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• W2012157201 abstract "Kunitz domain 1 (KD1) of tissue factor pathway inhibtor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nm, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine β-trypsin were obtained that diffracted to 1.8 Å. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Oγ of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1. Kunitz domain 1 (KD1) of tissue factor pathway inhibtor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nm, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine β-trypsin were obtained that diffracted to 1.8 Å. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Oγ of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1. Proteases and their inhibitors are important in the regulation of many physiologic processes such as fibrinolysis, blood coagulation, complement fixation, fertilization, angiogenesis, hippocampal plasticity, inflammatory response, and bone resorption and remodeling (1Bode W. Huber R. Biochim. Biophys. Acta. 2000; 1477: 241-252Crossref PubMed Scopus (246) Google Scholar, 2Chun T.H. Sabeh F. Ota I. Murphy H. McDonagh K.T. Holmbeck K. Birkedal-Hansen H. Allen E.D. Weiss S.J. J. Cell Biol. 2004; 167: 757-767Crossref PubMed Scopus (274) Google Scholar, 3Pang P.T. Teng H.K. Zaitsev E. Woo N.T. Sakata K. Zhen S. Teng K.K. Yung W.H. Hempstead B.L. Lu B. Science. 2004; 306: 487-491Crossref PubMed Scopus (893) Google Scholar, 4Busuttil S.J. Ploplis V.A. Castellino F.J. Tang L. Eaton J.W. Plow E.F. J. Thromb. Haemost. 2004; 2: 1798-1805Crossref PubMed Scopus (88) Google Scholar, 5Daci E. Udagawa N. Martin T.J. Bouillon R. Carmeliet G. J. Bone Miner. Res. 1999; 14: 946-952Crossref PubMed Scopus (72) Google Scholar). The protease inhibitors are grouped into the Kunitz (6Laskowski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1921) Google Scholar), Kazal (6Laskowski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1921) Google Scholar), serpin (7Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar), and mucus (8Wiedow O. Schroder J.M. Gregory H. Young J.A. Christophers E. J. Biol. Chem. 1990; 265: 14791-14795Abstract Full Text PDF PubMed Google Scholar) families. Two members of the Kunitz family of inhibitors that are involved in regulating coagulation and fibrinolysis are tissue factor pathway inhibitor (TFPI) 1The abbreviations used are: TFPI, tissue factor pathway inhibitor; KD1, Kunitz domain 1; FVIIa, factor VIIa; TF, tissue factor; ECM, extracellular matrix; MMP, matrix metalloproteinase; BPTI, bovine pancreatic trypsin inhibitor; APPI, amyloid precursor protein inhibitor; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; MES, 4-morpholineethanesulfonic acid. 1The abbreviations used are: TFPI, tissue factor pathway inhibitor; KD1, Kunitz domain 1; FVIIa, factor VIIa; TF, tissue factor; ECM, extracellular matrix; MMP, matrix metalloproteinase; BPTI, bovine pancreatic trypsin inhibitor; APPI, amyloid precursor protein inhibitor; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; MES, 4-morpholineethanesulfonic acid. and TFPI-2. TFPI-2, also known as matrix serine protease inhibitor or placental protein 5, features a domain organization similar to TFPI and contains three Kunitz-type inhibitory domains in tandem with a short acidic amino terminus and very basic carboxyl-terminal tail (9Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Crossref PubMed Scopus (185) Google Scholar, 10Miyagi Y. Koshikawa N. Yasumitsu H. Miyagi E. Hirihara F. Aoki I. Misugi K. Umeda M. Miyazaki K. J. Biochem. (Tokyo). 1994; 116: 939-942Crossref PubMed Scopus (94) Google Scholar). A variety of cells, including keratinocytes, dermal fibroblasts, smooth muscle cells, syncytiotrophoblasts, synovioblasts, and endothelial cells, synthesize and secrete TFPI-2 into the extracellular matrix (ECM) (11Rao C.N. Peavey C.L. Liu Y.Y. Lapiere J.C. Woodley D.T. J. Investig. Dermatol. 1995; 104: 379-383Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 12Herman M.P. Sukhova G.K. Kisiel W. Foster D. Kehry M.R. Libby P. Schoenbeck U. J. Clin. Investig. 2001; 107: 1117-1126Crossref PubMed Scopus (194) Google Scholar, 13Udagawa K. Miyagi Y. Hirahara F. Miyagi E. Nagashima Y. Minaguchi H. Misugi K. Yasumitsu H. Miyazaki K. Placenta. 1998; 19: 217-223Crossref PubMed Scopus (55) Google Scholar, 14Sugiyama T. Ishii S. Yamamoto J. Irie R. Saito K. Otuki T. Wakamatsu A. Suzuki Y. Hio Y. Ota T. Nishikawa T. Sugano S. Masuho Y. Isogai T. FEBS Lett. 2002; 517: 121-128Crossref PubMed Scopus (17) Google Scholar, 15Iino M. Foster D.C. Kisiel W. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 40-46Crossref PubMed Scopus (92) Google Scholar). TFPI-2 is found in three forms with Mr 27,000, Mr 30,000, and Mr 32,000 (16Rao C.N. Reddy P. Liu Y.Y. O'Toole E.A.O. Reeder D.J. Foster D.C. Kisiel W. Woodley D.T. Arch. Biochem. Biophys. 1996; 335: 45-52Crossref Scopus (87) Google Scholar). These three forms are believed to correspond to differentially glycosylated forms of TFPI-2 (16Rao C.N. Reddy P. Liu Y.Y. O'Toole E.A.O. Reeder D.J. Foster D.C. Kisiel W. Woodley D.T. Arch. Biochem. Biophys. 1996; 335: 45-52Crossref Scopus (87) Google Scholar). TFPI-2 exhibits inhibitory activity primarily toward trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor (TF) through its Kunitz domain 1 (KD1) and is believed to be the major inhibitor of plasmin in the ECM (17Chand H.S. Schmidt A.E. Bajaj S.P. Kisiel W. J. Biol. Chem. 2004; 279: 17500-17507Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, TFPI-2 has little inhibitory activity toward urokinase-type plasminogen activator, tissue-type plasminogen activator, or thrombin (18Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Crossref PubMed Scopus (138) Google Scholar). By inhibiting plasmin, TFPI-2 decreases the activation of the matrix metalloproteinases pro-MMP-1 and pro-MMP-3 and thereby suppresses formation of the active MMP-2. As a consequence of decreased MMP-2 activation, ECM degradation and tumor growth and metastasis are reduced (19Rao C.N. Mohanam S. Puppala A. Rao J.S. Biochem. Biophys. Res. Commun. 1999; 255: 94-98Crossref PubMed Scopus (87) Google Scholar, 20Rao C.N. Lakka S.S. Kin Y. Konduri S.D. Fuller G.N. Mohanam S. Rao J.S. Clin. Cancer Res. 2001; 7: 570-576PubMed Google Scholar, 21Konduri S.D. Rao C.N. Chandrasekar N. Tasiou A. Mohanam S. Kin Y. Lakka S.S. Dinh D. Olivero W.C. Gujrati M. Foster D.C. Kisiel W. Rao J.S. Oncogene. 2001; 20: 6938-6945Crossref PubMed Scopus (72) Google Scholar, 22Chand H.S. Du X. Ma D. Inzunza H.D. Kamei S. Foster D. Brodie S. Kisiel W. Blood. 2004; 103: 1069-1077Crossref PubMed Scopus (67) Google Scholar). Moreover, TFPI-2 expression is reported to be up-regulated in atherosclerotic coronary arteries, indicating that it may play a role in prevention of atherosclerotic plaque rupture (23Crawley J.T. Goulding D.A. Ferreira V. Severs N.J. Lupu F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 218-224Crossref PubMed Scopus (48) Google Scholar). Also, TFPI-2 has recently been shown to delay retinal degeneration by stimulating proliferation of the retinal pigment epithelium, although the mechanism by which this occurs is unclear (24Obata R. Yanagi Y. Tamaki Y. Hozumi K. Mutoh M. Tanaka Y. Eye. 2004; 18: 1-5Crossref PubMed Scopus (3) Google Scholar). Thus, understanding the protease specificity of KD1 is biologically significant. TFPI-2 inhibits proteases via the P1 arginine residue 2For comparison, the chymotrypsin amino acid numbering system is used throughout for trypsin, plasmin, and FVIIa. Residue 195 in chymotrypsin numbering corresponds to 195 in trypsin, 741 in plasmin, and 344 in FVIIa. Where necessary, the amino acid corresponding to a given protease is given in braces (e.g. {741}). Where insertions occur, thechymotrypsin numbering is followed by a capital letter, such as A. KD1 is numbered using the BPTI numbering system, such that residue 15 in BPTI numbering corresponds to residue 24 in KD1. Thus, the BPTI numbering and KD1 numbering differ by 9 throughout. 2For comparison, the chymotrypsin amino acid numbering system is used throughout for trypsin, plasmin, and FVIIa. Residue 195 in chymotrypsin numbering corresponds to 195 in trypsin, 741 in plasmin, and 344 in FVIIa. Where necessary, the amino acid corresponding to a given protease is given in braces (e.g. {741}). Where insertions occur, thechymotrypsin numbering is followed by a capital letter, such as A. KD1 is numbered using the BPTI numbering system, such that residue 15 in BPTI numbering corresponds to residue 24 in KD1. Thus, the BPTI numbering and KD1 numbering differ by 9 throughout. (Arg-15) in KD1 (17Chand H.S. Schmidt A.E. Bajaj S.P. Kisiel W. J. Biol. Chem. 2004; 279: 17500-17507Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Kamei S. Petersen L.C. Sprecher C.A. Foster D.C. Kisiel W. Thromb. Res. 1999; 94: 147-152Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although KD1 is specific for inhibiting plasmin, the other two Kunitz domains in TFPI-2 have no discernable inhibitory activity and may serve to bind to nearby proteins to localize TFPI-2 in the ECM. These two Kunitz domains may also serve as spacers to correctly position KD1 to interact with plasmin. As a prelude to crystal structure determinations, mutagenesis and molecular modeling of KD1 were employed to understand the specificity of interaction between KD1 and trypsin, plasmin, or FVIIa/TF (17Chand H.S. Schmidt A.E. Bajaj S.P. Kisiel W. J. Biol. Chem. 2004; 279: 17500-17507Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We now report the crystal structure of KD1 complexed with trypsin that precisely defines the interactions between KD1 and this protease. The crystal structures of KD1, plasmin, and FVIIa/TF were then used to refine the protease·inhibitor models built previously (17Chand H.S. Schmidt A.E. Bajaj S.P. Kisiel W. J. Biol. Chem. 2004; 279: 17500-17507Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Knowledge gained from these studies may help in the development of a potent and specific TFPI-2 KD1 molecule that can selectively inhibit plasmin without targeting other proteases. Such a molecule could have a large pharmacologic impact specifically in preventing tumor metastasis, retinal degeneration, and degradation of collagen in the ECM. Materials—Escherichia coli strain BL21(DE3) pLysS and pET28a expression vectors were products of Novagen Inc. (Madison, WI). Amicon Ultra-15 and Ultra-4 centrifugal filter devices (5000 molecular weight cutoff) were purchased from Millipore (Bedford, MA). Q-Sepharose FF, SP-Sepharose, and His-Trap HP columns were obtained from Amersham Biosciences. Novex® 4-20% Tris-glycine polyacrylamide gels were purchased from Invitrogen. Kanamycin and TPCK-trypsin were obtained from Sigma. Human thrombin was prepared as described previously (26Kisiel W. Smith K.J. McMullen B.A. Blood. 1985; 66: 1302-1308Crossref PubMed Google Scholar). All other reagents were of the highest purity commercially available. Expression and Purification of KD1—The first Kunitz-type domain of human TFPI-2 (KD1) was overexpressed as an amino-terminal His6-tagged fusion protein in E. coli strain BL21(DE3) pLysS using the T7 promoter system. The recombinant plasmid derived from pET28a, containing a His6 leader sequence followed by a thrombin cleavage site and the cDNA encoding the first Kunitz-domain of TFPI-2, was prepared according to standard procedures. The recombinant construct was examined for in-frame orientation and integrity by nucleic acid sequencing. The His6-tagged KD1 fusion protein was expressed in E. coli grown in rich media containing 10 mg/liter kanamycin and induced at 37 °C with 1 mm isopropyl thiogalactopyranoside at mid-log-phase (A600 = 0.4-0.7). The induced cells were harvested and lysed using a lysozyme-nucleotidase mix (0.2% lysozyme, 20 μg/ml DNase I, and 20 μg/ml RNase A in 10 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 1 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride). Cell lysis was carried out at room temperature for 2 h, and the lysate was subjected to centrifugation (20,000 × g for 15 min). The cell pellet was then resuspended in a detergent solution (3% Igepal® CA-630 in 10 mm Tris-HCl (pH 7.5) and 1 mm EDTA) and sonicated at 50% power, and inclusion bodies were collected by centrifugation (20,000 × g for 15 min). The inclusion bodies were then washed twice with water following brief sonication and centrifugation (20,000 × g for 15 min). The highly enriched inclusion bodies were then solubilized overnight in 50 mm Tris-HCl (pH 8.0) containing 8 m urea, 0.5 m NaCl, and 10 mm 2-mercaptoethanol. The suspension was centrifuged at 47,000 × g for 30 min, the supernatant was filtered (0.2 μ filters), and the filtrate was subsequently loaded onto a nickel-charged His-Trap column. The column was washed with equilibration buffer (50 mm Tris-HCl (pH 8.0) containing 6 m urea, 0.5 m NaCl, and 10 mm 2-mercaptoethanol), followed by equilibration buffer containing 25 mm imidazole. The His6-tagged KD1 fusion protein was eluted from the column in equilibration buffer containing 500 mm imidazole. The His-Trap purified protein was reduced by the addition of 50 mm dithiothreitol. This solution was incubated overnight with rocker shaking at 4 °C, diluted to a concentration of ∼0.5 mg/ml in 50 mm Tris-HCl (pH 9.0) containing 6 m urea and 0.02% azide, and dialyzed against 20 volumes of the same buffer at 4 °C. The refolding was then initiated by dialysis against 50 mm Tris-HCl (pH 9.0) containing 0.3 m NaCl, 2 m urea, 2.5 mm reduced glutathione, 0.5 mm oxidized glutathione, and 0.02% azide (buffer A) essentially as described by Stone et al. (27Stone M.J. Ruf W. Miles D.J. Edgington T.S. Wright P.E. Biochem. J. 1995; 310: 605-614Crossref PubMed Scopus (84) Google Scholar) for the refolding of human tissue factor. The dialysis was performed for 48 h at 4 °C, and the solution was subsequently dialyzed against fresh buffer A for another 48 h at 4 °C. The solution was then dialyzed extensively at 4 °C against 50 mm Tris-HCl (pH 9.0). The refolded protein solution was then filtered (0.2 μ filters) and applied to a Q-Sepharose FF column equilibrated at 4 °C with 50 mm Tris-HCl (pH 9.0). The protein was eluted from the column using a linear 0-1 m NaCl gradient, and the fractions were analyzed by SDS-PAGE. The fractions containing pure His6-tagged KD1 were pooled and digested with human thrombin at a 1:1000 enzyme/substrate molar ratio for 2 h at 37 °C. Complete digestion of KD1 by thrombin was confirmed by SDS-PAGE analysis of temporal aliquots. Thrombin-treated KD1 preparations were then applied to a His-Trap column to remove the His6 peptides followed by SP-Sepharose chromatography equilibrated with 50 mm MES (pH 6.0) buffer to remove traces of thrombin. The pure, His6 tag-free KD1 preparations were then dialyzed extensively against 20 mm Tris-HCl (pH 7.5), concentrated to >10 mg/ml in Amicon Ultra-15 (5000 molecular weight cutoff) filters, and stored at -80 °C. Each batch preparation was characterized with respect to protein concentration (A280), purity (SDS-PAGE analysis), and inhibition kinetics as previously described (17Chand H.S. Schmidt A.E. Bajaj S.P. Kisiel W. J. Biol. Chem. 2004; 279: 17500-17507Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Crystallography—For crystallographic purposes, the His6 tag-free preparations of KD1 were found to be essential to achieve high concentrations of KD1 without precipitation. Bovine trypsin (TPCK-treated) was dissolved in 1 mm HCl. For crystallization of the KD1·trypsin complex, the solution was set up at 27 mg/ml in a 1:1.1 molar ratio using sitting drops (equal volume of protein and mother liquor) subjected to vapor diffusion at 37 °C against a reservoir containing 100 mm Hepes (pH 7.5) with 40% (NH4)2SO4. Crystals were routinely obtained in 2 days. The crystals were soaked in 33% glycerol and flash frozen. X-ray Data Collection—The data set was collected at the Advanced Light Source (Berkeley, CA) using beam line 8.2.2 and an ADSC Q315 detector. One crystal diffracted to a resolution of 1.8 Å and belonged to the orthorhombic space group P212121with two molecules of the KD1·trypsin complex in the asymmetric unit. The data were processed and scaled using the programs DENZO and SCALEPACK (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). The statistics for data collection are summarized in Table I.Table IData collection and refinement statisticsKD1-TrypsinData collectionSpace groupP212121Unit cell dimensions (Å)a74.107b77.013c125.424Beam line/generatorAdvanced Light Source 8.2.2Resolution (Å)90.0-1.8Wavelength (Å)1.10Molecules/asymmetric unit2Measured reflections449,141Unique reflections63,596Redundancy7.1Overall completeness (%)aNumbers in parentheses represent data in the highest resolution shell, 1.802-1.847 Å94.8 (100)Rmerge(%)aNumbers in parentheses represent data in the highest resolution shell, 1.802-1.847 Å,bRmerge(I) = Σhkl((Σi|Ihkl,i - [Ihkl]|)/ΣiIhkl,i)7.3 (36.5)I/σ(I)aNumbers in parentheses represent data in the highest resolution shell, 1.802-1.847 Å23.8 (6.9)Refinement statisticsResolution (Å)10-1.8No. of atoms/residuesProtein572Metal2Water520Rcryst(%)cRcryst = Σhkl‖Fobs| - |Fcalc‖/Σhkl|Fobs|. Rfree was computed identically, except that 5% of the reflections were omitted as a test set23.0Rfree(%)cRcryst = Σhkl‖Fobs| - |Fcalc‖/Σhkl|Fobs|. Rfree was computed identically, except that 5% of the reflections were omitted as a test set29.5Root mean square deviationsBond length (Å)0.025Bond angle (°)2.108Ramachandran plotFavored84.6Allowed14.4Generously allowed0.6Disallowed0.4a Numbers in parentheses represent data in the highest resolution shell, 1.802-1.847 Åb Rmerge(I) = Σhkl((Σi|Ihkl,i - [Ihkl]|)/ΣiIhkl,i)c Rcryst = Σhkl‖Fobs| - |Fcalc‖/Σhkl|Fobs|. Rfree was computed identically, except that 5% of the reflections were omitted as a test set Open table in a new tab Structure Determination and Refinement—Most of the calculations were performed using the CCP4 suite (29Collaborative Computational Project Number 4 Acta Cryst. 1994; D50: 760-763Crossref Scopus (19668) Google Scholar). The starting phases for refinement were obtained by molecular replacement using the program EPMR (30Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (688) Google Scholar, 31Kissinger C.R. Gehlhaar D.K. Smith B.A. Bouzida D. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1474-1479Crossref PubMed Scopus (72) Google Scholar). The starting search model for KD1·trypsin was derived from the coordinates of the APPI·trypsin complex (Protein Data Bank code 1TAW, Ref. 32Scheidig A.J. Hynes T.R. Pelletier L.A. Wells J.A. Kossiakoff A.A. Protein Sci. 1997; 6: 1806-1824Crossref PubMed Scopus (122) Google Scholar). The rotation and translation search gave two trypsin·KD1 complexes per asymmetric unit. The model was rebuilt using the 2Fo - Fc and Fo - Fc electron density maps. Five percent of the data were kept out of refinement for cross-validation (33Kleywegt G.J. Brunger A.T. Structure (Lond.). 1996; 4: 897-904Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Subsequently, solvent molecules were added to the model. The final refinement statistics are given in Table I. The coordinates have been deposited into the Research Collaboratory for Structural Bioinformatics Protein Data Bank with accession code 1ZR0. Molecular Modeling—Three-dimensional structural information on complexes formed between KD1 and plasmin and between KD1 and FVIIa was obtained using molecular modeling strategies. The crystallographically determined structures of KD1 (this report), FVIIa/TF inhibited with a BPTI mutant (Ref. 34Zhang E. St. Charles R. Tulinsky A. J. Mol. Biol. 1999; 285: 2089-2104Crossref PubMed Scopus (93) Google Scholar, Protein Data Bank code 1FAK), FVIIa/TF (Ref. 35Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (684) Google Scholar, Protein Data Bank code 1DAN), and plasmin (Ref. 36Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar, Protein Data Bank code 1BML) served as templates in building these models. The relative positions of the inhibitor and proteinase domains were maintained, and minor adjustments were only made in the side chains. Hydrophobic/van der Waals contacts, hydrogen bonds, and ionic interactions were observed between each proteinase·inhibitor complex. All of these interactions were taken into consideration in evaluating each inhibitor·proteinase complex, and it was assumed that all potential hydrogen bond donors and acceptors would participate in these interactions. Bulk solvent was excluded from the proteinase· inhibitor complex, and, accordingly, it was anticipated that hydrogen bonds and ionic interactions that may play an important role in specificity could be accurately evaluated. The protocols for modeling these complexes have been described previously (37Bajaj M.S. Birktoft J.J. Steer S.A. Bajaj S.P. Thromb. Haemostasis. 2001; 86: 959-972Crossref PubMed Scopus (226) Google Scholar). KD1·Trypsin Crystallization and Structure Determination— His6-tagged KD1 was cloned and expressed in E. coli strain BL21(DE3)pLysS. The fusion protein was refolded/renatured in the presence of redox exchange buffer, the His6 tag was removed by thrombin proteolysis, and the His6 tag-free KD1 was purified to homogeneity. The KD1 fragment was then incubated in a 1.1:1 molar ratio with bovine trypsin containing ∼1 mm Ca2+, and crystals were obtained at 37 °C in 100 mm Hepes (pH 7.5) and 40% ammonium sulfate after 2-3 days. The crystals diffracted to 1.8 Å and were found to belong to the space group P212121 with unit cell dimensions of a = 74.107, b = 77.013, and c = 125.424. There were two KD1·trypsin complexes in the asymmetric unit. An initial structure was obtained by molecular replacement using the structure of APPI·trypsin (Protein Data Bank code 1TAW, Ref. 32Scheidig A.J. Hynes T.R. Pelletier L.A. Wells J.A. Kossiakoff A.A. Protein Sci. 1997; 6: 1806-1824Crossref PubMed Scopus (122) Google Scholar) as a search model and refined using CCP4/REFMAC to a final Rwork of 23.0 and Rfree of 29.5 (29Collaborative Computational Project Number 4 Acta Cryst. 1994; D50: 760-763Crossref Scopus (19668) Google Scholar). The refinement statistics and geometry of the final structure are summarized in Table I. KD1·Trypsin Interactions Involving the S1 Site and Gln-192 in Trypsin—The two trypsin molecules in the asymmetric unit are in close proximity with each other and interact via the 162-helix of one molecule and the carboxyl-terminal helix of the other through water molecules. Notably, one KD1·trypsin complex was very well ordered as compared with the other, which had some areas of increased disorder with high B-factors, particularly within the trypsin molecule loop regions. This increased flexibility within the second KD1·trypsin complex is attributable, in part, to the large solvent channel located next to it and its lack of contact with neighboring complexes in the crystal packing. Thus, all figures and results discussed in this study are based upon the more ordered and well-defined KD1·trypsin complex. The overall fold of the KD1·trypsin structure is similar to those of BPTI·trypsin (38Helland R. Otlewski J. Sundheim O. Dadlez M. Smalas A.O. J. Mol. Biol. 1999; 287: 923-942Crossref PubMed Scopus (84) Google Scholar, 39Grzesiak A. Helland R. Smalas A.O. Krowarsch D. Dadlez M. Otlewski J. J. Mol. Biol. 2000; 301: 205-217Crossref PubMed Scopus (67) Google Scholar) and APPI·trypsin (32Scheidig A.J. Hynes T.R. Pelletier L.A. Wells J.A. Kossiakoff A.A. Protein Sci. 1997; 6: 1806-1824Crossref PubMed Scopus (122) Google Scholar) and is shown in Fig. 1A. As is the case with all Kunitz-type inhibitors, the segment formed by residues 10-20 primarily mediates the protease inhibitory activity of KD1. This segment forms a hairpin-like structure, which is located outside the segment formed by residues 34-39 in KD1. A disulfide bridge (Cys-14-Cys-38) joins these two segments together along with several H-bonds forming a two-stranded antiparallel β-sheet. The P1 residue, Arg-15 of KD1, makes the primary contact with the S1 residue, Asp-189 of trypsin. The side chain NH1 and NH2 of Arg-15 in KD1 make H-bonds with the hydroxyl of Ser-190 and the carbonyl oxygen of Gly-219 in trypsin, as has been observed previously for the APPI·trypsin complex (32Scheidig A.J. Hynes T.R. Pelletier L.A. Wells J.A. Kossiakoff A.A. Protein Sci. 1997; 6: 1806-1824Crossref PubMed Scopus (122) Google Scholar). As noted in previous structures of BPTI·protease complexes, H-bonds between the carbonyl oxygen of Pro-13 in KD1 and nitrogen of Gly-216 in trypsin and between the backbone nitrogen of Arg-15 in KD1 and carbonyl oxygen of Ser-214 in trypsin were also observed (Fig. 1B). The negative charge that develops during complex formation at the carbonyl oxygen of Arg-15 in KD1 is stabilized by H-bonds involving the backbone nitrogens of Gly-193 and Ser-195 (Fig. 1B). In the KD1·trypsin complex, trypsin Gln-192 is well ordered in the structure. The side chain nitrogen of Gln-192 in trypsin forms an H-bond with the carbonyl oxygen of Cys-14 in KD1 as well as with a water molecule. This water molecule makes three additional H-bonds in a tetrahedral arrangement involving Nϵ of Arg-15 and the carbonyl oxygen of Cys-14 in KD1 and the carbonyl oxygen of Gly-219 in trypsin. Moreover, the side chain oxygen of trypsin Gln-192 is linked via well-ordered water molecules to the carbonyl oxygens of Ala-16 and Leu-34 in KD1, as well as to the side chain nitrogen of Asn-143, the hydroxyl of Tyr-151, and the carbonyl oxygens of Cys-220 and Ser-146 in trypsin (Fig. 1C). Such interactions further stabilize the KD1·trypsin complex. Hydrophobic Interactions That Stabilize the KD1 Structure and Its Interaction with Trypsin—KD1 consists of a hydrophobic core, which is composed of Leu-9, Tyr-11, Tyr-21, Tyr-22, Phe-33, and Tyr-35 (Fig. 2A). These residues are part of the 10-20 segment as well as the two β-strands in KD1 (Fig. 1A), and they are defined by the high-resolution electron density map shown in Fig. 2B. The hydrophobic residues Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 interact with a complementary hydrophobic patch in trypsin composed of Tyr-39, Phe-41, and Tyr-151. The details of these interactions are shown in Fig. 3 and appear to play a major role in the KD1·trypsin interaction.Fig. 3KD1·trypsin hydrophobic interactions. KD1 is shown as yellow ribbons, and trypsin is shown as magenta ribbons. Residues from KD1 are labeled in yellow, and those from trypsin are labeled in mage" @default.
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• W2012157201 title "Crystal Structure of Kunitz Domain 1 (KD1) of Tissue Factor Pathway Inhibitor-2 in Complex with Trypsin" @default.
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