The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5′-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2′-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2′-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

24The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. 28We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational 32 changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.of the mechanisms that permit the virus to invade cells and evade host immune restriction. SARS-CoV-2 is an enveloped, positive-sense single-stranded β-coronavirus with a large, complex RNA genome 2 . To hijack the host translation machinery for propagation, enzymes encoded by the genome of coronaviruses (CoVs) modify the 5'-end of virally encoded mRNAs by creating a cap 3 . 44RNA capping in CoVs involves activities of several nonstructural proteins (nsps): nsp13, a bifunctional RNA/NTP triphosphatase (TPase) and helicase; nsp14, a bifunctional 3'à5' mismatch exonuclease and mRNA cap guanine-N7 methyltransferase; nsp16, a ribose 2'-O methyltransferase; and an elusive guanylyl transferase 4-7 . 48Nsp16 forms an obligatory complex with nsp10 to efficiently convert client mRNA species from the Cap-0 ( me7 GopppA1) to the Cap-1 form ( me7 GopppA1m), by methylating the ribose 2'-O of the first nucleotide (usually adenosine in CoVs) of the nascent mRNA using SAM (S-adenosyl 52 methionine) as the methyl donor 4, 8 . This Cap-1 form serves to avoid induction of the innate immune response 9-11 . Hence, ablation of nsp16 activity should trigger an immune response to CoV infection and limit pathogenesis 9, 10 . It has been shown that live vaccination with nsp16defective SARS-CoV-1 or an immunogenic disruption of the nsp16-nsp10 interface protects mice 56 from an otherwise lethal challenge 12, 13 , making nsp16/nsp10 an attractive drug target.Crystal structures of SARS-CoV-1 nsp16/nsp10 in complex with SAM/SAH or sinefungin, but without an RNA substrate, have been reported 4,8,14 , so that key information about the catalytic 60 mechanism of mRNA capping in CoVs, and SARS-CoV-2 in particular, is still missing. To understand the determinants of RNA cap modification and help guide the development of SARS-CoV-2 antiviral therapies, we have now succeeded in solving the high-resolution structure (to 1.8

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2′-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2′-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2′-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

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