Rosas-Lemus Monica, Minasov George, Brunzelle Joseph S, Taha Taha Y, Lemak Sofia, Yin Shaohui, Shuvalova Ludmilla, Rosecrans Julia, Khanna Kanika, Seifert H Steven, Savchenko Alexei, Stogios Peter J, Ott Melanie, Satchell Karla J F
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
Center for Structural Biology of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
bioRxiv. 2024 Aug 14:2024.08.13.607777. doi: 10.1101/2024.08.13.607777.
Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.
冠状病毒非结构蛋白3（nsp3）在容纳复制转录复合体的双膜空泡中形成六聚体孔冠。类严重急性呼吸综合征（SARS）病毒中的nsp3具有其他冠状病毒nsp3蛋白中不存在的三个独特结构域。其中两个结构域，即SUD-N（大结构域2）和SUD-M（大结构域3），形成了由肽接头和结构域间二硫键连接的两个叶。我们解析了严重急性呼吸综合征冠状病毒（SARS-CoV）SUD-N/M的首个完整X射线结构，以及严重急性呼吸综合征冠状病毒2（SARS-CoV-2）SUD-N/M的一个突变变体的结构，该变体经过修饰以恢复因进化而自然丢失的结构域间二硫键的半胱氨酸。对所有结构的比较分析表明，SUD-N和SUD-M并非刚性结合，而是具有显著的旋转灵活性。系统发育分析支持穿山甲-SARS及密切相关病毒中也不存在二硫键半胱氨酸，这与穿山甲被认为是SARS-CoV-2出现过程中的中间宿主一致。这些半胱氨酸的缺失并不影响病毒复制或蛋白质翻译。
