Structural studies reveal unique features of nsp16 from SARS‐CoV‐2, a protein essential for immune system evasion and a possible drug target

RNA viruses have several mechanisms to modify the viral RNA genome to protect from host immune surveillance. SARS‐CoV‐2, the etiological agent of COVID‐19, and other coronaviruses encode capping proteins in its genome that modify the 5’ untranslated region of the viral genome and mRNA. Capping viral RNAs promotes translation, prevents degradation, and reduces the activation of host immune responses. This process is initiated by the viral proteins nsp13 and nsp12, which cleave the 5’ phosphate group and transfer a GMP from GTP to the 5′ end of the nascent (+)ssRNA. The nsp14‐nsp10 complex then catalyzes the transfer of a methyl group from S‐adenosylmethionine (SAM) to the N7 position of the GMP cap, generating Cap‐0. Finally, the nsp16‐nsp10 complex utilizes SAM to catalyze the 2′‐O‐methylation of the first ribonucleotide, generating Cap‐1. Several structures of the nsp16‐nsp10 complex have been solved previously for SARS‐CoV, MERS and SARS‐CoV‐2 with SAM, Cap‐0 and Cap‐0‐RNA bound to the complex as well as the products of the reactions. Recently, we determined the first structure of nsp16‐nsp10 with Cap‐0‐RNA and Cap‐1 RNA, revealing that Mn2+ coordinates the first four nucleotides of the bound RNA to orient the 2’‐OH of the ribose of the first nucleotide toward the methyl group of SAM for catalysis. Herein, we also show the first apo‐crystal structure of nsp16 determined in which neither SAM nor RNA substrates bound. This new structure and comparison to structures with substraces and products bound, will reveal critical movements of the enzyme for binding substates. This study shows an important set of structures of the viral 2’‐O‐methyltransferases revealing unique features of this complex that could be used for molecular dynamics studies and designing coronavirus‐specific inhibitors.

Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16.

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.

High-resolution structures of the SARS-CoV-2 2'-O-methyltransferase reveal strategies for structure-based inhibitor design.

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.