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

To further explore the role of ZBD, we tested the effect of four mutations (C25A and H29A in the RING-like module; H44A and C53A in the treble-clef zinc finger) expected to affect the ability to bind Zn1, Zn2 or Zn3, respectively. In agreement with the proposed structural role of these zinc ions, soluble His-tagged proteins containing these mutations could not be obtained and only low yields of GST-nsp10 fusion proteins carrying the same mutations could be recovered. For mutants C25A and H29A, band shift analysis revealed a complete loss of binding to a partially double-stranded DNA substrate containing a 5 0 single-stranded poly(dT) overhang (substrate 5 0 DNA-T10; Figure 5C , lane 3-4). These results complement previous findings, showing a complete loss of both ATPase and helicase activity for these mutants (37). In contrast, the level of nucleic acid binding by mutants H44A and C53A was comparable with that of the wild-type protein ( Figure 5C , lanes 5-6), consistent with nsp10-H44A retaining a limited level of ATPase and helicase activity (37) . On further testing, we observed that the addition of 40 mM EDTA altered the overall conformation of nsp10Á, as detected by changes in circular dichroism (Supplementary Figure S4A) , and reduced its binding to 5 0 -DNA-A10 (Supplementary Figure S4B ). In summary, these results reveal that ZBD interacts extensively with the HEL1 domain and that its integrity is an essential determinant of nsp10Á properties in in vitro assays.