Title: Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions
Abstract: The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases. The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases. West Nile virus (WNV) 1The abbreviations used are: WNV, West Nile virus; pNA, p-nitroanilide; HCV, hepatitis C virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Rt, retention time. is a member of the Flavivirus genus and is transmitted by mosquitoes, primarily Culex species (1Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (164) Google Scholar), between avian reservoir hosts and vertebrate dead-end hosts including humans and horses. Many viruses within this genus are medically important pathogens, including dengue, Japanese encephalitis, tick-borne encephalitis, and yellow fever viruses. WNV was first isolated in 1937 in Uganda's West Nile province and was subsequently found in regions of Africa, the Middle East, Europe, Russia, South-western Asia, and Australia (less severe subtype, Kunjin) (2Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar). Human infection is generally asymptomatic or causes a mild febrile disease, West Nile fever (1Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (164) Google Scholar). However, in a small number of cases, predominantly in the elderly, the infection with WNV results in encephalitis or meningitis that can be fatal (1Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (164) Google Scholar). Over the last decade, there has been an increase in the frequency of human outbreaks and severity of disease with recent epidemics in Israel (1998), Romania (1999), Russia (1999), and New York (1999) (3Lanciotti R.S. Roehrig J.T. Deubel V. Smith J. Parker M. Steele K. Crise B. Volpe K.E. Crabtree M.B. Scherret J.H. Hall R.A. MacKenzie J.S. Cropp C.B. Panigrahy B. Ostlund E. Schmitt B. Malkinson M. Banet C. Weissman J. Komar N. Savage H.M. Stone W. McNamara T. Gubler D.J. Science. 1999; 286: 2333-2337Crossref PubMed Scopus (1283) Google Scholar). Since the introduction of WNV into New York in 1999, it has spread rapidly throughout the United States (4,156 infections and 284 deaths in 44 states in 2002, 9862 infections and 264 deaths in 2003) (reported in 2000 by the CDC, Division of Vector-Borne Infectious Disease: West Nile Virus, Center for Disease Control and Prevention, www.cdc.gov/ncidod/dvbid/westnile/index.htm), Canada, and Mexico and has recently appeared in the United Kingdom (5Buckley A. Dawson A. Moss S.R. Hinsley S.A. Bellamy P.E. Gould E.A. J. Gen. Virol. 2003; 84: 2807-2817Crossref PubMed Scopus (178) Google Scholar). There is currently no vaccine or antiviral treatment available for human WNV infection. However, a chimeric live vaccine is in clinical trials and a veterinary vaccine is licensed for use in equines and exotic zoo birds (6Tesh R.B. Arroyo J. Travassos Da Rosa A.P. Guzman H. Xiao S.Y. Monath T.P. Emerg. Infect. Dis. 2002; 8: 1392-1397Crossref PubMed Scopus (124) Google Scholar). Flaviviruses are small enveloped viruses containing a single-stranded positive sense RNA genome of 10–11 kb with a single large open reading frame encoding a polyprotein precursor of ∼3400 amino acids. Gene expression requires both host and a virally encoded protease to process the polyprotein precursor into the individual functional proteins. They comprise three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (7Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1596) Google Scholar). The viral protease encoded within the N-terminal third of NS3 is responsible for cleavage at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 gene junctions and also at a site near the C terminus of the C protein to promote efficient generation of prM (Fig. 1) (8Chambers T.J. Weir R.C. Grakoui A. McCourt D.W. Bazan J.F. Fletterick R.J. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8898-8902Crossref PubMed Scopus (287) Google Scholar, 9Lobigs M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6218-6222Crossref PubMed Scopus (123) Google Scholar). Mutation of residues at any of these cleavage sites in the related yellow fever virus was shown to prevent efficient cleavage and abolished virus infectivity in cell culture (10Amberg S.M. Rice C.M. J. Virol. 1999; 73: 8083-8094Crossref PubMed Google Scholar, 11Lin C. Chambers T.J. Rice C.M. Virology. 1993; 192: 596-604Crossref PubMed Scopus (38) Google Scholar, 12Nestorowicz A. Chambers T.J. Rice C.M. Virology. 1994; 199: 114-123Crossref PubMed Scopus (70) Google Scholar, 13Chambers T.J. Nestorowicz A. Rice C.M. J. Virol. 1995; 69: 1600-1605Crossref PubMed Google Scholar), highlighting a vital role for the NS3 protease in replication. The essential nature of this protease in the virus life cycle is the basis for interest in NS3 as a possible target for developing antiviral inhibitors. NS3 is a multifunctional protein in which the N-terminal 184 amino acids encode for the protease and the C-terminal region encodes a nucleotide triphosphatase, an RNA triphosphatase, and a helicase (14Gorbalenya A.E. Donchenko A.P. Koonin E.V. Blinov V.M. Nucleic Acids Res. 1989; 17: 3889-3897Crossref PubMed Scopus (204) Google Scholar, 15Wengler G. Virology. 1991; 184: 707-715Crossref PubMed Scopus (162) Google Scholar, 16Li H. Clum S. You S. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar). The NS3 protease is a trypsin-like serine protease with a classic catalytic triad (His-51, Asp-75, and Ser-135) (17Bazan J.F. Fletterick R.J. Virology. 1989; 171: 637-639Crossref PubMed Scopus (250) Google Scholar). Protease activity has been shown for a number of related flaviviruses to be dependent on the association of NS2B as a cofactor (18Falgout B. Pethel M. Zhang Y.M. Lai C.J. J. Virol. 1991; 65: 2467-2475Crossref PubMed Google Scholar). A central 40 amino acid hydrophilic domain within the largely hydrophobic NS2B protein has been shown to be sufficient for cofactor activity (19Leung 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 (278) Google Scholar, 20Falgout B. Miller R.H. Lai C.J. J. Virol. 1993; 67: 2034-2042Crossref PubMed Google Scholar). The flanking hydrophobic domains within NS2B are likely to function in promoting membrane association of NS2B-NS3 (21Clum S. Ebner K.E. Padmanabhan R. J. Biol. Chem. 1997; 272: 30715-30723Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 22Brinkworth R.I. Fairlie D.P. Leung D. Young P.R. J. Gen. Virol. 1999; 80: 1167-1177Crossref PubMed Scopus (89) Google Scholar). NS2B-NS3pro has high specificity for substrate processing requiring a dibasic recognition sequence (P2-Lys, P1-Arg) that is conserved throughout the flaviruses (7Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1596) Google Scholar). This relatively unusual substrate specificity suggests that inhibitors could be somewhat selective over most host serine proteases (19Leung 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 (278) Google Scholar). Fundamental to the design of a specific inhibitor is a detailed understanding of the interactions between protease residues in the active site and substrates. The structure of the WNV NS3 protease is unknown, but there are reported crystal structures for related NS3 proteases of hepatitis C virus (HCV) with a cofactor (23Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and Dengue-2 virus without NS2B cofactor (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Therefore, the latter structure was of an inactive enzyme. In this study, a homology-modeled structure of the WNV NS3 protease, created through sequence comparison with the HCV and Dengue-2 virus NS3 protease structures, was used to predict residues in the S1 and S2 binding pockets that probably make important interactions with substrate residues (25Nall T. Chappell K.J. Stoermer M.J. Fang N-X. Tyndall J.D.A. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Identified residues were then mutated in a recombinant catalytically active construct by site-directed mutagenesis, and kinetic parameters for the mutant WNV proteases were determined and compared. General Methods—Protected amino acids and resins were obtained from Auspep, Novabiochem, and PepChem. Trifluoroacetic acid, piperidine, N,N-diisopropylethylamine, dichloromethane, and N,N-dimethylformamide (peptide synthesis grade) were purchased from Auspep. All of the other materials were of reagent grade unless otherwise stated. Crude peptides were purified by reversed-phase high pressure liquid chromatography on a Vydac C18 column (10–15 μm, 300 Å, 50 × 250 mm) using a gradient mixture of solvent (A) 0.1% trifluoroacetic acid/water and (B) 0.1% trifluoroacetic acid/10% water/90% acetonitrile. Analytical reversed-phase high pressure liquid chromatography was performed on a Waters system equipped with a 717 plus autosampler, 660 controller, and a 996 photodiode array detector using a reversed-phase Phenomenex Luna C18 column (5 μm, 100 Å, 250 × 4.6 mm). Purified peptides were characterized by analytical reversed-phase high pressure liquid chromatography (linear gradient 0–100% B over 30 min), mass spectrometry, and 1H NMR spectroscopy. The molecular mass of the peptides was determined by electrospray mass spectrometry on a Micromass LCT mass spectrometer. 1H NMR spectra were recorded on samples containing 4 mm peptide in Me2SO-d6 (550 μl) on a Bruker Avance 600 spectrometer at 298 K. Proton assignments were determined by TOCSY (80 ms of mixing time), DQF-COSY, ECOSY, and NOESY (350 ms of mixing time) spectra using the sequential assignment method (26Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). All of the spectra were processed on Silicon Graphics R10000 or R12000 workstations using XWINNMR, version 2.6 (27Xwinnmr, Version 2.6, C. B. B. G., Rheinstetten, GermanyGoogle Scholar). Homology Modeling—West Nile virus NS3 protease homology models were generated using the structures of Dengue-2 virus NS3 protease without cofactor (Protein Data Bank 1BEF) (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and with Bowman-Birk inhibitor (Protein Data Bank 1DF9, subunit B) (29Murthy H.M. Judge K. DeLucas L. Padmanabhan R. J. Mol. Biol. 2000; 301: 759-767Crossref PubMed Scopus (90) Google Scholar) and hepatitis C virus NS3 protease with bound cofactor (Protein Data Bank 1A1Q) (23Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Margosiak S. Dagostino E. Hostomska Z. Clin. Diagn. Virol. 1998; 10: 151-156Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Models were generated using the Modeler and Homology modules within InsightII (31Gussio R. Pattabiraman N. Zaharevitz D.W. Kellogg G.E. Topol I.A. Rice W.G. Schaeffer C.A. Erickson J.W. Burt S.K. J. Med. Chem. 1996; 39: 1645-1650Crossref PubMed Scopus (59) Google Scholar) on a Silicon Graphics R10000 workstation. Sequences were aligned using Align 2D, Structure Alignment (Homology module, InsightII), and ClustalW alignment (32Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55766) Google Scholar). Secondary structure predictions were conducted using the web-based PsiPred prediction for each of the three NS3 protease sequences (33Jones D.T. J. Med. Biol. 1999; 292: 195-202Google Scholar). Electrostatic potential mapping was performed on the WNV NS3 protease model using the Delphi module in InsightII (34Hicks R.P. Mones E. Kim H. Koser B.W. Nichols D.A. Bhattacharjee A.K. Biopolymers. 2003; 68: 459-470Crossref PubMed Scopus (30) Google Scholar). A four residue P3-P1′ substrate based on the NS4B-NS5 cleavage site (Leu-Lys-Arg-Gly) was docked into the active site of a homology model of the WNV NS3 protease using GOLD, version 2.1 (35Jones G. Willett P. Glen R.C. J. Mol. Biol. 1995; 245: 43-53Crossref PubMed Scopus (1391) Google Scholar). A specified distance constraint between the substrate residue Arg (P1) and WNV protease Tyr-150 (S1) was used based on our understanding of protease-inhibitor interactions (36Fairlie D.P. Tyndall J.D.A. Reid R.C. Wong A.K. Abbenante G. Scanlon M.J. March D.R. Bergman D.A. Chai C.L.L. Burkett B.A. J. Med. Chem. 2000; 43: 1271-1281Crossref PubMed Scopus (145) Google Scholar, 37Leung D. Abbenante G. Fairlie D.P. J. Med. Chem. 2000; 43: 305-341Crossref PubMed Scopus (890) Google Scholar). 2Tyndall, J. D. A., Nall, T., and Fairlie, D. P. (2005) Chem. Rev., in press. Docked structures were ranked for best-fitting conformations using GOLD, version 2.1 (35Jones G. Willett P. Glen R.C. J. Mol. Biol. 1995; 245: 43-53Crossref PubMed Scopus (1391) Google Scholar). Plasmid Construction—The expression plasmid pQE9 WNV CF40.Gly.NS3pro generated previously (25Nall T. Chappell K.J. Stoermer M.J. Fang N-X. Tyndall J.D.A. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) from WNV (strain NY99–4132) was used as a template for generating site-directed mutant constructs. Mutations (V115A, V115F, D129A, D129E, D129N, S135A, Y150A, Y150F, N152A, S160A, and S163A) in WNV NS3 were made using overlapping PCR. In the first round of PCR, two separate PCR reactions were performed with the primer pairs WNV CF40aa.FBamHI/WNV Mutation.R and WNV Mutation.F/WNV NS3pro.RHindIII to amplify products flanked by overlapping sequences incorporating the site-directed mutation. Table I shows the primer sequences. These products were then joined in the second round of PCR using the outer primers WNV CF40aa.FBamHI and WNV NS3pro.RHindIII. The resulting PCR products were digested with BamHI and HindIII and cloned into pQE9. The incorporation of the desired mutations was confirmed by automated sequence analysis.Table IPrimers used for construction of WNV CF40.Gly.NS3pro and site-directed mutantsPrimerSequenceWNV CF40aa.F′ (BamHI)5′-CGATGACGGCGGATCCACAGATATGTGGA TTGAGAGAACG-3′WNV NS3pro.R′ (HindIII)5′-GCCCCCAAGCTTACAGCATCTCAGGTTCGAAT-3′WNV NS3_V115A.F′5′-ACGAAACCAGGAGCGTTCAAAACACCT-3′WNV NS3_V115A.R′5′-AGGTGTTTTGAACGCTCCTGGTTTCGT-3′WNV NS3_V115F.F′5′-ACGAAACCAGGGTTCTTCAAAACACCT-3′WNV NS3_V115F.R′5′-AGGTGTTTTGAAGAACCCTGGTTTCGT-3′WNV NS3_D129A.F′5′-CCGTGACTTTGGCATTCCCCACTGGAA-3′WNV NS3_D129A.R′5′-TTCCAGTGGGGAATGCCAAAGTCACGG-3′WNV NS3_D129E.F′5′-GCCGTGACTTTGGAGTTCCCCACTGGA-3′WNV NS3_D129E.R′5′-TCCAGTGGGGAACTCCAAAGTCACGGC-3′WNV NS3_D129N.F′5′-GCCGTGACTTTGAACTTCCCCACTGGA-3′WNV NS3_D129N.R′5′-TCCAGTGGGGAAGTTCAAAGTCACGGC-3′WNV NS3_S135A.F′5′-CCCATCGGAACAGCAGGCTCACCAATA-3′WNV NS3_S135A.R′5′-TATTGGTGAGCCTGCTGTTCCAGTGGG-3′WNV NS3_Y150A.F′5′-GTGATTGGACTGGCTGGCAATGGAGTC-3′WNV NS3_Y150A.R′5′-GACTCCATTGCCAGCCAGTCCAATCAC-3′WNV NS3_Y150F.F′5′-GTGATTGGACTGTTTGGCAATGGAGTC-3′WNV NS3_Y150F.R′5′-GACTCCATTGCCAAACAGTCCAATCAC-3′WNV NS3_N152A.F′5′-GGGCTTTATGGAGCTGGAGTCATAATG-3′WNV NS3_N152A.R′5′-CATTATGACTCCAGCTCCATAAAGCCC-3′WNV NS3_S160A.F′5′-ATGCCCAACGGAGCATACATAAGTGCG-3′WNV NS3_S160A.R′5′-CGCACTTATGTATGCTCCGTTGGGCAT-3′WNV NS3_S163A.F′5′-GGCTCATACATAGCAGCGATAGTGCAG-3′WNV NS3_S163A.R′5′-CTGCACTATCGCTGCTATGTATGAGCC-3′ Open table in a new tab Enzyme Expression and Purification—The pQE9 vector was used for high level inducible expression of N-terminal hexahistidine-tagged recombinant proteins. Cultures of the Escherichia coli strain SG13009 transformed with the expression plasmid were grown in 500 ml of LB medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin at 37 °C until the A600 nm reached 0.5. The expression of the recombinant protein was induced by the addition of isopropyl-β-d-thiogalactopyranose to a final concentration of 0.3 mm and incubated for an additional 3 h at 22 °C. Cells were harvested by centrifugation and stored at -20 °C. For protein purification, cell pellets were thawed, resuspended in lysis buffer (5 ml/g wet pellet, 50 mm HEPES, pH 7.5, 300 mm NaCl, 10 mm imidazole, 5% glycerol) and protease inhibitors were added (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml benzamide, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml TCLK, all final concentrations) in an attempt to minimize proteolytic cleavage of recombinant protein. Subsequent analyses have found that these standard serine protease inhibitors do not inhibit WNV CF40.Gly.NS3pro (25Nall T. Chappell K.J. Stoermer M.J. Fang N-X. Tyndall J.D.A. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Resuspended cells were lysed by three passages through a French press (1000 Pascals) and then centrifuged (27,000 × g, 20 min, 4 °C). The recombinant proteases, each with a N-terminal hexahistidine tag, were purified by affinity chromatography on a Ni2+-nitrilotriacetic acid-agarose 1-cm column (Qiagen) that had been preequilibrated (50 mm HEPES buffer (30 ml), pH 7.5, 300 mm NaCl, 10 mm imidazole, 5% glycerol). Resin was then removed, mixed with the supernatant fraction of cell lysates, and incubated at 4 °C on a rotator for 30 min to allow the His-tagged protein to bind to the Ni2+ resin. The column was repacked and washed with 30 ml of buffer containing 20 mm imidazole, and the proteins were eluted into 6 × 1-ml fractions with buffer containing 100 mm imidazole. The pre- and post-isopropyl-β-d-thiogalactopyranose induction samples, soluble and insoluble fractions, and the elution fractions were analyzed by 12% SDS-PAGE. Substrate—The pNA substrate corresponding to the native WNV NS3 protease cleavage site Ac-KPGLKR-pNA (NS4A-NS5) was synthesized by a previously reported method (39Abbenante G. Leung D. Bond T. Fairlie D.P. Lett. Pept. Sci. 2001; 7: 347-351Google Scholar) (Ac-KPGLKR-pNA, Rt = 16.9 min). Electrospray mass spectrometry was [M + H] = 860.7 and [M + 2H]/2 = 430.8. The NMR spectral data are as follows: 1H NMR (Me2SO-d6) δ 10.58 (s, 1H, pNA-NH); 8.23 (d, 2H, pNA-ArH); 8.19 (t, 1H, GlyαNH); 8.18 (d, 1H, ArgαNH); 8.05 (d, 1H, Lys-1αNH); 7.95 (d, 1H, Lys-5αNH); 7.84 (d, 2H, pNA-ArH); 7.76 (d, 1H, LeuαNH); 7.68 (m, 6H,2× LyszNH3); 7.7–6.5 (4H,m,2× ArgNH2); 7.61 (t,1H, Arg-6ϵNH); 4.47 (m, 1H, Lys-1αCH); 4.36 (m, 1H, ArgαCH); 4.30 (m, 1H, LeuαCH); 4.26 (m, 1H, ProαCH); 4.24 (m, 1H, Lys-5αCH); 3.68 (m, 2H, GlyαCH2); 3.67 (m, 1H, ProδCH); 3.56 (m, 1H, ProδCH); 3.11 (m, 2H, ArgδCH2); 2.75 (m, 2H, Lys1ϵCH2); 2.73 (m, 2H, Lys-5ϵCH2); 2.05 (m, 1H, ProβCH); 1.93 (m, 1H, ProγCH); 1.85 (m, 1H, ProγCH); 1.82 (s, 3H, Ac-CH3); 1.80 (m, 1H, ProβCH); 1.75 (m, 1H, ArgβCH); 1.66 (m, 1H, Lys-5βCH); 1.65 (m, 1H, ArgβCH); 1.63 (m, 1H, Lys-1βCH); 1.56 (m, 1H, Lys-5βCH); 1.56 (m, 1H, ArgγCH); 1.56 (m, 1H, LeuγCH); 1.53 (m, 1H, LysγCH); 1.51 (m, 2H, Lys-1δCH2); 1.50 (m, 2H, Lys-5δCH2); 1.49 (m, 1H, Lys-1βCH); 1.49 (m, 1H, Lys-1γCH); 1.48 (m, 1H, LeuβCH); 1.47 (m, 1H, ArgγCH); 1.43 (m, 1H, LeuβCH); 1.32 (m, 1H, Lys-1γCH); 1.32 (m, 1H, Lys-5γCH); 0.87 (d, 3H, LeuδCH3); and 0.83 (d, 3H, LeuδCH3). High resolution electrospray mass spectrometry was calculated for C39H65N13O9 ([M + H]+ = 860.5101, found 860.5061; [M + 2H]/2 = 430.7587, found 430.7617). Enzyme Kinetics—The recombinant WNV protease (WNV CF40.Gly.NS3pro) and site-directed mutants (V115A, V115F, D129A, D129E, D129N, S135A, Y150A, Y150F, N152A, S160A, and S163A) were assayed against a hexapeptide substrate corresponding to P6-P1 of the known endogenous cleavage site NS4B-NS5 (Ac-KPGLKR-pNA) with a chromogenic pNA group in the P1′ position. The cleavage of the pNA chromophore from the peptide substrate by the WNV proteases produced a detectable color change at 405 nm, allowing protease activity to be measured. The assay was conducted in a 96-well plate with a final reaction volume of 200 μl containing 0.5 μm recombinant protease and 1 mm Ac-KPGLKR-pNA and with optimal processing conditions described by Nall et al. (25Nall T. Chappell K.J. Stoermer M.J. Fang N-X. Tyndall J.D.A. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) consisting of a final concentration of 50 mm Tris, pH 9.5, 30% glycerol, 1 mm CHAPS. After preincubation of buffered protease and substrate in separate wells (10 min, 37 °C), catalysis was initiated by mixing substrate with enzyme-buffer solution by automatic shaking. The optical density was measured at 405 nm every 30 s for 10 min in a SpectraMax 250 reader, and the average change in millioptical density/min was calculated. Assays were carried out in triplicate, and a control assay containing no enzyme was also conducted. Those proteases that were found to possess significant activity (greater than the negative control) were re-assayed in triplicate against eight different substrate concentrations ranging from 62.5 μm to 1 mm for determination of kinetic constants. To obtain accurate kinetic data for the D129A site-directed mutant, which possessed only slightly higher activity than the negative control, it was assayed in quadruplicate at the higher enzyme concentration of 2.5 μm and eight higher substrate concentrations ranging from 125 μm to 2 mm. Kinetic parameters were calculated from weighted non-linear regression of the initial velocities as a function of the eight substrate concentrations using GraphPad Prism 4 software. The kcat/Km values were calculated assuming that Michaelis-Menten kinetics v = Vmax[S]/([S] + Km). Triplicate measurements were taken for each data point. The data are reported as the means ± S.E. Protease Architecture and Design of Mutant Proteases—A structural homology model of the WNV NS3pro (25Nall T. Chappell K.J. Stoermer M.J. Fang N-X. Tyndall J.D.A. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) was reconstructed here based on the sequence alignments and the crystal structures of Dengue-2 virus NS3pro in complex with a Bowman-Birk inhibitor (29Murthy H.M. Judge K. DeLucas L. Padmanabhan R. J. Mol. Biol. 2000; 301: 759-767Crossref PubMed Scopus (90) Google Scholar) and of HCV NS3pro in complex with its cofactor NS4A (Fig. 2) (28Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar). The HCV NS3pro structure was included to enable the modeling of the WNV NS3pro in the context of its NS2B cofactor. The electrostatic surface map of WNV NS3pro is shown in Fig. 2A, highlighting acidic (red), basic (blue), and hydrophobic (white) regions with a previously reported substrate (P3-P1′ section, Leu-Lys-Arg-Gly corresponding to the WNV NS4B/NS5 cleavage site) docked in the active site. Based on homology with HCV NS3pro, the cofactor threads through the protease with the basic region in the upper left quadrant of Fig. 2A showing one end of the putative cofactor-binding domain. The substrate-binding active site is located in or on a largely hydrophobic region of the enzyme flanked by two acidic regions at Asp-129 and Asp-75 (shown in Fig. 2B). The model revealed that the S1 site was the largest pocket, that the S2 and subsites beyond were very shallow, and that the substrate must spreadeagle itself on the surface of the protease (Fig. 2B). Residues Val-115, Asp-129, Tyr-150, Ser-160, and Ser-163 are predicted to line the enzyme S1 subsite, whereas Asn-152 is predicted to be a key residue in the S2 subsite (Fig. 2, C and D), and these were chosen for site-directed mutagenesis. The S1 pocket was the major focus of our mutagenesis study as it appears to be the most promising site for targeting with a competitive inhibitor. Tyr-150 and Ser-163 are analogous to residues in the S1 subsite of the Dengue-2 virus NS3 protease that have been suggested to interact with P1 substituents of its substrate (24Murthy H.M. Clum S. Padmanabhan R. J. Biol. Chem. 1999; 274: 5573-5580Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 29Murthy H.M. Judge K. DeLucas L. Padmanabhan R. J. Mol. Biol. 2000; 301: 759-767Crossref PubMed Scopus (90) Google Scholar), and Tyr-150 has been identified by mutagenesis as important for the activity of Dengue-2 virus protease activity (38Valle R.P. Falgout B. J. Virol. 1998; 72: 624-632Crossref PubMed Google Scholar). Therefore, we decided to make the corresponding Y150A and Y150F mutant WNV NS3 proteases to test whether a π-cation interaction with the P1 Arg is crucial for substrate recognition and processing. The S160A and S163A mutants were made to test the possible hydrogen-bonding interaction of these residues in S1 with Arg at P1 of the substrate. Asp-129 is highly conserved among the flaviviruses and corresponds to Asp-189 of trypsin, a critical residue that sits at the base of the substrate binding pocket and forms an electrostatic bond with the P1 Lys or Arg of the trypsin cleavage site (30Graf L. Craik C.S. Patthy A. Roczniak S. Fletterick R.J. Rutter W.J. Biochemistry. 1987; 26: 2616-2623Crossref PubMed Scopus (138) Google Scholar). However, as noted above, Tyr-150 takes up this position in the flavivirus NS3 protease with Asp-129 located more peripherally at the outer edge of the S1 site (see Fig. 2A). Despite its high level of conservation across the flaviviruses, previous cell-based mutagenesis studies appeared to show that Asp-129 could be relatively freely substituted with retention of significant activity (38Valle R.P. Falgout B. J. Virol. 1998; 72: 624-632Crossref PubMed Google Scholar). In addition, crystallographic data for the Dengue-2 virus NS3 suggest that Asp-129 does not appear to interact with the P1 Lys but to only interact with a P1 Arg in one of two conformations (29Murthy H.M. Judge K. DeLucas L. Padmanabhan R. J. Mol. Biol. 2000; 301: 759-767Crossref PubMed Scopus (90) Google Scholar). We decided to test more accurately the importance of Asp-129 for substrate processing by making three mutant WNV CF40.Gly.NS3pro enzyme constructs (D129A, D129E, and D129N). Conservative mutants D129E (no change in charge) and D129N (isosteric replacement but loss of charge) together with D129A (loss of charge and bulk) at S1 would clarify whether a charged group is important for interaction with Arg at P1 of the substrate. The mutations of V115F and V115A were made to clarify the importance of Tyr-150 and determine the actual size of the S1 pocket. The rationale behind these two mutations was that V115F could fill the space available for the binding of a basic residue while being