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

As outlined above, the single-stranded part of the DNA substrate is bound to a nucleic acid-binding channel formed by domains 1A, 1B and 2A ( Figure 2C ). The backbone phosphates of the poly(dT) are located on top of domains 1A and 2A, with the thymine bases exposed to the solvent (Supplementary Figure S7A) . The majority of contacts with the bound DNA are made via the phosphodiester backbone and non-specific protein-base interactions as depicted in Figure 7 . Consistent with this observation, the base orientation varies in the four EAV nsp10-poly(dT) complexes of the asymmetric unit, while the position of the DNA backbone is rather rigid (Supplementary Figure S7B and C) . Several key residues from domains 1A and 2A contact the DNA backbone in the channel of the protein (Figure 7A and B) . Base T1, the most 5 0 one, is exposed to the solvent and protrudes outwards, causing a bend in the DNA backbone between T1 and T2. The bases T2 and T3 as well as T5 and T6 stack with each other at an average distance of 3.7 Å . In contrast, base T4 is almost perpendicular to T3, with its edge exposed to protein side chains that make specific contacts. Val271 in domain 1A forms van der Waals contacts with the base and the sugar ring of T4 and thus stabilizes the DNA conformation. Moreover, the binding is stabilized by several hydrogen bonds between His186, His339, Thr348, Ser351 and the backbone of the DNA, and by van der Waals contacts between Thr185, Leu227, Val230, Tyr338 and the phosphate groups of the DNA. While the interactions described above do not involve specific bases, six further interactions specific for thymine were found. For example, the backbone NH of Arg102 forms a hydrogen bond with the O4 atom of T6. The O2 and O4 atoms of base T3 form hydrogen bonds with the side chains of Asp350 and Tyr81. Also, several residues, such as Arg374 and Gln229, interact with both the base and the sugar ring. However, no interaction was observed between nsp10Á and position C2 0 of the ribose ring of the DNA substrate. This observation may explain why EAV nsp10 has the ability to unwind both DNA and RNA, in agreement with the substrate specificity observed for other helicases (52, 53) possessing or lacking the ability to interact with the 2 0 OH moiety of the RNA backbone.