Site-directed mutagenesis and kinetic studies of the West Nile Virus NS3 protease identify key enzyme-substrate interactions.
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.
Chappell
Keith J
KJ
Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Nall
Tessa A
TA
Stoermer
Martin J
MJ
Fang
Ning-Xia
NX
Tyndall
Joel D A
JD
Fairlie
David P
DP
Young
Paul R
PR
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics*; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics*; D014902:West Nile virus / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010455:Peptides; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases