The SARS-CoV-2 mRNA is capped at the 5′-end by methyltransferases (MTases) nsp14 and nsp16 (ref. 1). Nsp14 methylates the N7 atom of guanosine to generate the N7MeGpppA2’OH-RNA structure, which is then methylated at the 2′O atom of the initiating nucleotide by nsp16 to make the N7MeGpppN2’OMe-RNA structure1. Both nsp14 and nsp16 use S-adenosylmethionine (SAM) as the methyl donor and generate S-adenosylhomocysteine (SAH) as the reaction byproduct. Nsp14 harbors an exoribonuclease domain (ExoN) at the N-terminus and the N7-MTase domain at the C-terminus (Fig. 1a)2,3,4. 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-CoV5,6, solved to 3.2–3.4 Å resolution2. 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. We employ here fusion protein-assisted crystallization9,10 and report high-resolution crystal structures of the nsp14 N7-MTase-TELSAM fusion (TEL-MTase; Fig. 1b) in complex with SAM, SAH and SFG (Supplementary Table 1).

a, Domain organization of SARS-CoV-2 nsp14 and nsp10. b, The overall structure of TELSAM-MTase fusion in complex with SAM shown in a ribbon representation. The nsp14 N7-MTase domain and TELSAM are colored in cyan and yellow, respectively. The secondary structure elements for the N7-MTase domain are labeled. The residues not modeled in the structure are shown by dashed lines. A zinc ion (Zn) is shown as a sphere and colored gray. c, Cα trace superposition of nsp14 N7-MTaseSAM, nsp14 N7-MTaseSAH and nsp14 N7-MTaseSFG.

The MTase core in the three structures is nearly identical, superimposing with root-mean-square deviations (RMSDs) between 0.085 and 0.09 Å for 187 Cα atoms, showing it to be essentially invariant when bound to SAM, SAH or SFG (Fig. 1c). The MTase core consists of an atypical Rossmann fold, composed of a central five stranded β-sheet (β1′, β2′, β3′, β4′ and β8′) instead of the seven stranded β-sheet (β1–β7) typically associated with class I MTases11, including those from most viruses. Helices α1′, α2′, α3′ and αC, β-strands βA and βB, and a Zn2+ coordinated substructure are located on one side of the β-sheet, and two short helices αA and αB on the other (Fig. 1b). SAM, SAH and SFG are located at the C-terminal ends of strands β1′, β2′ and β3′, and are cradled by loops between β1′ and β2′, β2′ and αA, and β3′ and β4′ (Fig. 2a–c).

a, Structure of nsp14 MTase domain bound to SAM (left), with a detailed view of the interactions between them (right). The Fo – Fc difference electron density for SAM is shown in a pink mesh and contoured at 3σ level. Hydrogen bonds between the MTase domain and SAM are depicted as dashed lines and the water molecules are shown as red spheres. b, Structure of nsp14 MTase bound to SAH (left), with a detailed view of the interactions between them (right). c, Structure of nsp14 MTase domain bound to SFG (left), with a detailed view of the interactions between them (right).

In the full-length nsp14 structures2,7,8, 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. 1a–c). 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 domain12. 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. 1a). 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. 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. 1d).

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. 2a–c). 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. Asp352 is conserved in coronaviruses and its mutation to alanine in SARS-CoV has been shown to abrogate N7-MTase activity2,3,13. The tail portion is fixed by numerous interactions, including direct hydrogen bonds with the Arg310 side chain and the Gly333 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. 2a–c). In addition, the Pro335 ring is involved in van der Waals contacts with the nonpolar portion (atoms Cβ and Cγ) of SAM/SAH/SFG. Arg310, Asp331 and Asn386 are conserved in coronaviruses, and their mutation to alanine in SARS-CoV has been shown to abolish N7-MTase activity2,3,13. 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 activity2,3. 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. 2a–c). 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. 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.

All the amino acids at the interface are conserved in the SARS-CoV nsp14 N7-MTase. The crystal structure of SARS-CoV nsp14/nsp10 with SAM captured a subset of the interactions (Extended Data Fig. 2), but some key interactions, such as between Arg310, Gln313, Asn386 and the terminal carboxylate group of SAM were not observed, possibly because of the moderate resolution of the structure. Also, the configuration of the bound SAM is different, wherein the donor methyl group points in the opposite direction to what we observe here (Extended Data Fig. 2). Most importantly, the limited resolution of the SARS-CoV structure did not allow for the observation of water molecules, which form a crucial part of the N7-MTase-SAM interface (Extended Data Fig. 2).

Interestingly, because of the interconnection between the MTase domain and the ExoN domain (Extended Data Fig. 3), the MTase activity of the nsp14 is influenced by noncatalytic mutations in the ExoN domain13,14,15. To explore this further, we expressed and purified just the SARS-CoV-2 MTase domain (residues 289–527). We find that the MTase activity of the isolated MTase domain and the TEL-MTase fusion is nearly identical, showing that addition of TELSAM to the MTase domain does not impact its activity (Extended Data Fig. 4). But, consistent with the previous mutational and deletional analysis of SARS-CoV nsp14 (ref. 13,14,15), the activity of MTase domain and the TEL-MTase is reduced in the absence of the ExoN domain (Extended Data Fig. 4). This is probably due to a stabilizing allosteric effect of one domain on the other, as mutations in the MTase domain have also been found to reciprocally effect the ExoN activity3.

From isothermal titration calorimetry (ITC) analysis, SARS-CoV-2 nsp14/nsp10 complex binds SAM and SFG with similar affinities (Kd of 5.7 μM versus 4.4 μM), but binds SAH substantially better (Kd of 0.3 μM) (Supplementary Table 2 and Extended Data Fig. 5). The MTase domain and the TEL-MTase fusion bind SAM/SAH/SFG in a similar pattern, though the absolute affinities (Kd of around 22–25 µM for SAM/SFG and Kd of 5 µM for SAH) are lower than those observed with full-length nsp14/10 (Extended Data Fig. 6). This further reinforces the notion that the ExoN domain has a stabilizing allosteric effect on the MTase domain and helps to increase its affinity for SAM/SAH/SFG. Importantly, the residues that interact between the two domains are distant from the SAM/SAH/SFG binding site (Extended Data Fig. 3).

How to explain the higher affinity of SAH compared with SAM or SFG? In the nsp14 MTaseSAM structure, the donor methyl group of SAM (attached to its Sδ atom) abuts the Asn386 main chain carbonyl and seems to displace a water molecule that would normally be coordinated to the main chain carbonyl (Fig. 2a). Indeed, in the nsp14 MTaseSAH structure, we observe a well-ordered water molecule coordinated to the Asn386 main chain carbonyl at a position that would be incompatible with the methyl group of SAM (Fig. 2b). The entry of this water molecule may provide a partial explanation for the higher affinity of SAH relative to SAM, particularly the more favorable enthalpic contribution to binding (Supplementary Table 2 and Extended Data Fig. 5). It is less clear, however, why SAH would bind better than SFG. The amino group of SFG (attached to its Cδ) makes a direct hydrogen bond with the Asn386 main chain carbonyl and would seem to compensate for the loss of a water molecule (Fig. 2c). Whether this hydrogen bond is less favorable enthalpically than a coordinated water molecule to the Asn386 main chain carbonyl is uncertain at present.

An attractive feature of SARS-CoV-2 N7-MTase as a drug target is its high conservation of sequence across other coronaviruses and nearly total conservation of sequence across all the strains of SARS-CoV-2 (Extended Data Fig. 7a,b). Interestingly, we find that the affinity of SAH for nsp14 is substantively better than for SAM or SFG; positing SAH as the scaffold of choice for the design of more potent SAM competitors. Indeed, when Devkota et al. added a nitrile group to position 7 of the adenine base of SAH, it further improved its potency and binding (Kd of 0.05 µM)6 and a bulky aromatic substituent at the same place led to single-digit nanomolar inhibitors16. Notably, the N7-MTase-SAM/SAH/SFG interface also contains a conserved cysteine (Cys387) at 3.9 Å and 4.6 Å from the N7 and N6 atoms of the adenine base, respectively (Fig. 2), allowing for a suitable ‘warhead’ on the adenine base to make a covalent bond with the conserved cysteine. Such covalent inhibitors have been designed previously for other MTases17, including one that forms a covalent bond with Cys449 in the active site of protein arginine methyltransferase 5 (PRMT5)18.

Overall, the high-resolution structures of SARS-CoV-2 nsp14 N7-MTase presented here will aid in the development of new antivirals against SARS-CoV-2 and other pathogenic coronaviruses.