Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

Xu, Xiaoling; Lou, Zhiyong; Ma, Yanlin; Chen, Xuehui; Yang, Zhangsheng; Tong, Xiaohang; Zhao, Qi; Xu, Yuanyuan; Deng, Hongyu; Bartlam, Mark; Rao, Zihe

More recently, strong immunolabeling of the nsp4 C-terminal was observed on the double membrane vesicles from SARS-CoV infected cells [34] . The highly conservation of this fragment among all coronavirus species and its involvement in assembly of the DMVs suggests that this cytoplasmic fragment of nsp4 may play an important role in coronavirus replication complex assembly. Here, we report the crystal structure of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59, together with the structure of a C425S site-directed mutant. The structure covers an 89 amino acid region from T408 to Q496 and is shown to possess a new fold. Two wild-type monomers in one asymmetric unit are linked by a Cys425-Cys425 disulfide bond to form a dimer, which could be observed and easily dissociate in reducing solution in vitro. Mutagenesis of Cys425 to Ser broke the disulfide bond, but two mutant monomers interact with each other via their cross-linked C-termini to form a strikingly different ''close'' conformation compared to the ''open'' conformation of the wild-type dimer, in which the C-termini of the wild-type monomers are oppositely oriented. Through analysis of these two conformations of the dimer and the cellular localization of nsp4C, we conclude that nsp4C exists as a monomer in the cytosol. This structure may serve as a basis for the functional studies of nsp4 from coronaviruses, thus providing preliminary structural insights into the membrane anchoring of coronavirus nsp4.