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

We proceeded to solve the crystal structure of nsp10Á in complex with a nucleic acid substrate. Nidovirus RNA helicases, including EAV nsp10, were previously found to lack the ability to discriminate between RNA and DNA substrates, a property shared with only a few other helicases (34, 35) . This substrate promiscuity allowed us to use a partially double-stranded DNA substrate (5 0 DNA-T10) containing a 5 0 single-stranded poly(dT) overhang for crystallographic studies. The binding of this substrate was deduced from an increase of the protein's Stokes radius in gel filtration chromatography (Supplementary Figure S5) . The binary complex diffracted to a resolution of 2.65 Å in space group P1 and was solved by molecular replacement (Table 1) . Continuous electron density was found in the enzyme's binding pocket ( Figure 2C ), which apparently corresponded to seven thymidine residues. This part presents in an extended conformation and lies in a channel formed by domains 1A, 1B and 2A, with its 5 0 end in domain 2A and its 3 0 end in domain 1A. The remaining three unpaired thymidines and the entire double-stranded portion of the substrate could not be located. The asymmetric unit contained four nsp10Á-DNA binary complexes with a Matthews coefficient of 2.73 Å 3 /Da, corresponding to a solvent content of 55%. These complexes shared a remarkably similar spatial arrangement with the RMSD of their Ca atoms being only 0.8 Å . Several connecting residues between subdomains were missing in the structure of the complex, indicating apparent structural flexibility of these residues.