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

Abstract

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.

A high-throughput radioactivity-based assay for screening SARS-CoV-2 nsp10-nsp16 complex

“…This necessitates antiviral development 36 . The 2′-O-MTase nsp16 has been proposed as an appealing target for development of anti-coronaviral therapeutics 8, 11,28, 37 . Deletion of SARS-CoV nsp16 coding-region resulted in a blockade of viral RNA synthesis 18 , and nsp16 mutants have shown a strong attenuation in infected mice 19 .…”

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

“…Protein expression, purification, and crystallization. Recombinant nsp16 and nsp10 proteins with 6xHis-tags removed were purified from Escherichia coli and crystallized as previously described (10).…”

“…2A). We previously showed that the cap binding site, also called the High Affinity Binding Site (HBS), is bordered by flexible loops that adopt an open conformation upon Cap-0 analog binding (10). Interactions of the nsp16 residues Tyr6828, Tyr6930, Lys6935, Thr6970, Ser6999 and Ser7000 with the Cap-0 analog and Cap-0-RNA are similar in both structures.…”

“…To support drug discovery efforts, researchers around the globe have determined crystal structures of the SARS-CoV-2 nsp16-nsp10 in complex with ligands (8)(9)(10)(11)(12), complementing prior structural biology observations for this enzyme from SARS-CoV and MERS-CoV (13)(14)(15). This structural information has facilitated a detailed examination of the SARS-CoV-2 nsp16-nsp10 substrate binding sites (10,11). It is well established that the 2¢-O-MTase in SARS-CoV-2 and other RNA viruses can be activated by divalent metal ions (13,14,16).…”

A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19

“… 17 Various structures of nsp10-nsp16 from SARS-CoV-2 in complex with SAM, SAH, RNA, and sinefungin have been recently deposited in the Protein Data Bank (PDB) that provide vast amounts of information on the interaction of substrates with nsp16 and could be used in structure-guided drug discovery. 27 , 28 …”