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

Full-length EAV nsp10 and a series of truncated variants were overexpressed in and purified from E. coli. After extensive crystallization trials, diffracting crystals could only be obtained for a truncated form of nsp10 (aa 1-402) lacking the 65 C-terminal residues. For simplicity, we will hereafter refer to this protein as nsp10Á, which was used throughout this study unless otherwise specified. To verify that nsp10Á, which contained all characteristic SF1 helicase sequences (motifs), is enzymatically active, we performed in vitro enzyme assays to compare full-length and truncated nsp10. In agreement with previously published results (35) , full-length nsp10 displayed only weak ATPase activity in the absence of nucleic acid, but was strongly stimulated by the addition of poly-uridine (polyU). In the absence of polyU nsp10Á showed a 5-fold higher ATPase activity than the full-length protein ( Figure 1A ), yet this increased ATP turnover did apparently not translate into increased helicase activity. Unwinding of a partially double-stranded DNA substrate by nsp10Á was incomplete, but went to completion when using full-length nsp10 ( Figure 1B ). As expected, replacement of the conserved lysine of the Walker A motif, which is essential for ATP hydrolysis (35) , with glutamine (mutant K164Q) completely abolished ATPase and consequentially also helicase activity. This confirmed that the observed activities could be completely attributed to the recombinant EAV proteins used, rather than to potential trace amounts of contaminating bacterial enzymes.