Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis.
Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis.
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通过基于结构的诱变切换双组分 NS2B-NS3 黄病毒蛋白酶的底物特异性。
DOI：
10.1128/jvi.02719-06
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发表时间：
2007
影响因子：
5.4
通讯作者：
Strongin,AlexY
中科院分区：
文献类型：
--
作者：
Shiryaev,SergeyA;Ratnikov,BorisI;Aleshin,AlexanderE;Kozlov,IgorA;Nelson,NicholasA;Lebl,Michal;Smith,JeffreyW;Liddington,RobertC;Strongin,AlexY
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 Arg76and Pro131-Thr132limit the P1′-P2′ subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu76and Lys131-Pro132widen 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.
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DOI：
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