Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m500588200
ISSN1083-351X
AutoresJun Li, Siew Pheng Lim, David Beer, Viral Patel, Daying Wen, Christine Tumanut, David C. Tully, Jennifer Williams, Jan Jiřiček, John P. Priestle, Jennifer L. Harris, Subhash G. Vasudevan,
Tópico(s)HIV Research and Treatment
ResumoRegulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1′ and P3′. In contrast, the P2′ and P4′ substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences. Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1′ and P3′. In contrast, the P2′ and P4′ substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences. Dengue virus is the etiologic agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is the most prevalent arthropod-transmitted infectious disease in humans. Dengue consists of four closely related but antigenically distinct viral serotypes (DEN1-4), 1The abbreviations used are: DEN1-4, dengue serotypes 1-4; ACMC, 7-amino-3-carbamoylmethyl-4-methyl coumarin; AMC, 7-amino-4-methyl coumarin; Boc, tert-butoxycarbonyl; Bz, benzoyl; CF40-Gly-NS3pro185, NS2B (amino acids 1394-1440) fused to NS3 protease domain (amino acids 1476-1660); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; cNS2B, NS2B 40-amino acid core sequence; Nle, norleucine; NS3pro, NS3 protease domain (amino acids 1476-1660); SBzl, thiobenzyl ester. 1The abbreviations used are: DEN1-4, dengue serotypes 1-4; ACMC, 7-amino-3-carbamoylmethyl-4-methyl coumarin; AMC, 7-amino-4-methyl coumarin; Boc, tert-butoxycarbonyl; Bz, benzoyl; CF40-Gly-NS3pro185, NS2B (amino acids 1394-1440) fused to NS3 protease domain (amino acids 1476-1660); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; cNS2B, NS2B 40-amino acid core sequence; Nle, norleucine; NS3pro, NS3 protease domain (amino acids 1476-1660); SBzl, thiobenzyl ester. of the genus Flavivirus (1.Gubler D.J. Clin. Microbiol. Rev. 1998; 11: 480-496Crossref PubMed Google Scholar, 2.Monath T.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2395-2400Crossref PubMed Scopus (641) Google Scholar). Following primary infection, lifelong immunity develops that prevents repeated assault by the same serotype but does not provide protection from a virus of a different serotype (3.Halstead S.B. Rojanasuphot S. Sangkawibha N. Am. J. Trop. Med. Hyg. 1983; 32: 154-156Crossref PubMed Scopus (212) Google Scholar). Dengue diseases are endemic in the tropics and subtropics, and the viruses are maintained in a cycle that involves humans and the Aedes aegypti mosquito. Infection with dengue viruses produces a spectrum of clinical illness ranging from a nonspecific viral syndrome to severe and fatal hemorrhagic disease (1.Gubler D.J. Clin. Microbiol. Rev. 1998; 11: 480-496Crossref PubMed Google Scholar, 2.Monath T.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2395-2400Crossref PubMed Scopus (641) Google Scholar). Currently there is no antiviral drug or vaccine available against dengue viruses, and the pathogenesis of the disease is poorly understood. As with other members of the Flaviviridae family, the genomes of the dengue viruses consist of a positive single-stranded RNA of ∼10,700 bases in length (4.Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1564) Google Scholar). Co-translational processing and post-translational processing of the polyprotein give rise to three structural proteins and at least seven non-structural proteins (4.Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1564) Google Scholar). The correct processing of these proteins is essential for virus replication and requires host proteases such as signalase and furin (5.Stadler K. Allison S.L. Schalich J. Heinz F.X. J. Virol. 1997; 71: 8475-8481Crossref PubMed Google Scholar) and a two-component viral protease, NS2B/NS3 (4.Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1564) Google Scholar). Previous studies have shown that the N-terminal part of NS3 contains trypsin-like protease domain (6.Preugschat F. Yao C.W. Strauss J.H. J. Virol. 1990; 64: 4364-4374Crossref PubMed Google Scholar) and that the activity of NS3 was dependent on at least 40 amino acids of NS2B (6.Preugschat F. Yao C.W. Strauss J.H. J. Virol. 1990; 64: 4364-4374Crossref PubMed Google Scholar, 7.Falgout B. Pethel M. Zhang Y.M. Lai C.J. J. Virol. 1991; 65: 2467-2475Crossref PubMed Google Scholar, 8.Zhang L. Mohan P.M. Padmanabhan R. J. Virol. 1992; 66: 7549-7554Crossref PubMed Google Scholar). The preferred NS3 protease-cleavage sites in the viral polyprotein have two basic amino acid residues (Arg-Arg, Arg-Lys, Lys-Arg, or occasionally Gln-Arg) at the P2 and P1 positions, followed by a Gly, an Ala, or a Ser at the P1′ position (4.Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1564) Google Scholar). The crystal structure of the DEN-2 NS3pro in the absence of NS2B has been determined at 2.1-Å resolution by Murthy et al. (9.Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and shows a shallow substrate binding site, indicating a lack of significant interactions beyond P2-P2′. The NS3pro domain in the absence of NS2B is an inefficient protease as demonstrated by the low turnover rate of the small chromogenic substrate N-α-benzoyl-l-Arg-p-nitroanilide (10.Yusof R. Clum S. Wetzel M. Murthy H.M. Padmanabhan R. J. Biol. Chem. 2000; 275: 9963-9969Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Although NS2B is required for efficient enzymatic activity of the NS3pro, the structure of the latter without the cofactor resembles that of the related hepatitis C NS3 protease bound to its activating peptide NS4A. The exact mechanism by which the NS2B cofactor stimulates the protease is not currently known. However, it is plausible that NS2B resembles NS4A and interacts directly with the NS3 protease domain, causing a conformational change that extends the binding pockets (10.Yusof R. Clum S. Wetzel M. Murthy H.M. Padmanabhan R. J. Biol. Chem. 2000; 275: 9963-9969Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The aim of the current study was to elucidate and compare the substrate specificity of NS3 protease from all four serotypes. We performed functional substrate profiling of the P1-P4 and P1′-P4′ for the DEN1-4 protease complexes using tetrapeptide and octapeptide positional scanning peptide libraries. As a consequence, we expanded the earlier findings on DEN2 NS3 to a broader extent (P4-P4′) and discovered that its substrate preference was shared by enzymes of the other three serotypes. Materials—Dengue virus serotype 1 (strain Hawaii), and serotype 4 (H241) were purchased from American Type Culture Collection (Manassas, VA). Dengue virus serotype 3 (strain S221/03, GenBank™ accession number AY662691) was obtained from a dengue patient and was a kind gift from Dr. Eng Eong Ooi (Environmental Health Institute, Singapore). The plasmids pGEM-T-(E-NS3) and pET15b-NS3NS5 containing, respectively, the NS2B/NS3 and NS3 cDNAs from Dengue virus serotype 2 (strain TSV01, GenBank™ accession number AY037116) were kind gifts from James Cook University, Queensland, Australia. The dengue virus NS3 protease substrate peptide Boc-Gly-Arg-Arg-AMC was purchased from Bachem (Bubendorf, Switzerland). Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA). Reverse Transcription PCR of DEN1, DEN3, and DEN4 cDNA Fragments Encoding NS2B/NS3—C6/36 cells were inoculated with the DEN1, DEN3, or DEN4 virus and incubated at 28 °C for 5-7 days. Cell culture media were collected and spun at 14,000 rpm to remove cell debris. Viral RNAs were obtained by extracting 2 ml of the clarified culture media with 1 ml of TRIzol LS Reagent (Invitrogen) according to the manufacturer's instructions. First strand cDNA synthesis for the NS2B/NS3 sequences was performed using the primers DEN1 reverse (5′-TGTTGTGGAAGTTTCCCTATTTC-3′), DEN3 reverse (5′-TGGTGTTATTACTGTTGTGGC-3′), or DEN4 reverse (5′-GTAAGTTGGCAAACTGGCAATC-3′) with Superscript II (Invitrogen) at 45 °C for 1 h, followed by PCR with Pfu polymerase (Stratagene) and the primers DEN1 forward (5′-TGGCTATGGTACTGTCAATTG), DEN3 forward (5′-CCATTCTTGGCTTTGGGATTC-3′), or DEN4 forward (5′-CCATTATGGCTGTGTTGTTTG-3′) along with the corresponding reverse primers. Preparation of CF40-Gly-NS3pro185 Expression Constructs—All DEN1-4 CF40-Gly-NS3pro185 expression constructs comprised the 40-amino acid hydrophilic core sequence of serotype-specific NS2B (cNS2B; amino acids 1394-1440) linked via a flexible Gly4SerGly4 linker to the N-terminal 185 amino acids of NS3 (NS3pro185; amino acids 1476-1660) (11.Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) and cloned into the vector pET15b (Novagen, Madison, WI). To obtain the cNS2B sequence, PCR was carried out using the primers DEN1-4 NS2B forward (DEN1, 5′-TATGCTCGAGGCCGATTTATCACTGGAGAAA-3′; DEN2, 5′-TATACTCGAGGCTGATTTGGAACTGGAGAG-3′; DEN3, 5′-TATGCTCGAGGCGGACCTCACTGTAGAAAAA-3′; and DEN4, 5′-TATGCTCGAGGCAGACCTGTCACTAGAGAAG-3′; restriction enzyme sites are underlined) and DEN1-4 NS2B reverse (DEN1, 5′-CCCGCCTCCACCACTACCTCCGCCCCCGAGTGTGTCATCTCTCTCTTCAT-3′; DEN2, 5′-CCCGCCTCCACCACTACCTCCGCCCCCCAGTGTTTGTTCTTCTTCTTCA-3′; DEN3, 5′-CCCGCCTCCACCACTACCTCCGCCCCCTAGGATATTCTCAGTCTCATCAT-3′; and DEN4, 5′-CCCGCCTCCACCACTACCTCCGCCCCCTATCATATTGGTTTCCTCGATGT-3′). To obtain the NS3pro185 sequence, PCR was carried out using the primers DEN1-4 NS3 forward (DEN1, 5′-GGGGGCGGAGGTAGTGGTGGAGGCGGGTCAGGAGTGCTATGGGACAC-3′; DEN2, 5′-GGGGGCGGAGGTAGTGGTGGAGGCGGGGCCGGAGTATTGTGGGATGT-3′; DEN3, 5′-GGGGGCGGAGGTAGTGGTGGAGGCGGGTCCGGCGTTTTATGGGA CG-3′; and DEN4, 5′-GGGGGCGGAGGTAGTGGTGGAGGCGGGTCAGGAGCCCTGTGGGAC-3′) and DEN1-4 NS3 reverse (DEN1, 5′-ATCGATGATCATTACCTAAACACCTCGTCCTCAATC-3′; DEN2, 5′-TAATGGATCCTTACTTTCGAAAGATGTCATCTTCA-3′; DEN3, 5′-GGCGGATCCTTATGCATTTGTTTGCGCTATTCC-3′; and DEN4, 5′-GGCGGATCCTTACTTTCGAAAAATGTCCTCATCC-3′; restriction enzyme sites are underlined). DNA templates were either DEN1, DEN3, and DEN4 NS2B/NS3 PCR products or the plasmids pGEM-T-(E-NS3) for DEN2 cNS2B and pET15b-NS3NS5 for DEN2 NS3pro185. DEN1-4 CF40-Gly-NS3pro185 chimeric sequences were generated in an overlap PCR reaction with the two PCR products (cNS2B and NS3pro185) and the primers DEN1-4 NS2B forward and NS3 reverse. DEN1 CF40-Gly-NS3pro185 was digested with XhoI/BclI, and DEN2-4 CF40-Gly-NS3pro185 were digested with XhoI/BamHI. The constructs were then cloned into the XhoI/BamHI sites in pET15b. All constructs were verified by automated sequencing (PE Applied Biosystems, Foster City, CA). Expression and Purification of DEN 1-4 CF40-Gly-NS3pro185—Competent Escherichia coli BL21-CodonPlus-(DE3) (Stratagene) were transformed with pET15b-DEN 1-4 CF40-Gly-NS3pro185 expression vectors and grown in 500 ml Luria-Bertani broth containing ampicillin (100 μg/ml), chloramphenicol (50 μg/ml), and 0.2% (w/v) glucose at 37 °C with shaking until A595 reached ∼0.5. Cells were centrifuged in a Sorvall SLA 3000 rotor at 5000 × g for 10 min and resuspended in 500 ml of Luria-Bertani media with ampicillin and chloramphenicol. Cultures were induced with 0.4 mm isopropyl β-d-thiogalactopyranoside, and growth was continued for a further 16 h at 16 °C. The resulting cells were pelleted and resuspended in 30 ml of cold lysis buffer (50 mm HEPES, pH 7.5, 300 mm NaCl, and 5% glycerol). Cells were passed through a cell disruptor twice at 20,000 p.s.i. (Basic Z model; Constant Systems Ltd.), and debris was removed by centrifugation at 35,000 × g for 30 min. The protein solution was filtered by 0.22-μm filter and loaded onto a 5-ml HiTrap chelating heparin (Amersham Biosciences) column equilibrated with the lysis buffer. The resin was washed with 10 column volumes of lysis buffer before the bound proteins were eluted from the column with lysis buffer and a linear gradient of imidazole from 20-300 mm in the same buffer. The peak fractions were analyzed by 10% SDS-PAGE. The positive fractions were pooled, desalted, and concentrated with spin concentrators (Amicon Ultra-15 ml; Millipore, Billerica, MA) with a molecular mass cutoff of 10,000 Da. SDS-PAGE Gels and Western Analysis—Protein samples were resolved on a 12% SDS-polyacrylamide gel, transblotted onto Hybond-C membranes (Amersham Biosciences), blocked with 3% nonfat skim milk in phosphate-buffered saline, and then probed with anti-NS3 polyclonal (a gift from James Cook University) or anti-His monoclonal (1:1000 dilution; Qiagen, Valencia, CA) antibodies for 1 h at room temperature. After extensive washes in 0.05% Tween 20 in phosphate-buffered saline, a secondary anti-mouse antibody conjugated to horseradish peroxidase (1:5000 dilution; Sigma) was applied to the blots for at least 1 h at room temperature. Washes were repeated, and membrane-bound antibodies were detected with an ECL chemiluminescence kit (Amersham Biosciences). Profiling of P4-P1 and P1′-P4′ Specificities with Substrate Libraries—For P4-P1 substrate specificity determination, two-position fixed positional scanning tetrapeptide libraries were synthesized and assayed as described previously (12.Pinilla C. Appel J.R. Blanc P. Houghten R.A. BioTechniques. 1992; 13 (901-902): 904-905Google Scholar, 13.Rano T.A. Timkey T. Peterson E.P. Rotonda J. Nicholson D.W. Beckeer J.W. Chapman K.T. Thornberry N.A. Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 14.Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (466) Google Scholar, 15.Harris J.L. Alper P.B. Li J. Rechsteiner M. Backes B.J. Chem. Biol. 2001; 8: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Assays were carried out in 384-well plates on SpectraMax Gemini EM or XS microtiter plate reader (Molecular Device). The final reaction mixtures (30 μl) contained 50 mm Tris-HCl (pH 8.5), 20% glycerol, 1 mm CHAPS, and ∼150 μm total substrate. After the addition of enzymes (1-3 μm CF40-Gly-NS3pro185 proteases) to the tetrapeptide coumarin library, reaction mixtures were incubated at 37 °C, and the liberated coumarin fluorophore was monitored at a λex of 380 nm and a λem of 450 nm. Initial fluorescent velocities in relative fluorescent units per second were calculated as a fraction of the highest velocity in the library set and plotted into a two-dimensional format with DecisionSite (Spotfire). The octapeptide donor quencher positional scanning library was synthesized and assayed as described previously (16.Petrassi H.M. Williams J.A. Li J. Tumanut C. Ek J. Nakai T. Masick B. Backes B.J. Harris J.L. Bioorg. Med. Chem. Lett. 2005; 15: 3162-3166Crossref PubMed Scopus (39) Google Scholar). Briefly, CF40-Gly-NS3pro185 proteases (0.5-2 μm) were incubated in 96-well plates with 100 μl reaction containing the same buffer as described above with ∼100 μm total substrates (16.Petrassi H.M. Williams J.A. Li J. Tumanut C. Ek J. Nakai T. Masick B. Backes B.J. Harris J.L. Bioorg. Med. Chem. Lett. 2005; 15: 3162-3166Crossref PubMed Scopus (39) Google Scholar, 17.Shipway A. Danahay H. Williams J.A. Tully D.C. Backes B.J. Harris J.L. Biochem. Biophys. Res. Commun. 2004; 324: 953-963Crossref PubMed Scopus (64) Google Scholar). The reactions were monitored at a λex of 320 nm and a λem of 380 nm, and initial velocities were analyzed and graphed in DeltaGraph. Steady-state Kinetics of Fluorogenic and Chromogenic Peptide Substrates—Five fluorogenic tetrapeptide substrates with the 7-amino-3-carbamoylmethyl-4-methyl coumarin (ACMC) leaving group (Bz-Nle-Lys-Arg-Arg-ACMC, Bz-Nle-Lys-Thr-Arg-ACMC, Bz-Nle-Thr-Arg-Arg-ACMC, Bz-Thr-Lys-Arg-Arg-ACMC, and Bz-Thr-Thr-Arg-Arg-ACMC) were synthesized using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase peptide synthesis techniques (14.Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (466) Google Scholar, 15.Harris J.L. Alper P.B. Li J. Rechsteiner M. Backes B.J. Chem. Biol. 2001; 8: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The thiobenzyl ester substrate, Bz-Nle-Lys-Arg-Arg-SBzl, was purchased from Peptides International. After high performance liquid chromatography purification, the concentration of aliquots of each fluorogenic substrate was determined using total hydrolysis with trypsin, and the released ACMC fluorophore was read at a λex of 380 nm and a λem of 450 nm. The concentration of each substrate was then calculated with standard ACMC solutions. The concentration of the SBzl substrate was also determined using total hydrolysis with trypsin; the released SBzl moiety was monitored spectrophotometrically at 324 nm in the presence of 0.5 mm 4,4′-dithiodipyridine, and concentration was determined using the extinction coefficient of 19,800 m-1 cm-1 for the SBzl-thiopyridine conjugate. Active site titration for purified CF40-Gly-NS3pro185 proteases was performed by inhibition with freshly reconstituted aprotinin (18.Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd Ed. Wiley-VCH, Inc., New York2000: 313-317Google Scholar, 19.Kuzmic P. Sideris S. Cregar L.M. Elrod K.C. Rice K.D. Janc J.W. Anal. Biochem. 2000; 281: 62-67Crossref PubMed Scopus (51) Google Scholar). For kinetic studies, CF40-Gly-NS3pro185 proteases were incubated with various concentrations of individual ACMC, AMC, or SBzl peptide substrates at 37 °C. The proteolytic reaction was monitored as an increase in fluorescence at a λex of 380 nm and a λem of 450 nm for the ACMC and AMC substrates or an increase in absorbance at 324 nm in the presence of 0.5 mm 4,4′-dithiodipyridine for the SBzl substrate. Typical reaction mixtures (100 μl) contained 50 mm Tris-HCl, pH 8.5, 20% glycerol, 1 mm CHAPS, 10 nm enzyme, and fluorogenic/chromogenic peptide substrates ranging from 0.5 μm to 1 mm. Initial fluorescence or absorbance velocities (relative fluorescence units per minute or relative absorbance units per minute) were converted to m·s-1 from a standard ACMC or AMC calibration curve or to an extinction coefficient of 19,800 m-1 cm-1 for the SBzl-thiopyridine conjugate. The progression curves were fitted into a Michaelis-Menten equation by nonlinear regression using GraphPad Prism. Steady-state kinetic constants of each substrate were determined from duplicate measurements and reported as mean ± S.E. Model of Substrate Binding to the NS3pro Structure—The P4-P4′ octapeptide (Nle-Lys-Arg-Arg-Ser-Gly-Ser-Gly) was fitted to the active site of the enzyme using the crystal structure of the dengue NS3 protease complex with the mung bean Bowman-Birk inhibitor (Protein Data Bank code 1DF9) (20.Murthy H.M. Judge K. DeLucas L. Padmanabhan R. J. Mol. Biol. 2000; 301: 759-767Crossref PubMed Scopus (90) Google Scholar) as a guide. The side chains of residues 44-51 of the inhibitor, representing P4-P4′, were mutated to the sequence of the octapeptide and manually fitted to dengue NS3 protease with the molecular modeling program Maestro (Schrödinger LLC, Portland, OR), seeking to maximize electrostatic interactions, hydrogen bond formation, and hydrophobic interactions. The main chain coordinates were not moved, nor were any atoms of the protein altered. In Vitro Expression and Purification of DEN1-4 CF40-Gly-NS3pro185—Clum et al. showed that the expression of the core hydrophilic domain of the DEN2 New Guinea C strain NS2B (cNS2B; 40 amino acids) as an N-terminal fusion was sufficient for activating the NS3 protease domain (21.Clum S. Ebner K.E. Padmanabhan R. J. Biol. Chem. 1997; 272: 30715-30723Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The introduction of a flexible, protease-resistant, nine-amino acid linker (Gly4SerGly4) generated a soluble and catalytically active protease complex (11.Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). In the study presented here, similar constructs were expressed based on this strategy by cloning the chimeric CF40-Gly-NS3pro185 cDNAs derived from dengue serotypes 1-4 into the pET15b vector from Novagen (see "Experimental Procedures"). Expression of the recombinant DEN2 CF40-Gly-NS3pro185 protease as an N-terminal His tag fusion protein in E. coli followed by affinity purification led to high yields (typically 15-20 mg from a 1-liter culture) of soluble protein, of which >95% were full-length (Fig. 1). The identity of CF40-Gly-NS3pro185 was confirmed with Western analyses using anti-His and anti-NS3 antibodies (Fig. 1). The minor lower band detected by the anti-NS3 antibody (Fig. 1B, asterisk), but not by the anti-His antibody (Fig. 1C), is presumably the heterodimeric protein without the hexahistidine tag. CF40-Gly-NS3pro185 proteases for DEN1, DEN3, and DEN4 were similarly expressed and purified, except that the yield of the DEN4-derived enzyme was 10-fold lower (data not shown). Active site titration with aprotinin was used to accurately assess the active enzyme concentration (11.Leung D. Schroder K. White H. Fang N.X. Stoermer M.J. Abbenante G. Martin J.L. Young P.R. Fairlie D.P. J. Biol. Chem. 2001; 276: 45762-45771Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Aprotinin binds to the four CF40-Gly-NS3pro185 proteases with high affinity (Ki = 79, 25, 88, and 6.4 pm for DEN1-4 CF40-Gly-NS3por185, respectively). Characterization of Enzymatic Activity of DEN1-4 CF40-Gly-NS3pro185 on a Fluorogenic Tripeptide Substrate—The activities of the four CF40-Gly-NS3pro185 enzymes were characterized using the fluorogenic peptide Boc-GRR-AMC, which had been shown previously to be cleaved by the DEN2-NGC cNS2B/NS3 protease complex (10.Yusof R. Clum S. Wetzel M. Murthy H.M. Padmanabhan R. J. Biol. Chem. 2000; 275: 9963-9969Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The Km, Vmax, and kcat/Km or substrate specificity for DEN2 CF40-Gly-NS3pro185 using Boc-GRR-AMC were determined by varying the substrate concentration from 1000 to 10 μm using 12 serial dilutions (see "Experimental Procedures"). The steady-state kinetic parameters obtained were kcat = 0.13 ± 0.02 s-1, Km = 150 ± 15 μm, and kcat/Km = 840 ± 100 m-1 s-1 (Table I).Table ISteady-state kinetic parameters for the dengue CF40-Gly-NS3pro185 proteasesSerotypeSubstrateaSingle letter amino acid abbreviations are used in the middle sequences. The lowercase "n" stands for norleucine.kcatKmkcat/Kms−1μmm−1 s−1DEN1Boc-GRR-AMC0.03 ± 0.01120 ± 30290 ± 20Bz-nKRR-ACMC0.32 ± 0.016.2 ± 0.851,800 ± 5,500Bz-nKTR-ACMC0.97 ± 0.07202 ± 294,820 ± 370Bz-nTRR-ACMC0.28 ± 0.0271 ± 154,000 ± 600Bz-TKRR-ACMC0.05 ± 0.016.6 ± 1.57,520 ± 1,570Bz-TTRR-ACMC0.21 ± 0.03360 ± 77580 ± 60DEN2Boc-GRR-AMC0.13 ± 0.02150 ± 15840 ± 100Bz-nKRR-ACMC1.4 ± 0.112 ± 2112,100 ± 18,500Bz-nKTR-ACMC1.4 ± 0.134 ± 1040,300 ± 10,200Bz-nTRR-ACMC0.76 ± 0.0346 ± 616,700 ± 2,000Bz-TKRR-ACMC0.20 ± 0.0111 ± 118,300 ± 2,100Bz-TTRR-ACMC0.17 ± 0.0176 ± 92,200 ± 200DEN3Boc-GRR-AMC0.09 ± 0.02125 ± 16690 ± 150Bz-nKRR-ACMC0.61 ± 0.0212 ± 151,700 ± 5,700Bz-nKTR-ACMC1.1 ± 0.1180 ± 206,160 ± 370Bz-nTRR-ACMC0.58 ± 0.0253 ± 510,800 ± 800Bz-TKRR-ACMC0.10 ± 0.0113 ± 37,400 ± 1,300Bz-TTRR-ACMC0.33 ± 0.02220 ± 231,540 ± 90DEN4Boc-GRR-AMC0.07 ± 0.01180 ± 40380 ± 95Bz-nKRR-ACMC2.8 ± 0.17.3 ± 0.9380,000 ± 40,000Bz-nKTR-ACMC4.9 ± 0.252 ± 593,900 ± 7,100Bz-nTRR-ACMC1.1 ± 0.0272 ± 415,600 ± 600Bz-TKRR-ACMC0.43 ± 0.018.6 ± 0.749,200 ± 3,300Bz-TTRR-ACMC0.48 ± 0.07210 ± 602,250 ± 380Bz-nKRR-AMC2.9 ± 0.18.6 ± 1.8340,000 ± 72,000Bz-nKRR-SBzl300 ± 256.0 ± 2.950,200,000 ± 24,800,000a Single letter amino acid abbreviations are used in the middle sequences. The lowercase "n" stands for norleucine. Open table in a new tab The activities of the purified DEN1, DEN3, and DEN4 CF40-Gly-NS3pro185 were characterized using the same Boc-GRR-AMC fluorogenic peptide. All four proteases exhibited comparable Km values, but the kcat and kcat/Km values showed greater variations, with the values for DEN1 protease being lower and, hence, the least active (Table I). This observation was consistent also for a DEN1 protease cloned from a clinical isolate obtained during the Dengue fever outbreak in the Indonesian city of Jakarta (data not shown). Profiling of P4-P1 Specificities of DEN1-4 CF40-Gly-NS3pro185—Sequence analysis of the NS3 proteases from all four distinct serotypes indicated that they share greater than 60% identity in their primary sequences (Fig. 2). To explore the substrate structure-activity relationship, the P4-P1 substrate specificities of recombinant NS3 proteases from DEN1-4 were examined using tetrapeptide positional scanning synthetic combinatorial libraries of the general structure Ac-XXXX-7-amino-4-carbamoylmethyl coumarin (Fig. 3) (14.Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (466) Google Scholar, 15.Harris J.L. Alper P.B. Li J. Rechsteiner M. Backes B.J. Chem. Biol. 2001; 8: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 17.Shipway A. Danahay H. Williams J.A. Tully D.C. Backes B.J. Harris J.L. Biochem. Biophys. Res. Commun. 2004; 324: 953-963Crossref PubMed Scopus (64) Google Scholar). The tetrapeptide substrates were synthesized and assayed as mixtures of peptides in a positional scanning format where two positions were fixed with a specific amino acid and two positions were randomized with 19 amino acids (X represents all natural amino acids with the exception of Cys and Met and the inclusion of Nle). Specifically, the tetrapeptide substrates used in this study represented each combination of the P1 position fixed as a specific amino acid with either a fixed P2, a fixed P3, or a fixed P4 position for a total of 1083 wells (361 wells for P1 × P2, 361 wells for P1 × P3, and 361 wells for P1 × P4), with each well containing 361 substrates as a mixture (19 randomized amino acids × 19 randomized amino acids × 1 fixed amino acid × 1 fixed amino acid). The total number of substrates in the library was 130,321 (19 × 19 × 19 × 19). Cleavage of the peptide-7-amino-4-carbamoylmethyl coumarin bond results in an increase in fluorescence that can be directly monitored. The total concentration of substrates in each well was ∼150 or ∼0.4 μm for each substrate. The relative rates for the mixture of substrates are represented in a two-dimensional matrix, with each square in the matrix representing both the identity of the two fixed amino acids (x-axis and y-axis) and the relative activity as indicated on a gray scale in which white represents no activity and black represents the highest activity (Fig. 3B). The activity of the enzyme across all three sub-libraries (P1 × P2, P1 × P3, and P1 × P4) was normalized to the highest activity as indicated by the white-to-black scale below each of the two-dimensional graphs. The enzymatic activity was also represented in histogram form (Fig. 3B), where the P1 position is fixed as arginine, the
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