Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase

Deng, Zengqin; Lehmann, Kathleen C.; Li, Xiaorong; Feng, Chong; Wang, Guoqiang; Zhang, Qi; Qi, Xiaoxuan; Yu, Lin; Zhang, Xingliang; Feng, Wenhai; Wu, Wei; Gong, Peng; Tao, Ye; Posthuma, Clara C.; Snijder, Eric J.; Gorbalenya, Alexander E.; Chen, Zhongzhou

Nsp10 of the EAV-Bucyrus isolate (NCBI Reference Sequence NC_002532) is composed of amino acids 2371-2837 of replicase pp1ab, which will throughout this study be referred to as nsp10 residues 1-467. The full-length nsp10 sequence or a C-terminally truncated version comprising residues 1-402 (nsp10Á) were cloned into a modified pET28a vector with a tobacco etch virus (TEV) protease cleavage site. Mutations were generated using the QuikChange protocol and confirmed by DNA sequencing. The proteins were overexpressed at 37 C in Escherichia coli strain BL21 (DE3) grown to an OD 600 of $0.8 in Luria-Bertani medium in the presence of 50 mg/ml kanamycin. Protein expression was induced with 0.2 mM isopropyl b-D-1-thiogalactopyranoside for 12 h at 16 C. Cell pellets were resuspended in lysis buffer (20 mM HEPES, pH 7.0, for nsp10Á or pH 8.0 for full-length nsp10, 500 mM NaCl and 30 mM imidazole), supplemented with protease inhibitor cocktail (Roche) and disrupted by sonication. Lysates were clarified at 20 000g for 30 min and the soluble fraction was applied to a Ni 2+ chelating column. After sample loading, the column was washed (20 mM HEPES, pH 7.0 or 8.0, 500 mM NaCl and 60 mM imidazole) and the protein was eluted (20 mM HEPES, pH 7.0 or 8.0, 500 mM NaCl and 400 mM imidazole). Proteins intended for ATPase or helicase assays were dialysed against storage buffer (20 mM HEPES, pH 7.0 or 8.0, 100 mM NaCl, 50% glycerol) and stored at À20 C. Truncated protein for crystallization studies was digested with 10% (w/w) TEV protease to remove the His-tag. Further purification was performed by size-exclusion chromatography using a Superdex 200 column (GE Healthcare) with GF buffer (20 mM HEPES, pH 7.0, 500 mM NaCl). The peak fraction was collected and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.