Substitution of these water molecules will be an important feature to take into account in the design of SAM competitive inhibitors of the SARS-CoV-2 N7-MTase.Īll the amino acids at the interface are conserved in the SARS-CoV nsp14 N7-MTase. Many of these are ‘good waters’ in that they bridge the MTase and SAM/SAH/SFG and can be considered as extensions of the MTase amino acids in the SAM/SAH/SFG binding pocket. Indeed, the entire nsp14 MTase-ligand interface is defined by an unusually large number of well-ordered water molecules that mediate hydrogen bonds between the ligand and the protein (Fig. Thus, although Asp331 is not involved in a direct hydrogen bond with the ligand, its interaction via a water molecule makes it crucial for N7-MTase activity 2, 3. Arg310, Asp331 and Asn386 are conserved in coronaviruses, and their mutation to alanine in SARS-CoV has been shown to abolish N7-MTase activity 2, 3, 13. In addition, the Pro335 ring is involved in van der Waals contacts with the nonpolar portion (atoms Cβ and Cγ) of SAM/SAH/SFG. The tail portion is fixed by numerous interactions, including direct hydrogen bonds with the Arg310 side chain and the Gl圓33 and Trp385 main chain atoms, as well as intricate water-mediated interactions with Gln313, Asp331 and Asn386 side chains and the Ile332 and Trp385 main chains (Fig. Asp352 is conserved in coronaviruses and its mutation to alanine in SARS-CoV has been shown to abrogate N7-MTase activity 2, 3, 13. The ribose sugar makes direct hydrogen bonds with the Asp352 side chain, as well as water-mediated interactions with both the Gln354 side chain and main chain. The adenine base of SAM, SAH and SFG is ensconced in a cavity formed by the Ala353, Phe367, Tyr368, Cys387 and Val389 side chains, while the N1 and N6 atoms make hydrogen bonds with the backbone amide and carboxyl groups of Tyr368, respectively, and the N3 atom makes a hydrogen bond with the amide group of Ala353 (Fig. The most notable difference is in residues 467–482, which fold into helix αC and β-strand βB in SARS-CoV-2 nsp14 (Extended Data Fig. Excluding the hinge region, the SARS-CoV-2 and SARS-CoV nsp14 N7-MTase cores superimpose with a RMSD of 0.67 Å for 183 Cα atoms. Intriguingly, this β-sheet is disordered in our three structures, suggesting that its interactions with the ExoN domain are required for its folding and stability (Extended Data Fig. The β-sheet extends from the MTase core and interacts with the ExoN domain, and flexibility of the hinge has been suggested to allow for the movement between the MTase core and the ExoN domain 12. ![]() In the full-length nsp14 structures 2, 7, 8, a characteristic of the MTase fold is a ‘hinge’ region composed of a three-stranded β-sheet (β5′, β6′ and β7′ residues 402–433) and an interdomain loop (residues 288–299) that precedes the MTase core (Extended Data Fig. 1b) in complex with SAM, SAH and SFG (Supplementary Table 1). ![]() We employ here fusion protein-assisted crystallization 9, 10 and report high-resolution crystal structures of the nsp14 N7-MTase-TELSAM fusion (TEL-MTase Fig. Although the SARS-CoV-2 nsp14/nsp10 has been imaged by cryo-EM, the resolution of these structures is limited to 2.5–3.9 Å and they do not capture interactions with SAM, SAH or sinefungin (SFG) 7, 8. The nsp14 N7-MTase is an attractive target for the development of antivirals, but most structure-guided efforts thus far have depended on crystal structures of nsp14/nsp10 from SARS-CoV 5, 6, solved to 3.2–3.4 Å resolution 2. Nsp14 harbors an exoribonuclease domain (ExoN) at the N-terminus and the N7-MTase domain at the C-terminus (Fig. Both nsp14 and nsp16 use S-adenosylmethionine (SAM) as the methyl donor and generate S-adenosylhomocysteine (SAH) as the reaction byproduct. Nsp14 methylates the N7 atom of guanosine to generate the N7MeGpppA 2’OH-RNA structure, which is then methylated at the 2′O atom of the initiating nucleotide by nsp16 to make the N7MeGpppN 2’OMe-RNA structure 1. The SARS-CoV-2 mRNA is capped at the 5′-end by methyltransferases (MTases) nsp14 and nsp16 (ref.
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