Title: Insights to Substrate Binding and Processing by West Nile Virus NS3 Protease through Combined Modeling, Protease Mutagenesis, and Kinetic Studies
Abstract: West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection. West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection. West Nile virus (WNV) 4The abbreviations used are: WNV, West Nile virus; Aib, 2-aminoisobutyric acid; Aoc, l-2-aminooctanoic acid; BSQV, Bussuquara virus; Cha, l-cyclohexylalanine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Chg, l-cyclohexylglycine; Cit, l-citrulline; hArg, l-homoarginine; JE, Japanese encephalitis virus; MVE, Murray Valley encephalitis; Nle, l-norleucine; Nva, l-norvaline; Orn, l-ornithine; pNA, para-nitroanilide; Tbg, L-tert-butylglycine; YF, yellow fever virus; ZIKV, Zika virus; Den, dengue virus; SLEV, St. Louis encephalitis virus; LGTV, Langat virus; TBE, tick-borne encephalitis virus. 4The abbreviations used are: WNV, West Nile virus; Aib, 2-aminoisobutyric acid; Aoc, l-2-aminooctanoic acid; BSQV, Bussuquara virus; Cha, l-cyclohexylalanine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Chg, l-cyclohexylglycine; Cit, l-citrulline; hArg, l-homoarginine; JE, Japanese encephalitis virus; MVE, Murray Valley encephalitis; Nle, l-norleucine; Nva, l-norvaline; Orn, l-ornithine; pNA, para-nitroanilide; Tbg, L-tert-butylglycine; YF, yellow fever virus; ZIKV, Zika virus; Den, dengue virus; SLEV, St. Louis encephalitis virus; LGTV, Langat virus; TBE, tick-borne encephalitis virus. is a member of the Flavivirus genus, which contains many significant human pathogens, including dengue virus (Den), Japanese encephalitis virus (JE), and yellow fever virus (YF), and was first isolated in 1937 from Uganda's West Nile province. WNV has subsequently been found in regions of Africa, the Middle East, Europe, Russia, western Asia, and Australia (less severe subtype Kunjin) and most recently in North America (1van der Pensaert M.B. Meulen K.M. Nauwynck H.J. Arch Virol. 2005; 150: 637-657Crossref PubMed Scopus (123) Google Scholar). WNV is transmitted by Culex mosquitoes from avian reservoir hosts to vertebrate dead end hosts, including humans and horses (2Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (163) Google Scholar). Human infection is generally asymptomatic or causes a mild febrile disease, West Nile fever. However, more recent infections of WNV have also been associated with higher rates of severe neurological disease and fatalities, particularly among the elderly (2Hayes C.G. Ann. N. Y. Acad. Sci. 2001; 951: 25-37Crossref PubMed Scopus (163) Google Scholar). Since the introduction of WNV into New York in 1999, the virus has spread rapidly throughout North America, infecting over 19,000 people and causing more than 700 fatalities (see the Center for Disease Control and Prevention site on the World Wide Web at www.cdc.gov/ncidod/dvbid/westnile/index.htm). Currently there is no vaccine or antiviral therapy for the prevention or treatment of human WNV infection (1van der Pensaert M.B. Meulen K.M. Nauwynck H.J. Arch Virol. 2005; 150: 637-657Crossref PubMed Scopus (123) Google Scholar). WNV is a small, enveloped virus with a single-stranded, positive sense 11-kb RNA genome, which encodes a single polyprotein precursor. This polyprotein must be cleaved co- and post-translationally to produce 10 functional proteins: three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Translation of the viral polyprotein is membrane-associated with host proteases cleaving junctions within the lumen of the endoplasmic reticulum and the Golgi, whereas a viral protease encoded within NS3 cleaves at the junctions NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 and also internal sites within C, NS3, and NS4A (Fig. 1) (3Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (303) Google Scholar). Cleavage at these sites by the NS3 protease is essential for viral replication, so the protease is a potential therapeutic target (4Tyndall J.D. Nall T. Fairlie D.P. Chem. Rev. 2005; 105: 973-999Crossref PubMed Scopus (345) Google Scholar, 7Nall T.A. Chappell K.J. Stoermer M.J. Fang N.X. Tyndall J.D. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). NS3 is a multifunctional protein, the protease comprising the N-terminal third and nucleotide triphosphatase, RNA triphosphatase, and helicase components comprising the remainder (8Gorbalenya A.E. Donchenko A.P. Koonin E.V. Blinov V.M. Nucleic Acids Res. 1989; 17: 3889-3897Crossref PubMed Scopus (203) Google Scholar, 10Li H. Clum S. You S. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar). NS3 is a trypsin-like serine protease with a classical catalytic triad (His-51, Asp-75, Ser-135) (11Bazan J.F. Fletterick R.J. Virology. 1989; 171: 637-639Crossref PubMed Scopus (247) Google Scholar) and is highly specific for substrates with dibasic P1 and P2 components and a small amino acid at P1′. This recognition sequence is highly conserved throughout flaviruses (12Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1578) Google Scholar); however, different flaviviruses prefer either Lys or Arg at P2. Although Den and YF NS3 proteases predominantly recognize Arg at P2, WNV protease recognizes Lys at P2 (Table 1). The activity of flavivirus NS3 proteases is dependent on an NS2B cofactor, with truncation studies in Den2 having shown that a central 40-amino acid hydrophilic domain is sufficient for activity (13Falgout 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 (14Clum S. Ebner K.E. Padmanabhan R. J. Biol. Chem. 1997; 272: 30715-30723Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).TABLE 1Flavivirus cleavage sequences and cofactor homology Mosquito-borne flaviviruses include West Nile virus (WNV), St. Louis encephalitis (SLEV), dengue virus subtypes 1-4 (Den1, Den2, Den3, Den4), yellow fever virus (YF), Japanese encephalitis virus (JE), Murray Valley encephalitis (MVE), Zika virus (ZIKV), and Bussuquara virus (BSQV). Tick-borne flaviviruses include Langat virus (LGTV) and tick-borne encephalitis virus (TBE). The first column shows the alignment of the region of NS2B involved in substrate binding, whereas the next four columns show the native flavivirus cleavage sequences (P4-P1′). The final column shows the degree of homology between the various flaviviruses and the WNV NS2B 40-amino acid cofactor domain and the NS3 protease domain. Residues shown in green and yellow designate homology. The residue shown in boldface type and designated by the asterisk is believed to interact with P2. A P2 arginine residue is shown in blue, and a P2 lysine is shown in red. Due to their pivotal roles in both normal physiology and disease, proteases are increasingly attracting interest as pharmaceutical targets (15Leung D. Abbenante G. Fairlie D.P. J. Med. Chem. 2000; 43: 305-341Crossref PubMed Scopus (887) Google Scholar). Since early successes in human immunodeficiency virus chemotherapy (human immunodeficiency virus-protease inhibitors) and in the treatment of high blood pressure (angiotensin-converting enzyme (ACE) inhibitors), a large number of new protease inhibitors have entered clinical trials (16Abbenante G. Fairlie D.P. Med. Chem. 2005; 1: 71-104Crossref PubMed Scopus (246) Google Scholar). One reason for the drug potential of proteases is the relatively predictable way in which they recognize their substrates and inhibitors in extended (β-strand) conformations (4Tyndall J.D. Nall T. Fairlie D.P. Chem. Rev. 2005; 105: 973-999Crossref PubMed Scopus (345) Google Scholar, 17Fairlie 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). There are no known examples of proteolytic processing of peptide α-helices, β-sheets, or β-turns. Greater access to three-dimensional structures for proteases (over 1500 in the Protein Data Bank) has also facilitated hybrid structure/substrate-based drug design (5Loughlin W.A. Tyndall J.D.A. Glenn M.P. Fairlie D.P. Chem. Rev. 2004; 104: 6085-6117Crossref PubMed Scopus (207) Google Scholar). Recently reported crystal structures for NS2B/NS3 proteases of both WNV and Den2 (6D'Arcy A. Chaillet M. Schiering N. Villard F. Pheng Lim S. Lefeuvre P. Erbel P. Acta Crystallogr. Sect. F. 2006; 62: 157-162Crossref PubMed Scopus (21) Google Scholar) provide new structural insights to flaviviral proteases in ligand-bound conformations. An earlier homology model of the WNV protease (7Nall T.A. Chappell K.J. Stoermer M.J. Fang N.X. Tyndall J.D. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), derived from the crystal structures of a highly homologous dengue NS3 protease without NS2B cofactor (18Murthy H.M. Judge K. DeLucas L. Padmanabhan R. J. Mol. Biol. 2000; 301: 759-767Crossref PubMed Scopus (90) Google Scholar) and a less homologous hepatitis NS3 with bound NS4A cofactor (19Kim 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 (673) Google Scholar), differs significantly from the crystal structure of WNV NS2B-NS3. This has prompted a reexamination now of some of the previous mutagenesis data (20Chappell K.J. Nall T.A. Stoermer M.J. Fang N.X. Tyndall J.D. Fairlie D.P. Young P.R. J. Biol. Chem. 2005; 280: 2896-2903Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The reported WNV protease crystal structure shows an N-capped tetrapeptide aldehyde inhibitor bound in the substrate-binding cleft as a loop (instead of the commonly observed β-strand conformation) with the "P5" capping benzoyl residue sitting on top of the P1 residue. It therefore seemed unlikely that this ligand had the same binding mode as substrates beyond P1 and P2. In previous kinetic studies using short hexapeptide p-nitroanilide substrates derived from endogenous polypeptide cleavage sites, we found no preference of WNV protease for specific residues except at P1 and P2 (7Nall T.A. Chappell K.J. Stoermer M.J. Fang N.X. Tyndall J.D. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). However, more recent studies using nonnative dengue hexapeptide and decapeptide 5L. Juliano, personal communication. 5L. Juliano, personal communication. and tetrapeptide and octapeptide (21Li J. Lim S.P. Beer D. Patel V. Wen D. Tumanut C. Tully D.C. Williams J.A. Jiricek J. Priestle J.P. Harris J.L. Vasudevan S.G. J. Biol. Chem. 2005; 280: 28766-28774Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) substrate sequences have suggested opportunities for enhancing substrate affinity for flaviviral proteases using nonnative or nonproteinogenic amino acids with hydrophobic (Nle, Leu) residues at P4 and a basic (Lys > Arg) residue at P3, a feature not seen in suboptimal native sequences. Using a combination of computer docking of substrates into the enzyme crystal structure, site-directed mutagenesis of the protease (Fig. 1), and kinetic studies of the processing of tetrapeptide substrates, we have focused the present study on increasing substrate affinity and processing efficiency, identifying the enzyme residues likely to be involved in binding to the P2-P4 positions of substrates, and taking early steps toward potent substrate-based nonpeptidic inhibitors by incorporating unnatural amino acids in tetrapeptide substrates. para-Nitroanilide (pNA) Substrate Synthesis—pNA substrates were synthesized according to the general method of Abbenante et al. (22Abbenante G. Leung D. Bond T. Fairlie D.P. LIPS. 2001; 7: 347-351Google Scholar) and characterized by analytical high performance liquid chromatography, mass spectrometry, and NMR (see supplemental material). Modeling of Substrates into the NS3 Crystal Structure—The crystal structure of West Nile virus NS2B/NS3 protease (Protein Data Bank code 2fp7) was prepared for docking by adding protons using InsightII (version 2000; Accelrys Inc.). Substrates were assembled using the Biopolymer and Sketcher modules within InsightII and minimized using Discover. All substrate docking experiments were conducted using GOLD version 2.1.2 (23Jones G. Willett P. Glen R.C. J. Mol. Biol. 1995; 245: 43-53Crossref PubMed Scopus (1379) Google Scholar). Hydrogen bonding and distance constraints were used to align the substrate within the active site as follows. The P1 Arg residue was positioned as observed for the corresponding residue in the aldehyde inhibitor complex (Protein Data Bank code 2fp7) (24Erbel P. Schiering N. D'Arcy A. Renatus M. Kroemer M. Lim S.P. Yin Z. Keller T.H. Vasudevan S.G. Hommel U. Nat. Struct. Mol. Biol. 2006; 13: 372-373Crossref PubMed Scopus (423) Google Scholar), using a distance constraint of 3.5 ± 1.5 Å between the Arg ζ-carbon and the aromatic γ-carbon of Tyr-161 in the S1 pocket. This positions the positive charge of the arginine optimally for a π-cation interaction but also enables a charge-charge interaction with Asp129. A hydrogen bond between the P2 Lys z-NH3+ and the Asn-152 side-chain carbonyl oxygen was used to anchor P2 in the shallow solvent-exposed S2 pocket, as predicted in earlier modeling work (7Nall T.A. Chappell K.J. Stoermer M.J. Fang N.X. Tyndall J.D. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and later verified experimentally (24Erbel P. Schiering N. D'Arcy A. Renatus M. Kroemer M. Lim S.P. Yin Z. Keller T.H. Vasudevan S.G. Hommel U. Nat. Struct. Mol. Biol. 2006; 13: 372-373Crossref PubMed Scopus (423) Google Scholar). Hydrogen bond constraints for critical substrate backbone-enzyme interactions between NS3-Gly-153 carbonyl oxygen and substrate P3 NH, Gly-153 NH and substrate P3 carbonyls, and Gly-151 carbonyl oxygen and Arg P1 αNH were also used. No constraints on the position of P3 and P4 side chains were used. For the larger 2-naphthoyl residue in 2-naphthoyl-KKR-pNA, the docking (Fig. 2) produced poses with a high degree of steric clash in the vicinity of S4. In these cases, the docking poses were minimized with the enzyme backbone either fixed or tethered and the enzyme side chains and the entire substrate allowed to move using Discover (Accelrys). Plasmid Construction—The expression plasmid pQE9 WNV CF40.Gly.NS3pro, previously generated from WNV (strain NY99-4132) (10Li H. Clum S. You S. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar), was used as a template for site-directed mutagenesis. The site-directed mutations in NS2B (V75A, V75F, N84A, N84D, N84E, N84L, N84S, Q86A, Q86E, Q86L, L87A, and L87F) and NS3 (T111F, T111L, V154F, V154L, I155F, M156A, I162F, A164S, A164V, and V166L) were inserted by amplifying the entire plasmid with PCR using Phusion™ polymerase (Finnzymes) together with a pair of partially overlapping primers (supplemental material). The template plasmid was digested with DpnI at 37 °C for 1 h. The PCR-amplified plasmid was then used to transform Escherichia coli strain XL10 Gold competent cells, which were grown in the presence of 100 μg/ml ampicillin, the expression plasmid was purified, and sequences were confirmed by automated analysis. Enzyme Expression and Purification—The pQE9 vector was used to allow high level, inducible expression of N-terminal His6-tagged recombinant proteins. Cultures of E. coli strain SG13009 transformed with the expression plasmids containing the site-directed mutations were grown in 2 × 25 ml of LB medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin at 37 °C until the A600 reached 0.5. Expression of the recombinant protein was induced by the addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 0.3 mm and incubated for an additional 3 h at 22°C. Cells were then harvested by centrifugation at 4500 × g for 10 min and stored at -20 °C. For protein purification, the cell pellets were thawed and resuspended in 1 ml of lysis buffer (50 mm HEPES, pH 7.5, 300 mm NaCl, 10 mm imidazole, 5% glycerol). To prevent proteolytic cleavage of protein during lysis and purification, the following protease inhibitors were added to give the final concentrations of 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml benzamidine, and 1 mm phenylmethylsulfonyl fluoride. Resuspended cells were lysed by sonication, and insoluble products were pelleted by centrifugation at 27,000 × g for 20 min. The recombinant proteases were purified by affinity chromatography using an N-terminal His6 tag on Ni2+ nitrilotriacetic acid-agarose. Resin (0.5 ml) was pre-equilibrated with 10 ml of column buffer (50 mm HEPES, pH 7.5, 300 mm NaCl, 10 mm imidazole, 5% glycerol), and then the resin was removed and mixed with the supernatant of the cell lysates. These mixtures were incubated at 4 °C on a shaker for 30 min to allow the His-tagged protein to bind to the Ni2+ column. Resin was pelleted at low speed (100 × g), and the buffer was removed. The resin was washed with 3 × 5 ml of column buffer containing 50 mm imidazole, and the proteins were eluted into a single 300-μl fraction with column buffer containing 500 mm imidazole. Purification was confirmed by 12% SDS-PAGE. Enzymatic Characterization and Substrate Analysis—Purified recombinant protease, WNV CF40.Gly. NS3pro, and site-directed mutants were assayed against tetrapeptide (Ac-LKKR-pNA) and hexapeptide (Ac-LQYTKR-pNA) substrates corresponding to P6-P1 of cleavage sites in the endogenous substrates but with a chromogenic pNA group at the P1′ position. Cleavage of pNA from the peptides by WNV protease produced a yellow color that allowed monitoring at 405 nm. The assay was conducted in a 96-well plate, with a final reaction volume of 200 μl containing 0.25 or 0.5 μm recombinant protease, using optimized conditions (final concentration of 50 mm glycine-NaOH, pH 9.5, 30% glycerol, and 1 mm CHAPS) (7Nall T.A. Chappell K.J. Stoermer M.J. Fang N.X. Tyndall J.D. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Eight different substrate concentrations, each in triplicate, were used for determining kinetic constants. After preincubation in separate wells (10 min, 37 °C), catalysis was initiated by mixing substrate with enzyme-buffer solution by automatic shaking for 5 s. The optical density was measured at 405 nm every 11-30 s for 210 s to 30 min (depending on activity) in a SpectraMax 250 reader, and the average change in millioptical density/min was calculated. For low substrate concentration, where there was a visible loss in activity over time, only the first five points were used to calculate the average change in millioptical density/min. Kinetic parameters were calculated from weighted nonlinear regression of the initial velocities as a function of the eight substrate concentrations using Graphpad Prism 4® software. The parameters kcat, Km, and kcat/Km were calculated assuming Michaelis-Menten equilibrium kinetics, v = Vmax[S]/([S] + Km). Triplicate measurements were taken for each data point, and means ± S.E. are reported. Predicted Substrate-Enzyme Interactions—The reported crystal structure of WNV NS3 protease in association with the cofactor domain of NS2B has increased the understanding of the mechanism of substrate binding and protease activity. Since the enzyme was also bound to a tetrapeptide-aldehyde inhibitor, it was possible to observe enzyme residues that interact with P1 and P2 side chains. However, because the inhibitor was not bound in the extended β-strand conformation typical of substrate-protease interactions (4Tyndall J.D. Nall T. Fairlie D.P. Chem. Rev. 2005; 105: 973-999Crossref PubMed Scopus (345) Google Scholar, 17Fairlie 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), enzyme contacts with substrate beyond P1 and P2 positions could not be deduced from that crystal structure. In this work, we sought to dissect critical substrate interactions with enzyme at S2, S3, and S4. Cleavage sites for WNV protease vary considerably between P6 and P3 in native polypeptide substrates (e.g. DPNRKR ↓ GW (NS2A-NS2B), LQYTKR ↓ GG (NS2B-NS3), FASGKR ↓ SQ (NS3-NS4A), KPGLKR ↓ GG (NS4A-NS5)), with polar, acidic, basic, and hydrophobic residues all tolerated at P6-P3 positions. To predict protease residues that are important for substrate binding, we conducted molecular modeling experiments using GOLD to dock tetrapeptide pNA substrates into the crystal structure of the WNV NS2B/NS3 protease (Protein Data Bank code 2fp7). The tetrapeptide substrate Ac-LKRR-pNA spanning P4-P1 had been previously identified (21Li J. Lim S.P. Beer D. Patel V. Wen D. Tumanut C. Tully D.C. Williams J.A. Jiricek J. Priestle J.P. Harris J.L. Vasudevan S.G. J. Biol. Chem. 2005; 280: 28766-28774Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) as optimal for dengue protease and, since we knew that WNV elicited a preference for Lys over Arg at P2, our docking studies began with the tetrapeptide substrate Ac-LKKR-pNA. Docking flexible molecules such as peptides into rigid solid state structures of proteases is notoriously difficult due to inadequate sampling of conformational space and insufficiently minimized docked poses (25Stoermer M.J. Med. Chem. 2006; 2: 89-112Crossref PubMed Scopus (27) Google Scholar) and because GOLD does not allow for cooperative interactions or enzyme flexibility. To generate more valid docking results, hydrogen bonding and distance constraints were used to restrict the substrate to β-strand-like conformations that are more biologically relevant (4Tyndall J.D. Nall T. Fairlie D.P. Chem. Rev. 2005; 105: 973-999Crossref PubMed Scopus (345) Google Scholar, 5Loughlin W.A. Tyndall J.D.A. Glenn M.P. Fairlie D.P. Chem. Rev. 2004; 104: 6085-6117Crossref PubMed Scopus (207) Google Scholar). The crystal structure of WNV protease suggests that in addition to substrate-binding residues within NS3, residues within the NS2B cofactor also interact with substrate. Molecular docking (Fig. 2A) suggests that the P3 Lys side chain does not occupy a well defined S3 pocket in the enzyme but instead is largely solvent-exposed and binds in a shallow groove extending toward S1. This hydrophobic region is perhaps the reason that the four endogenous cleavage sequences contain a range of residues at P3 (Arg, Thr, Gly, and Leu). In all cases, there are hydrophobic elements in proximity to the main chain that are able to interact with the hydrophobic wall of the groove (e.g. Ile-155) as well as being able to accommodate both charged and polar side-chain termini directed outward into solvent. Asn-152 is hypothesized to be the S2 hydrogen bond acceptor of the P2 Lys side chain (7Nall T.A. Chappell K.J. Stoermer M.J. Fang N.X. Tyndall J.D. Young P.R. Fairlie D.P. J. Biol. Chem. 2004; 279: 48535-48542Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). On the opposite side of the substrate binding cleft to S2 is a hydrophobic surface patch consisting of Val-154, Met-156, and cofactor residue Leu-87. The hydrophobicity of these residues is highly conserved within the Flavivirus genus, and they most likely constitute one side of the shallow S4 pocket. The cofactor residue Gln-86 is observed in the crystal structure to participate in a hydrogen bond with the P3 Lys-NH3+ of an aldehyde inhibitor bound in an unusual conformation, but, since this residue is poorly conserved among the flaviviruses, the side chain is probably not important for substrate binding. Farther away, the cofactor residue Val-75 may make interactions with P5 or P6. However, it is difficult to predict how substrate might extend into and bind at this position. Also of interest is the cofactor residue Asn-84, which appears to make a hydrogen bond with the P2 Lys. This residue is semiconserved within the Flavivirus genus as either a polar or negatively charged residue (Asn, Ser, Thr, Asp, and Glu). Docking of 2-naphthoyl-KKR-pNA generally resulted in two docking poses. One had the bulky naphthoyl residue in the S1 pocket, resulting in a nonextended conformation reminiscent of the turnlike structure of the inhibitor aldehyde in the published crystal structure. The other had the aromatic residues interacting with hydrophobic enzyme residues at S4 but with some resultant steric clashes. Docking with GOLD used an explicit rigid protein method that is frequently inadequate for proteases, since they often display a high degree of active site plasticity (25Stoermer M.J. Med. Chem. 2006; 2: 89-112Crossref PubMed Scopus (27) Google Scholar). In this case, we took the docked poses where the 2-naphthoyl residue occupied the conventional extended conformation in the S4 site and used a combination of molecular dynamics and energy minimization to investigate possible induced fit binding modes. Fig. 2B shows one minimized docking pose where the small S4 pocket has been enlarged by a subtle movement of the side chains of Ile-155 and Val-154 in NS3 and Val-75, Val-77, and Leu-87 of NS2B. After these movements, the now slightly deeper S4 pocket is additionally defined by two residues (Phe-116 from NS3 and Phe-85 from NS2B) that make favorable aromatic-aromatic interactions with the naphthoyl ring. Regarding the orientation of the substrate in the active site of the enzyme, the model suggests that the carbonyl carbon of the substrate scissile amide was 2.5-2.8 Å from the catalytic serine hydroxyl and in an orientation reminiscent of a Michaelis complex, despite no explicit restraints being used to fix it. Following these substrate-docking modeling experiments, we prepared a number of site-directed mutants with residue substitutions in both NS3 and NS2B to test the predicted interactions. In parallel, we synthesized a library of chromogenic pNA substrates, designed around the optimal substrate used for our docking studies, and examined their processing kinetics by both wild type and mutant NS2B/NS3 West Nile Virus proteases. Cofactor-Substrate Correlations—The crystal structure of WNV NS2B/NS3 protease revealed that Asn-84 of the NS2B cofactor is within hydrogen bonding distance of the P2 Lys of the bound ligand. Asn-84 is located within a highly conserved region of the cofactor, the Gly residue on the N-terminal side is completely conserved, and the third residue on the C-terminal side is a highly conserved hydrophobic Leu or Ile. Although the residue homologous with NS2B Asn-84 in other flaviviruses is variable, it is always polar or negatively charged (Table 1). This is of particular interest, because there appears to be an association between this residue and either Lys or Arg at P2 in native cleavage sequences. An Asn residue is at this position in WNV and St. Louis encephalitis (SLEV) proteases, corresponding to a preferred Lys at P2 of native substrates. However, in the proteases of all four serotypes of dengue, there is either Ser or Thr at this position matched by Arg at P2 in native substrates. The presence of Gln at P2 for one of the crucial cleavage sites (NS2B-NS3) suggests a requirement for a hydrogen-bonding pair and not a charge-charge interaction pair. Specific partnering between cofactor and substrate is also se