od of Hya (b-hydroxyaspartic
`acid) L63, O of Leu-L65, and O of Gly-L64; the prefix ‘‘L’’ is
`for the light chain.
`Binding Site of EPR-1. Recent studies suggest that the
`region (Leu-L83–Leu-L88) between the two EGF domains of
`factor Xa is the binding site for EPR-1 (13, 14). The structure
`of this region was not visible in either the apo (15) or
`DX-9065a-bound factor Xa (16) structure because of disorder.
`In both form 1 and 2 crystals, the region appears as a extended
`segment (Fig. 3). Although both EGF domains have very
`similar structures to those of factor IXa (29), the relative
`arrangement of these two domains is very different because of
`the different lengths and structures of the inter-EGF domain
`region. This difference is also true among the three crystal-
`lographically independent molecules in our two crystal forms
`(Fig. 4). In all three molecules, there is no interaction between
`the first and the second EGF domains, in contrast to the
`ball-and-socket-like interaction in factor IXa (29). Because a
`hexa-peptide of the sequence Leu-Phe-Thr-Arg-Lys-Leu,
`which is present in the region between the two EGF domains,
`recapitulates the inflammatory response, this extended struc-
`ture is thought to be required for recognition by EPR-1 (13).
`EPR-1 may contribute to the complex formation of prothrom-
`binase by binding and stabilizing this exposed and extended
`region between the two EGF domains.
`
`FIG. 1. Chemical formulae of the FX-2212a inhibitor (2S)-(39-
`amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid and the
`DX9065a (2S)-{4-[1-acetimidoyl-(3S)-pyrrolidinyl]oxyphenyl}-3-(7-
`amidino-2-naphthyl)propionic acid. Schematic drawing of the inter-
`actions between two inhibitors, DX9065a and FX-2212a, and factor
`Xa. Hydrogen bonds are shown as thin dashed lines, and hydrophobic
`interactions are shown as thick dashed lines. In the case of Q192, the
`aliphatic chain portion of Q192 makes the hydrophobic interaction.
`The symbol ‘‘’’’ indicates that the two interacting aromatic groups are
`not stacked but are perpendicular to each other.
`
`determine the Ki value, inhibition assays were performed at
`different substrate concentrations ranging from 0.0 to 0.4 mM.
`Des[1–44] factor Xa in complex with FX-2212 was crystal-
`lized into two different crystal forms. The crystallization
`conditions were found by sparse matrix methods (19) by using
`Crystal Screens (Hampton Research, Riverside, CA) and were
`optimized. Form 1 crystals were obtained from a solution
`containing 5 mgyml Des[1–44] factor Xa, 1 mM FX-2212, 10%
`polyethylene glycol 3350, 50 mM Mes pH 6.0, 100 mM Li2SO4,
`and 4 mM CaCl2 by vapor phase equilibration. The crystals
`appeared within 1 month. Form 2 crystals were obtained from
`a solution containing 5 mgyml Des[1–44] factor Xa, 1 mM
`FX-2212, 10% polyethylene glycol 3350, 50 mM malate–
`imidazole (pH 5.5), 250 mM sodium acetate, and 4 mM CaCl2
`also by vapor phase equilibration. This crystal form appeared
`within 1 week. Form 1 crystals belonged to space group P21
`(a 5 58.3 Å, b 5 105.2 Å, c 5 63.2 Å, b 5 103.4°) with two
`molecules per asymmetric unit, and form 2 crystals belonged
`to space group P212121 (a 5 61.5 Å, b 5 65.8 Å, c 5 81.4 Å)
`with one molecule in an asymmetric unit. X-ray diffraction
`data from a form 1 crystal to 2.4-Å resolution were collected
`on an R-axis IIc area detector (Rigaku Co., Tokyo), and data
`from a form 2 crystal were collected to 2.3-Å resolution at the
`X12B beam line at the Brookhaven National Laboratory. Both
`data sets were collected under flash freezing conditions (100
`K) by using 15% glycerol and 7.5% 2,3-butanediol as a
`cryoprotectant, respectively. The data reduction statistics from
`the DENZO and the SCALEPACK (20) processing are given in
`Table 1.
`The structures were solved by the molecular replacement
`method by using the AMORE program (21, 22) with the data of
`a resolution from 15.0 to 3.5 Å. The human Des[1–45] factor
`Xa structure (ref. 15; Protein Data Bank Identification:
`1HCG) was used as the search model. The two best and the
`next best solutions of the rotation search had the signals ’15
`times and 5 times higher than the background, respectively, for
`the form 1 crystal and 13 times and 6 times, respectively, for
`the form 2 crystal. Corresponding numbers for the translation
`
`

`
`6632
`
`Biochemistry: Kamata et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`FIG. 2. The scheme of the synthesis of FX-2212. N-tert-Butoxycarbonyl-N-3-(tert-butyldimethylsilyloxy)propyl-4-pyridylamine 3: 2 was prepared
`from 3-bromo-1-propanol and tert-butyldimethylsilyl chloride. To a solution of 4-tert-butoxycarbonylaminopyridine (400 mg, 2.1 mmol) in
`N,N9-dimethylformamide (DMF) (4 ml) was added 60% NaH (83 mg, 2.1 mmol) and 2 (1.0 g, 4.2 mmol) in DMF (6 ml), and the mixture was stirred
`for 12 h. After work up and purification, 375 mg of 3 was obtained. N-tert-Butoxycarbonyl-N-3-hydroxypropyl-4-pyridylamine 4: To a solution of
`3 (375 mg, 1.0 mmol) in tetrahydrofuran (THF) (10 ml) was added acetic acid (175 ml, 3.1 mmol) and 1 M tetrabutylammonium fluoride in THF
`(3.1 ml, 3.1 mmol) at room temperature, and the mixture was stirred for 3 h. After work up and purification, 213 mg of 4 was obtained.
`N-tert-Butoxycarbonyl-N-3-iodopropyl-4-pyridylamine 5: 4 (212 mg, 0.84 mmol) in CH2Cl2 (6 ml) was treated with triphenylphosphine (550 mg, 2.1
`mmol), iodine (426 mg, 1.7 mmol), and imidazole (143 mg, 2.1 mmol) at 0°C for 0.5 h. After work up and purification, 272 mg of 5 was obtained.
`Ethyl 5-(N-tert-butoxycarbonyl-N-4-pyridylamino)-2-(39-cyano-3-biphenylyl)pentanoate 6: After ethyl 39-cyanophenyl-3-phenylacetate (295 mg, 1.1
`mmol) in THF (3 ml) was added dropwise to a mixture of 1 M lithium bis(trimethylsilyl) amide in THF (1.16 ml, 1.2 mmol) and
`hexamethylphosphoramide (750 ml, 4.3 mmol) in THF (3 ml) and stirred for 0.5 h at 278°C, 5 in THF (4 ml) was added to the mixture, and the
`reaction mixture was allowed to reach room temperature. After work up and purification, 153 mg of 6 was obtained. Ethyl 2-(39-amidino-3-
`biphenylyl)-5-(4-pyridylamino)pentanoate dihydrochloride 7: After treating 6 (40 mg, 0.08 mmol) in ethanol (1 ml) with 4 M HCl–dioxane (10 ml)
`at room temperature for 2 days, the mixture was concentrated. The residue was dissolved in ethanol (10 ml) and bubbled with NH3 gas until
`saturation at 0°C. After stirring at room temperature for 3 days, the reaction mixture was concentrated in vacuo. The residue was purified to give
`25 mg of 7. FX-2212, 2-(39-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid dihydrochloride 8: 7 (20 mg, 0.04 mmol) was dissolved in 2 M
`HCl (4 ml), and the mixture was refluxed for 2 h. After concentration and purification, 15 mg of FX-2212 was obtained as white solids. The identity
`of the compound was checked by NMR and MS.
`
`Inhibitor Binding. The racemic mixture of FX-2212 inhibits
`factor Xa activity by 50% (IC50) at 272 nM but shows very weak
`inhibition for thrombin activity even at 100 mM (data not
`
`Table 1. Diffraction data and refinement statistics
`Crystal form 2
`Crystal form 1
`2.3
`2.4
`46,925
`78,773
`14,542
`26,594
`30–2.4 Å 91.2% 30–2.3 Å 96.2%
`2.53–2.4 Å 58.4% 2.4–2.3 Å 95.2%
`5.7
`8.5
`
`Resolution, Å
`Measurements, n
`Unique reflections
`Data completeness
`
`14,081
`24,386
`8.0–2.4 Å 20.6% 8.0–2.3 Å 19.6%
`29.4
`28.7
`
`Rsym on intensity*, %
`Reflections used in
`refinement
`R value†
`R-free‡, %
`rms deviation from ideal
`bond length, Å
`rms deviation from ideal
`bond angle, °
`B value for nonhydrogen
`protein atoms, Å2
`rms deviation in B value
`of bonded atoms, Å2
`Nonhydrogen atoms, n
`Water molecules, n
`*Rsym 5 SuI(h) 2 ^I(h)&uyS I(h).
`†R 5 SuFo 2 FcuySFo with FyS F . 2.
`‡R-free, R value for 10% of the data, which were not included during
`crystallographic refinement.
`
`0.008
`
`1.31
`
`24.1
`
`1.74
`5,433
`302
`
`0.007
`
`1.24
`
`15.6
`
`1.64
`2,790
`211
`
`shown). Although the racemic mixture was present in the
`crystallization solutions, only the S-isomer of FX-2212 (FX-
`2212a) binds to factor Xa. The binding of FX-2212a to factor
`Xa involves three interaction sites (Figs. 1 and 5). The first
`interaction involves the biphenylamidine group occupying the
`S1 pocket. The second is between the pyridine ring of FX-
`2212a and the factor Xa-specific aryl-binding site, which is
`comprised of Tyr-99, Trp-215, and Phe-174. The third inter-
`action between the carboxyl group of FX-2212a and residues
`in the catalytic site is mediated through water molecules. In all
`three crystallographically independent factor Xa molecules,
`the binding mode of FX-2212a to the factor Xa active site is
`almost preserved, despite the fact that the crystal packing
`interactions around the binding site are different.
`The binding in the S1 site involves the formation of salt
`bridges between the amidino group of FX-2212a and the
`carboxyl group of Asp-189 in twin–twin geometry (Fig. 5b).
`The biphenyl ring makes hydrophobic interactions with resi-
`dues in the S1 pocket. The first phenyl ring, which contains the
`amidino group, makes hydrophobic interactions with the main
`chain of Ala-190–Cys-191 and Trp-215–Gly-216. The second
`phenyl ring, which links the benzamidine group to the pen-
`tanoic acid, makes hydrophobic interactions with the side
`chain of Gln-192. This interaction causes the main chain of
`Gln-192 to move ’1.0 Å toward the biphenyl ring.
`In the second interaction site, the pyridine ring of FX-2212a
`is located in the center of the aryl-binding site and parallel to
`the indole ring of Trp-215. Hydrophobic interactions are made
`mainly between the pyridine ring of FX-2212a and Trp-215. In
`addition to this hydrophobic interaction, the nitrogen atom of
`the pyridine ring forms hydrogen bonds to O of Thr-98 and O
`
`

`
`Biochemistry: Kamata et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`6633
`
`crystallographically independent molecules (Fig. 5a). Al-
`though the interactions at the open side of the inhibitor binding
`pocket are different among the three independent complexes
`(shown in orange in Fig. 5b), the hydrogen bonding network at
`the covered side of the pocket between FX-2212a and the
`factor Xa remains the same (shown in green in Fig. 5b). The
`first conserved water molecule, Wat B (790 and 670 in the form
`1, 519 in the form 2), mediates the binding between one oxygen
`of the carboxyl group of FX-2212a and Oh of Tyr-99. Two
`other conserved water molecules are located in the hydrogen
`bonding range from Wat B. One is Wat C (789 and 649 in form
`1, 520 in form 2), which makes hydrogen bonds to N« of His-
`57 and to N« of Gln-61, and the other is Wat D (791 and 648
`in form 1, 518 in form 2), which makes hydrogen bonds to Og
`of Ser-195 and to O of Ser-214. The side chain of the catalytic
`Ser-195 rotates by ’130° from the position in the apo-factor
`Xa structure, in which the Og of Ser-195 makes a hydrogen
`bond with N« of His-57. Also, Wat E (793 and 652 in form 1,
`521 in form 1) makes hydrogen bonds to O of Phe-41, N of Gly-
`193, and Wat C. These conserved water molecules may rep-
`resent the potential site to introduce modification groups in
`designing new inhibitors.
`
`DISCUSSION
`In contrast to the abundance of crystal structures of complexes
`between thrombin and chemical inhibitors (33–45), our struc-
`ture is only the second structure of the inhibitor-bound factor
`Xa. The structure of the DX-9065a-bound factor Xa was the
`first inhibitor complex structure published (13). DX-9065a was
`developed by Daiichi Pharmaceutical Co., Ltd., Tokyo, and
`inhibits factor Xa with Ki 5 41 nM; IC50 5 92 nM at pH 8.4
`(46, 47) and Ki 5 103 nM; IC50 5 208 nM at pH 7.4 (data not
`shown). Although the chemical structure and the binding
`mode of this inhibitor are different from those of FX-2212a
`(Fig. 1), neither inhibitors interact directly with the S2 and S3
`sites, in contrast to many thrombin inhibitors. Because the S2
`site is entirely blocked by the large side chain of Tyr-99, which
`is consistent with the preference of glycine for the P2 site, the
`S2 site does not seem to be available for the binding of factor
`Xa inhibitors. The S3 site, in which the P3 residue of the
`substrate makes an antiparallel b-ladder with Gly-216, was not
`involved in inhibitor binding either.
`
`FIG. 3. Ribbon drawing of the Des[1–44] factor Xa-FX-2212a
`complex structure (only one molecule in the form 1 crystal is shown).
`The light chain consists of the first (yellow) and the second EGF
`domains (orange). The trypsin-like catalytic domain is shown in blue.
`FX-2212a (red ball and stick) is bound to the active site. One calcium
`ion (pink) each is bound to the first EGF domain and the catalytic
`domain. The binding region for the effector protease receptor-1 is
`shown in magenta.
`
`of Ile-175 through a water molecule, ‘‘A’’ (558 and 672 in the
`crystal form 2, 578 in the crystal form 1). This water, Wat A,
`also is preserved in the apo factor Xa structure (518 in 1HCG).
`The carboxyl acid of the inhibitor is directed toward the
`catalytic triad and makes interactions with residues in the
`active site and other residues through water molecules (Figs. 1
`and 5b). It is clear from the electron density maps that only the
`S-isomer of FX-2212a binds to the factor Xa in all three
`
`FIG. 4. The extended flexible structures of the effector protease receptor-1 binding site in three crystallographically independent molecules:
`two molecules (green and red) in the form 1 crystal and one molecule (blue) in the form 2 crystal. The second EGF domains were superimposed.
`
`

`
`6634
`
`Biochemistry: Kamata et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`(a) Stereo view of the electron density for FX-2212a in difference electron density maps (contoured at 1.6s) calculated after modeling
`FIG. 5.
`the first EGF domain and the simulated annealing refinement. The final structure is superimposed. (b) Binding interactions of FX-2212a (magenta
`ball and stick) with Des[1–44] factor Xa in the form 1 crystal. The Ca backbone is shown in blue, and residues involved in interaction are shown
`as a yellow ball-and-stick model. Conserved hydrogen bonds in the three crystallographically independent molecules are shown in green and a unique
`hydrogen bond in this interaction is shown in orange.
`
`The lack of availability of the S2 site may be compensated
`by the interaction to Gln-192: The second phenyl ring, which
`links the benzamidine group to the pentanoic acid, makes
`hydrophobic interactions with the aliphatic portion of the side
`chain of Gln-192. The rotation around the bond between the
`two phenyl rings allows the second phenyl ring to be positioned
`in parallel to the side chain amide group of Gln-192 without
`breaking the twin–twin geometry interaction between the
`amidino group and Asp-189. Gln-192, which defines the pref-
`erence of the P3 site, has moved closer toward the inhibitor
`than in the apo-factor Xa structure. In the DX-9065a-bound
`factor Xa structure, Gln-192 also moves and makes hydropho-
`bic interactions with the naphthyl ring, but the more rigid ring
`structure does not seem to allow the twin–twin geometry
`interaction between the amidino group and Asp-189 at the
`same time.
`The structure of Des[1–44] factor Xa in complex with the
`new synthetic inhibitor FX-2212a provides us useful informa-
`tion for directing a search for the next generation of inhibitors
`with improved properties.
`
`We thank Kyeong-Kyu Kim and Jarmila Jancarik (University of
`California, Berkeley) for useful suggestions, and Malcolm Capel
`(NSLS beam line X12B) for help with data collection. This work has
`been supported by the director, Office of Energy Research, Office of
`Biological and Environmental Research, U.S. Department of Energy
`
`(under contract DE-AC03-76SF00098) and Banyu Pharmaceutical
`Co., Ltd., Tokyo.
`
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