SARS-CoV-2 Mpro cleaves full-length human TRMT1. A) Overview of the structure of the SARS-CoV-2 Mpro homodimer (PDB 7BB2) with substrate peptide residues (P4-P3-P2-P1-P1′-P2′-P3′-P4′) illustrated in the Mpro active site (inset); proteolytic cleavage takes place between substrate residues P1 and P1′ (dotted line). B) The TRMT1(527-534) sequence found in a linker region between the TRMT1 SAM methyltransferase (MTase) and Zinc Finger (ZF) domains is consistent with the SARS-CoV-2 Mpro cleavage consensus sequence. C) Western blots of recombinantly expressed full-length TRMT1 incubated with 10 µM catalytically inactive (Cys145Ala) or active (Wild-type) SARS-CoV-2 Mpro at 37°C. Incubation with WT Mpro results in proteolysis of FL TRMT1 and the appearance of cleavage products corresponding the ZF domain (observed with both anti-TRMT1(609-659) and anti-TRMT1(460-659) antibodies) and the MTase domain (observed with only anti-TRMT1(460-659) antibody). D) Western blots of endogenous human TRMT1 in HEK293T cell lysate incubated with 10 µM of either catalytically inactive (Cys145Ala) or active (Wild-type) Mpro at 37°C. Endogenous FL TRMT1 is stable in human cell lysate over the course of a 10-hour incubation with C145A Mpro (left) and is rapidly proteolyzed upon incubation with WT Mpro (right).

Structure of human TRMT1(526-536) peptide bound to SARS-CoV-2 Mpro. A) TRMT1 peptide bound in Mpro active site showing substrate binding pockets S1, S2, S4, and S3′. Fo-Fc omit electron density map of TRMT1 peptide bound to Mpro contoured at 2σ. TRMT1 Gln P1, an ultra-conserved residue in Mpro cleavage consensus which is critical for Mpro-mediated proteolysis, is nestled in the S1 subsite pocket of the Mpro active site. B) Direct hydrogen bond contacts formed between Mpro residues (white) and the bound TRMT1 peptide (light blue) are illustrated as yellow dashed lines. Mpro Phe140, His163, and Glu166 recognize the TRMT1 P1 Gln530 sidechain; additional sidechain and backbone hydrogen bond contacts include Mpro Thr24-TRMT1 Thr534, Mpro Thr26-TRMT1 Asn532, Mpro Asn142-TRMT1 Asn532, Mpro Glu166-TRMT1 Arg528, and Mpro Gln189-TRMT1 Leu529, consistent with canonical Mpro-peptide substrate contacts in the active site. The TRMT1 Gln530-Ala531 peptide bond is positioned between catalytic dyad residues His41 and Cys145Ala (maroon).

Analysis of Mpro-peptide structures illustrates two distinct substrate binding modes. A) Comparison of known Mpro substrate cleavage sequences and the P2′ θ,′ backbone dihedral angles measured in the corresponding C145A Mpro-peptide structures for each substrate. We included all known C145A Mpro-viral peptide structures in this analysis, except those that were missing the P3′ residue or had poorly-defined electron density for the C-terminal portion of the peptide; structures used in this analysis are PDB IDs: 7MGS, 7T8M, 7DVW, 7T9Y, 7TA4, 7TA7, 7TC4, and 8D35. Additionally, since a C145A Mpro-nsp6/7 structure was not available, we used an H41A Mpro-nsp6/7 structure (PDB 7VDX) for this analysis. B) Section of an Mpro-bound peptide substrate showing residues P1′, P2′, and P3′, with the key P2′ θ,′ dihedral angle illustrated with a curved arrow; the four backbone atoms that define the P2′ θ,′ dihedral angle are labeled and highlighted with blue circles (P2′N–P2′Cα–P2′C–P3′N). C) Alignment of peptide substrate backbones in the Mpro active site reveals two distinct binding modes at the C-terminal end of the bound peptides characterized by P2′ θ,′ dihedral angles ý 157° (nsp4/5, nsp5/6, nsp8/9, nsp9/10, nsp10/11, nsp15/16) or :: 116° (TRMT1, nsp6/7). Peptide overlays were generated by aligning SARS-CoV-2 Mpro-peptide substrate structures in PyMOL. The location of the P2′ θ,′ dihedral angle in the substrate peptide backbone is denoted with a star. D) Alignment of nsp4/5- and TRMT1-bound Mpro structures showing divergent C-terminal peptide substrate binding modes in the Mpro active site. The backbone geometry of nsp4/5 (P2′ θ,′ = 168°) positions the P3′ Phe sidechain away from the Mpro surface (‘P3′-out’ conformation), while the TRMT1 backbone geometry (P2′ θ,′ = 115°) positions the P3′ Phe sidechain toward the Mpro active (‘P3′-in’ conformation) site where it displaces Mpro Met49 to open and occupy the S3′ pocket.

Human TRMT1 peptides are cleaved with similar catalytic efficiencies to known Mpro substrates. A) Kinetics of nsp4/5 and TRMT1 peptide cleavage by Mpro. To initiate the reaction, 50nM enzyme was added to 100-0.097 µM peptide. Each fluorogenic peptide was conjugated with a quenching moiety, and upon peptide cleavage, the fluorescence of the cleavage product was measured to determine initial rates of the reaction. nsp4/5 rates were faster than those observed with the TRMT1 peptide. B) The catalytic efficiency of TRMT1 is similar to that reported of nsp8/9, though both of these substrates exhibit a large difference from nsp4/5. This suggests that TRMT1 is a feasible substrate of Mpro. *nsp8/9 kinetic data are from MacDonald et al. (15); these data were measured under similar assay conditions to our nsp4/5 and TRMT1 data and our nsp4/5 kinetic parameters agree closely with those measured by MacDonald et al.. C) Illustration of changes in Mpro Met49, Asn142, and Gln189 residue positioning in TRMT1-bound (white) versus nsp4/5-bound (orange) structures. The TRMT1 peptide is shown in blue; nsp4/5 peptide is not shown. D) No major changes in catalytic efficiency are observed for nsp4/5 and TRMT1 peptide cleavage upon mutagenesis of key Mpro residues involved in TRMT1 binding and recognition.

Molecular dynamics (MD) simulations confirm dominant peptide binding conformations and suggest discrimination in cleavage kinetics result catalytic steps that follow initial binding and nucleophilic attack. A) Distribution of the sum of the minimum distance for P3′ Phe residue in nsp4/5 or TRMT1 from three residues (Thr25, Met49, Cys44) which form the S3′ subsite; P3′-in and P3′-out conformations are illustrated above the distribution plot. The much larger proportion of TRMT1 at smaller distances reflects the peptide’s preference for binding in the P3′-in conformation where TRMT1 P3′ Phe occupies the S3′ pocket during the majority of the MD simulation. B) Distribution of the attack angle of the nucleophilic Mpro Cys145 sulfur atom and the substrate carbonyl carbon atom in the to-be-cleaved amide bond (S–C=O angle 8, top illustration) during the course of the MD simulation. Although nsp4/5 has a higher proportion of attack angles observed closer to the optimal 90 degrees compared to TRMT1, consistent with faster nsp4/5 cleavage kinetics, this small preference is insufficient to explain the 200-fold faster cleavage kinetics of nsp4/5 observed in experimental proteolysis assays.

The Mpro-targeted TRMT1 cleavage site sequence (human TRMT1 residues 526-536) is highly conserved in primates and most mammals, with the notable exception of rodents, where the glutamine Q530 residue most critical for Mpro-directed cleavage is substituted to a lysine in Muroidea. Sequence logo plots of the cleavage site in TRMT1(526-536), produced with WebLogo3. The human reference sequence is in black and orange residues show the differences. A, B, and C panels are from primates, mammals, and rodents, respectively.