title: Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis.
abstract: The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1'-P2' positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1' or P2'. Structure-based modeling suggests that Arg(76) and Pro(131)-Thr(132) limit the P1'-P2' subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu(76) and Lys(131)-Pro(132) widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3'-P4' positions were fixed and the P1' and P2' positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.
authors.affiliation: The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, USA.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Shiryaev
authors.forename: Boris I
authors.initials: BI
authors.lastname: Ratnikov
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Aleshin
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kozlov
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Nelson
authors.forename: Michal
authors.initials: M
authors.lastname: Lebl
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Smith
authors.forename: Robert C
authors.initials: RC
authors.lastname: Liddington
authors.forename: Alex Y
authors.initials: AY
authors.lastname: Strongin
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014902:West Nile virus / Q000201:enzymology* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases