Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2′-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn²⁺, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2′-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2′-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.

病毒信使 RNA 的加帽对于有效翻译、病毒复制和防止宿主细胞先天反应系统检测至关重要。SARS-CoV-2 基因组编码 2'- O-甲基转移酶 nsp16，当它与共激活因子 nsp10 结合时，使用S-腺苷甲硫氨酸 (SAM) 作为供体将甲基转移到 mRNA 的第一个核糖核苷酸上。病毒mRNA加帽的最后一步。在这里，我们提供了生化和结构证据，证明该反应需要二价阳离子，最好是 Mn ²⁺和冠状病毒特异性四残基插入物。我们确定了 SARS-CoV-2 2'- O的 X 射线结构-甲基转移酶（nsp16-nsp10 异二聚体）与其反应底物、产物和二价金属阳离子复合。这些结构快照显示，金属离子和插入物稳定了加帽 RNA 和 nsp16 之间的相互作用，导致活性位点中的核糖核苷酸精确排列。将 2' - O-甲基转移酶的可用结构与来自不同生物的加帽 RNA 进行比较表明，冠状病毒 nsp16 特有的四残基插入改变了结合槽中加帽 RNA 的骨架构象，从而促进了催化作用。该插入片段在冠状病毒中高度保守，并且在哺乳动物甲基转移酶中不存在使该区域成为结构指导的选择性冠状病毒抑制剂药物设计的有希望的位点。