Roles of the P1, P2, and P3 Residues in Determining Inhibitory Specificity of Kallistatin toward Human Tissue Kallikrein
2000; Elsevier BV; Volume: 275; Issue: 49 Linguagem: Inglês
10.1074/jbc.m005605200
ISSN1083-351X
AutoresVincent Chen, Lee Chao, Julie Chao,
Tópico(s)Enzyme function and inhibition
ResumoKallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386–388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys ≥ P1 Tyr > P1 Leu ≥ P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr99 and Trp219 of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp98J and hydrogen bond with Gln174 of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin. Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386–388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys ≥ P1 Tyr > P1 Leu ≥ P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr99 and Trp219 of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp98J and hydrogen bond with Gln174 of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin. methylcoumarinamide polyacrylamide gel electrophoresis structurally conserved region Kallistatin is a serpin that inhibits tissue kallikrein by forming a covalent serpin-proteinase complex (1Zhou G.X. Chao L. Chao J. J. Biol. Chem. 1992; 267: 25873-25880Abstract Full Text PDF PubMed Google Scholar). The inhibitory activity of kallistatin toward tissue kallikrein is abolished upon heparin binding to kallistatin (1Zhou G.X. Chao L. Chao J. J. Biol. Chem. 1992; 267: 25873-25880Abstract Full Text PDF PubMed Google Scholar). An unexpected finding is that kallistatin contains phenylalanine at the P1 position (2Chai K.X. Chen L.M. Chao J. Chao L. J. Biol. Chem. 1993; 268: 24498-24505Abstract Full Text PDF PubMed Google Scholar), since tissue kallikrein is known to have primary specificity for arginine and methionine at the P1 residue (3Muller-Esterl W. Iwanaga S. Nakanishi S. Trends Biochem. Sci. 1986; 11: 336-339Abstract Full Text PDF Scopus (142) Google Scholar). Whether tissue kallikrein or other serine proteinase(s) is the target enzyme of kallistatin in vivo has not been determined. Kallistatin acts as a multifunctional serpin that performs various functions at different tissues. In addition to acting as a tissue kallikrein inhibitor, kallistatin is a potent vasodilator and regulator of vascular remodeling independently of its interaction with tissue kallikrein (4Chen L.M. Chao L. Chao J. Hum. Gene Ther. 1997; 8: 341Crossref PubMed Scopus (45) Google Scholar, 5Chao J. Stallone J.N., J.N. Liang Y.M. Chen L.M. Wang D.Z. Chao L. J. Clin. Invest. 1997; 100: 1-7Crossref PubMed Scopus (75) Google Scholar, 6Miao R.Q. Murakami H. Song Q. Chao L. Chao J. Circ. Res. 2000; 86: 418-424Crossref PubMed Scopus (32) Google Scholar). Tissue kallikrein is a serine proteinase that specifically cleaves low molecular weight kininogen at Met-Lys and Arg-Ser to generate the vasoactive kinin peptide (3Muller-Esterl W. Iwanaga S. Nakanishi S. Trends Biochem. Sci. 1986; 11: 336-339Abstract Full Text PDF Scopus (142) Google Scholar). The physiological functions of tissue kallikrein are principally mediated by kinin, which binds to bradykinin B1 and B2 receptors. Binding of kinin to their receptors activates second messengers and mediates a road spectrum of biological effects including blood pressure reduction, muscular contraction, vascular permeability, neutrophil chemotaxis and pain, inflammatory cascades, and vascular cell growth (7Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). Low levels of tissue kallikrein are associated with hypertension and diabetes (8Margolius H.S. Annu. Rev. Pharmacol. Toxicol. 1989; 29: 343-364Crossref PubMed Google Scholar). Elevated levels of tissue kallikrein, however, are correlated with the pathogenesis of asthma, arthritis, and inflammatory articular diseases (9Worthy K. Figueroa C.D. Dieppe P.A. Bhoola K.D. Int. J. Exp. Pathol. 1990; 71: 587-601PubMed Google Scholar, 10Forteza R. Botvinnikova Y. Ahmed A. Cortes A. Gundel R.H. Wanner A. Abraham W.M. Am. J. Respir. Crit. Care. Med. 1996; 154: 36-42Crossref PubMed Scopus (46) Google Scholar, 11Volpe-Junior N. Donadi E.A. Carvalho I.F. Reis M.L. Inflamm. Res. 1996; 45: 198-202Crossref PubMed Scopus (13) Google Scholar). Tissue kallikrein is also identified in colon and breast cancer cells and may be involved in malignant transformation by stimulating proliferation of tumor cells and increasing vascular permeability (12Hermann A. Bluchinger P. Rehbock J. Biol. Chem. Hoppe-Seyler. 1995; 376: 365-370Crossref PubMed Scopus (23) Google Scholar, 13Rehbock J. Buchinger P. Hermann A. Figueroa C. J. Cancer Res. Clin. 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J. 1997; 323: 167-171Crossref PubMed Scopus (16) Google Scholar, 18Chagas J.R. Portaro F.C. Hirata I.Y. Almeida P.C. Juliano M.A. Juliano L. Prado E.S. Biochem. J. 1995; 306: 63-69Crossref PubMed Scopus (59) Google Scholar, 19Deshpande M.S. Burton J. J. Med. Chem. 1992; 35: 3094-3102Crossref PubMed Scopus (7) Google Scholar, 20Fiedler F. Eur. J. Biochem. 1987; 163: 303-312Crossref PubMed Scopus (43) Google Scholar, 21Chagas J.R. Hirata I.Y. Juliano M.A. Xiong W. Wang C. Chao J. Juliano L. Prado E.S. Biochemistry. 1992; 31: 4969-4974Crossref PubMed Scopus (43) Google Scholar). Furthermore, several recent reports used extending synthetic peptides derived from the reactive center sequence of kallistatin to analyze the enzymatic specificity of tissue kallikrein (22Pimenta D.C. Juliano M.A. Juliano L. Biochem. J. 1997; 327: 27-30Crossref PubMed Scopus (16) Google Scholar, 23Bourgeois L. Brillard-Bourdet M. Deperthes D. Juliano M.A. Juliano L. Tremblay R.R. Dube J.Y. Gauthier F. J. Biol. Chem. 1997; 272: 29590-29595Crossref PubMed Scopus (49) Google Scholar, 24Pimenta D.C. Chao J. Chao L. Juliano M.A. Juliano L. Biochem. J. 1999; 339: 473-479Crossref PubMed Scopus (26) Google Scholar). These studies showed that the P2 residue with a bulky and hydrophobic side chain, particularly Phe, is a critical determinant for tissue kallikrein specificity. Crystallographic analysis of porcine tissue kallikrein suggested that the hydrophobic phenyl side chain of Phe at P2 could be optimally accommodated into a hydrophobic crevice between Tyr99 and Trp215 of tissue kallikrein (25Bode W. Chen Z. Bartles K. Kutzbach C. Schmidt-Kastner G. Bartunik H. J. Mol. Biol. 1983; 164: 237-282Crossref PubMed Scopus (223) Google Scholar, 26Katz B.A. Liu B. Barnes M. Springman E.B. Protein Sci. 1998; 7: 875-885Crossref PubMed Scopus (58) Google Scholar). 1The chymotrypsinogen nomenclature is used to number amino acid residues of human tissue kallikrein (26Katz B.A. Liu B. Barnes M. Springman E.B. Protein Sci. 1998; 7: 875-885Crossref PubMed Scopus (58) Google Scholar). The residue numbering of human tissue kallikrein is indicated in brackets whenever appropriate. However, the knowledge on P3 specificity of tissue kallikrein is limited. Although some reports suggest that the P3 residue does not have much effect on tissue kallikrein specificity, the role of the P3 site has not been established (19Deshpande M.S. Burton J. J. Med. Chem. 1992; 35: 3094-3102Crossref PubMed Scopus (7) Google Scholar, 20Fiedler F. Eur. J. Biochem. 1987; 163: 303-312Crossref PubMed Scopus (43) Google Scholar, 27Kettner C. Mirabelli C. Pierce J.V. Shaw E. Arch. Biochem. Biophys. 1980; 202: 420-430Crossref PubMed Scopus (30) Google Scholar). A previous study has used various serpin mutants to investigate the inhibitory specificity toward a number of serine proteinases (28Patston P.A. Gettins P.G.W. Thromb. Haemostasis. 1994; 72: 166-179Crossref PubMed Scopus (13) Google Scholar). With the heparin-regulated activity and its specific inhibitory activity toward tissue kallikrein, kallistatin is an ideal model to study tissue kallikrein specificity by structural and functional analysis. In this study, we used kallistatin as a model to explore the inhibitory specificity of the P1, P2, and P3 residues toward human tissue kallikrein. We have created 57 kallistatin mutants with various amino acid substitutions at the P1, P2, or P3 position (residue 388, 387, or 386) and compared their inhibitory specificity toward human tissue kallikrein. The interaction of the P2 and P3 residues with the reactive site of human tissue kallikrein was further assessed by molecular modeling of kallistatin-tissue kallikrein complex. This study presents a comprehensive picture for the P1, P2, and P3 specificity of human tissue kallikrein. These results provide useful insights for future development of specific and potent tissue kallikrein inhibitors and for discovery of other physiological targets of kallistatin. Escherichia coli strain TOP10, the pTrc-His B expression vector, was purchased from Invitrogen (San Diego, CA); the restriction enzymes, T4 kinase, calf intestinal alkaline phosphatase, Klenow fragment, and isopropylthio-β-galactoside were from Life Technologies, Inc.; Taq polymerase was from PerkinElmer Life Sciences; nickel-nitrilotriacetic acid-agarose was from Qiagen (Santa Clarita, CA); the POROS® HE/1 column was from PerSeptive Biosystems (Cambridge, MA); heparin was from The Upjohn Co.; d-Val-Leu-Arg-methylcoumarinamide (MCA)2 and phenylmethylsulfonyl fluoride were from Enzyme System Products (Livermore, CA); human tissue kallikrein was purified as described previously (29Shimamoto K. Chao J. Margolius H.S. J. Clin. Endocrinol. Metab. 1980; 51: 840-848Crossref PubMed Scopus (93) Google Scholar); anti-kallistatin monoclonal antibody was generated as described previously (30Chao J. Schmaier A. Chen L.M. Yang Z. Chao L. J. Clin. Lab. Med. 1996; 127: 612-620Abstract Full Text PDF PubMed Scopus (106) Google Scholar). A prokaryotic expression vector, pTrc His, was used to produce recombinant kallistatins. A hexahistidine sequence was added to the expression vector at the amino termini of the recombinant human kallistatin so that the protein purification could be achieved by a metal affinity chromatography. The P1, P2, and P3 variants were constructed as described previously (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar). Briefly, degenerate 5′ oligonucleotides, 5′-CGATCAAATTCNNNTCTGCCCAGA-3′, 5′-CGATCAAANNNTTCTCTGCCCAGA-3′ and 5′-CGATCNNNTTCTTCTCTGCCCAGA-3′, containing degenerate codons at residue 388, 387, and 386, respectively, were used to generate pools of P1, P2, and P3 mutant fragments by polymerase chain reaction. The degenerate nucleotides are denoted by underlines. The mutant fragments were then cloned into the expression vector, pTrc-KS (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar). The mutants with different P2 and P3 residues were identified by DNA sequencing. The method for the expression of the kallistatin variants were the same as described before (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar) except that volume was scaled down to 25 ml of cell culture. After centrifugation at 4,000 × g for 5 min at 4 °C, cells were suspended in 1 ml of buffer containing 20 mm sodium phosphate, 0.5m NaCl, pH 7.8, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride, 2 mmbenzamidine, 50 μg/liter soybean trypsin inhibitor, 1 μm of leupeptin, 10 mm β-mercaptoethanol, and 0.5% Triton X-100. Cell lysates were prepared by adding 1 mg/ml lysozyme and incubated on ice for 30 min. The DNA and RNA were digested by adding 5 μg/ml of DNase I and 10 μg/ml of RNase A and incubated on ice for another 30 min. Cell lysates were centrifuged for 10 min at 15,000 × g and the supernatant was collected. To purify recombinant kallistatins, aliquots of 50% slurry of nickel-nitrilotriacetic acid-agarose (Qiagen) were added into each tube and mixed gently for 30 min at 4 °C. The supernatants were removed after centrifugation at 15,000 × g for 10 s. The resins were washed four times using 500 μl of wash buffer (20 mm sodium phosphate, pH 6.8, 500 mm NaCl, 20 mm imidazole, and 0.2% Triton X-100) and pelleted. The recombinant proteins were eluted from the resin by 60 μl of elution buffer (20 mm sodium phosphate, pH 8.0, 160 mmimidazole). The concentration of each kallistatin variant was determined by enzyme-linked immunosorbent assay (30Chao J. Schmaier A. Chen L.M. Yang Z. Chao L. J. Clin. Lab. Med. 1996; 127: 612-620Abstract Full Text PDF PubMed Scopus (106) Google Scholar). The binding assay for the wild-type and mutant kallistatins toward tissue kallikrein was performed according to the method described previously (1Zhou G.X. Chao L. Chao J. J. Biol. Chem. 1992; 267: 25873-25880Abstract Full Text PDF PubMed Google Scholar). Briefly, about 0.5 μg of recombinant kallistatins were incubated with 20,000 cpm of 125I-labeled human tissue kallikrein in 20 μl of 20 mm sodium phosphate buffer, pH 8.0, at 37 °C for 90 min. The binding reaction was stopped by adding 3× SDS-sample buffer and boiling for 5 min. The samples were resolved in 10% SDS-PAGE and analyzed by autoradiography. The relative intensity of the complexes formed by recombinant kallistatins and tissue kallikrein was calculated by densitometric analysis of the autoradiogram using NIH Image, version 1.47. The mutants with stronger binding activity were then expressed in a large quantity and purified to apparent homogeneity for further characterization. The mutants displaying better tissue kallikrein binding activity were expressed in 1 liter of cell culture, and then soluble cell lysates were isolated and purified by nickel affinity and heparin affinity chromatography as described previously (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar). Protein purity was assessed by Coomassie Blue staining following SDS-PAGE. Kallistatin concentrations of the mutants were determined by a specific enzyme-linked immunosorbent assay. The ability of native and recombinant kallistatins to form SDS-stable complexes with tissue kallikrein was assessed by incubating 2 μmconcentrations of different kallistatins with a 1 μmconcentration of the serine proteinase in 50 mm Tris-HCl, pH 8.0, and 0.1 m NaCl, at 37 °C for 2 h. Reactions were quenched by adding SDS sample buffer containing 100 mmdithiothreitol and boiled for 2 min, and samples were analyzed by SDS-PAGE. For monitoring the complex formation of kallistatin with chymotrypsin, a time course of the reaction (2, 5, 10, 15, and 60 min) was performed under the same condition as mentioned above in the absence or presence of 120 units/ml heparin. The stoichiometry of inhibition (SI) values for the inhibition of human tissue kallikrein or chymotrypsin were determined as described previously (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar). Different concentrations of the recombinant kallistatins were incubated with 25 nm tissue kallikrein or 50 nm chymotrypsin in 50 mm Tris-HCl, pH 8.0, 0.1 m NaCl, and 0.1% bovine serum albumin in the absence or presence of 50 units/ml heparin. The reaction was carried out at 37 °C for a period of time sufficient to ensure that complex formation was complete (24 h for tissue kallikrein and 3 h for chymotrypsin). The residual amidolytic activity was measured by adding 20 μl of the reaction mixture into 30 μm of substrate (Val-Leu-Arg-MCA for tissue kallikrein and Ala-Ala-Pro-Phe-MCA for chymotrypsin) in 2 ml of 50 mm Tris, pH 8.0, and 0.1 NaCl. The rate of substrate hydrolysis was monitored at 380-nm excitation and 460-nm emission. The inhibition stoichiometry was obtained from the abscissa intercept of a linear regression fit of the residual enzymatic activity versus the molar ratio of inhibitor to enzyme. The association rate constants of the P1, P2, and P3 mutants to human tissue kallikrein were determined under pseudo-first order condition as described previously (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar). The association rate constants (ka) were calculated as described previously (31Chen V.C. Chao L. Chao J. Biochim. Biophys. Acta. 2000; 1479: 237-246Crossref PubMed Scopus (32) Google Scholar). The amounts of kallistatins were employed at least a 10-fold molar excess over serine proteinases. At least five inhibitor concentrations were examined for each reaction. The effects of heparin on the association rate were also demonstrated by preincubation of the recombinant kallistatins with 20 units/ml heparin at 37 °C for 5 min followed by kinetic assays. Serine proteinases, their respective substrates, and reaction buffers used in the assays were as follows: human tissue kallikrein (3 nm), rat tissue kallikrein (3 nm), and activated protein C (8 nm) with 30 μmVal-Leu-Arg-MCA in 20 mm sodium phosphate, pH 8.0; human plasma kallikrein (3.5 nm), trypsin (2 nm), and plasmin (10 units/ml) with 30 μmPro-Phe-Arg-MCA in 20 mm sodium phosphate, pH 8.0, 0.1 m NaCl; human thrombin (1 nm) with 30 μm Phe-Pro-Arg-MCA in 20 mm sodium phosphate, pH 8.0, 0.1 m NaCl; bovine pancreatic chymotrypsin (2 nm) with 30 μm N-succinyl-Ala-Ala-Pro-Phe-MCA in 20 mm sodium phosphate, pH 8.0, 0.1 m NaCl; human neutrophil cathepsin G (70 nm) with 1.0 mm N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in 50 mm Tris-HCl, pH 7.5, 100 mm NaCl; and human neutrophil elastase (20 nm) with 1.0 mmGlu-Pro-Arg-p-nitroanilide in 50 mm Tris-HCl, pH 7.5, 100 mm NaCl. All of the reaction buffers contained 0.1% bovine serum albumin. The atomic coordinates of the intact serpins, α1-antitrypsin (2psi.pdb), ovalbumin (1ova.pdb), antithrombin (1ant.pdb), and cleaved formed of protein C inhibitor (1pai.pdb) were obtained from the Brookhaven Protein Data Bank. A molecular model was created using the homology modeling module, Composer, in the SYBYL (version 6.5; Tripos, Inc.). The topologically equivalent residues across these serpins were determined first based on sequence homology. A structural alignment of the serpins was then performed with the equivalent residues as a starting point. This alignment determined the structurally conserved regions (SCRs) as well as the average framework of the SCRs. SCRs of kallistatin were generated by using fragments of the homologs to construct the backbone of the SCRs and a rule-based procedure to generate the side chains. The structurally variable regions were constructed by using fragments from known structures that are compatible with the rest of the model and then using sequence information to postulate the best single fragment to use in the final model. Since the reactive center loop is a highly variable region among serpins, there is no good template for modeling the reactive center loop of kallistatin. We chose the atomic coordinates of antithrombin to model the reactive center loop of kallistatin, P16–P5′, because the reactive loop of antithrombin has the same number of residues as kallistatin, and the hinge region of the reactive loop was partially inserted into the A β-sheet as predicted for serpins (32Wei A. Rubin H. Cooperman B.S. Christianson D.W. Nat. Struct. Biol. 1994; 1: 251-258Crossref PubMed Scopus (167) Google Scholar). The kallistatin model was then refined by side chain torsion relieving and energy minimization. The backbone of the whole model was constrained and then energy minimized by steepest descent until the maximum derivative was less than 50 kcal/(mol·Å). The constraint was then removed, and additional minimization was performed until the maximum derivative was less than 5 kcal/(mol·Å) using a steepest descent algorithm. Finally, conjugate gradient minimization continued until the maximum derivative was less than 0.1 kcal/(mol·Å). The atomic coordinates of the x-ray structure of human tissue kallikrein were generously provided by Dr. Katz (26Katz B.A. Liu B. Barnes M. Springman E.B. Protein Sci. 1998; 7: 875-885Crossref PubMed Scopus (58) Google Scholar). The coordinates of the "kallikrein loop," consisting of 14 residues from 95 [102] to 98K [115], are not available in this molecular model. The "kallikrein loop" was created by homology modeling using the program Composer, in the SYBYL (version 6.5; Tripos). The coordinates of the kallikrein loop from a theoretical molecular model of prostate-specific antigen (1pfa.pdb), obtained from the Protein Data Bank, were used as a template to model that of human tissue kallikrein. The method for generating the kallikrein loop of prostate-specific antigen was described in previous studies (33Villoutreix B.O. Getzoff E.D. Griffin J.H. Protein Sci. 1994; 3: 2033-2044Crossref PubMed Scopus (73) Google Scholar). The final model of tissue kallikrein was then refined by side chain torsion relieving and energy minimization. The backbone of the whole model was constrained and then energy-minimized by steepest descent until the maximum derivative was less than 50 kcal/(mol·Å). The constraint was then removed, and additional minimization was performed until the maximum derivative was less than 5 kcal/(mol·Å) using a steepest-descent algorithm. Finally, conjugate gradient minimization continued until the maximum derivative was less than 0.1 kcal/(mol·Å). The structures of porcine tissue kallikrein-bovine pancreatic trypsin inhibitor complex (1kai.pdb) were used as templates to model human tissue kallikrein-kallistatin complex. Previous studies pointed out that the structures of the backbone atoms between residues P2 and P2′ of the serine proteinase inhibitors are highly conserved (34Greer J. J. Mol. Biol. 1981; 153: 1043-1053Crossref PubMed Scopus (37) Google Scholar, 35Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-451Crossref PubMed Scopus (1009) Google Scholar). The P2-P2′ residues in bound bovine pancreatic trypsin inhibitor were, therefore, selected and changed the corresponding sequence of kallistatin. After flanking residues were deleted, the four-residue peptide was used to model the docking conformation of the reactive site. The reactive center loop of kallistatin was modeled based on the crystal structure of antithrombin, whose P1 Arg residue pointed inward in an orientation not appropriate for interaction with the S1 site of serine proteinase. Therefore, the atomic coordinates of backbone and side chain structure of the computational mutated P2-P2′ peptide from bovine pancreatic trypsin inhibitor were used to force the corresponding region in the reactive loop of the kallistatin model to adopt the canonical structure of the peptide. The structure of the reactive center loop was then torsion relieved and energy minimized by the method mentioned above. After the model of human tissue kallikrein was superimposed onto the structure of porcine tissue kallikrein and energy-minimized, the reactive center loop of kallistatin was fit manually into the reactive crevice of tissue kallikrein using the docked bovine pancreatic trypsin inhibitor peptide as starting guideline. Torsion angles and steric clashes of the residues in the complex were adjusted to low energy rotamers to maximize steric fit. The complex was then energy-minimized as described previously. To evaluate the local interactions of mutated residues of kallistatin in a complex, the P2 or the P3 residue was computationally changed to the desired amino acids. The side chain of the mutated residue was adjusted to a low energy rotamer, and the complex was then minimized for 200 iterations of steepest descent. In the present study, we used intact recombinant kallistatin to investigate its reactive center loop, P1, P2, and P3 residues (residues 388, 387, and 386), toward specificity of tissue kallikrein by site-directed mutagenesis, protein engineering, and molecular modeling. The reactive center loop of a serpin can adopt an inhibitory conformation that fits optimally into the reactive pocket of its target serine proteinase. Several recent studies have used extended synthetic peptides imitating the reactive center sequences of serpins, instead of short peptide, to study the specificity of tissue kallikrein (22Pimenta D.C. Juliano M.A. Juliano L. Biochem. J. 1997; 327: 27-30Crossref PubMed Scopus (16) Google Scholar, 23Bourgeois L. Brillard-Bourdet M. Deperthes D. Juliano M.A. Juliano L. Tremblay R.R. Dube J.Y. Gauthier F. J. Biol. Chem. 1997; 272: 29590-29595Crossref PubMed Scopus (49) Google Scholar). The reactive loop of a macromolecule, such as kallistatin or kininogen, rather than small synthetic peptides can generate more extended interactions with tissue kallikrein. Therefore, the conformation of the reactive loop or an elongated synthetic peptide should play a more significant role than that of a small synthetic peptide in binding with the proteinase. Kallistatin is an appealing model to study the inhibitory specificity toward human tissue kallikrein, since it has a unique P1 Phe residue and specific inhibitory activity for human tissue kallikrein. We have created 57 kallistatin variants with mutations at either the P1, P2, or P3 position for protein expression in a prokaryotic expression system. Each recombinant kallistatin was initially expressed and partial purified by a nickel affinity agarose column in a miniscale. The purity of recombinant kallistatins was approximately 40–80% estimated by Coomassie Blue staining in SDS-PAGE. The concentrations of kallistatin mutants were determined by enzyme-linked immunosorbent assay. The same amounts of the partially purified recombinant proteins were used to determine which variants exhibit relatively strong binding activity toward human tissue kallikrein by kallikrein-binding assay. A variant with inhibitory activity can form an 82-kDa SDS- and heat-stable complex with 125I-labeled tissue kallikrein. The binding assay showed that wild-type kallistatin (P1 Phe) and the variants containing Arg, Leu, Lys, Met, and Tyr at P1 are capable of forming 85-kDa complexes with tissue kallikrein (Fig. 1). The intensity of the complex is determined mainly by relative concentrations of kallistatin and tissu
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