METTL3-14 domain architecture and structure.

(A) Domain architecture of METTL3 and METTL14. ZnF = zinc finger, NHM = N-terminal α-helical motif, CTM = C-terminal motif, RGG = arginine-glycine-glycine motif. (B) Crystal structure of the MTase domains of METTL3-14. Ribbon representations (left) are coloured as in panel (A). Surface renderings (right) are coloured according to the electrostatic potential. SAM and the putative RNA binding site are indicated.

Bisubstrate analogues as transition state mimics for METTL3.

(A) METTL3-catalysed transfer of the methyl group of SAM to the N6-atom of A in a GGACU-motif containing mRNA and the production of m6A and SAH. The inset shows the design principle of bisubstrate analogues (BAs) as transition state analogues. The point of linkage in the BA is indicated with a double arrow (red) between the N6-atom of adenosine and 5’N of the SAM analogue. (B) Chemical structures of the BAs used in this study. Substrate adenosine = black; SAM analogue = blue, linker = red. Compound names are as previously published: BA1/2/3/4/6, ref. 34; Compound 12, ref. 36; GA*, ref. 37.

BAs for METTL3-14 characterized in this study.

Crystal structures of METTL3-14 show that BAs bind in the METTL3 active site.

(A) Superposition of the crystal structures of METTL3-14 bound to SAM and the four BAs. METTL3 backbone is shown as ribbon, side chains involved in polar interactions with SAM or intramolecularly are shown as sticks, waters as red spheres. SAM (cyan) is shown as sticks and indicated, and BAs are shown as transparent sticks (BA1 = yellow, BA2 = magenta, BA4 = salmon, BA6 = palegreen). Black dashes indicate polar contacts between METTL3 and SAM in the crystal structure. (B) Outline of METTL3/SAM interactions from a LigPlot+ analysis.42 Black dashed lines indicate polar contacts between METTL3 and SAM in the crystal structure, residues forming the binding pocket environment are shown in grey. (C) Structure of METTL3-BA2. Figure composition as in (A). BA2 is coloured magenta, its SAM and adenosine moieties are indicated. The green dashes indicate a hydrogen bond that does not form with BA2, but is likely to have favourable geometry to form between D395 and adenosine-N6 (i.e., the NH2 group) of the natural RNA substrate. (D) Outline of METTL3/BA2 interactions from a LigPlot+ analysis, as in (B). The SAM analogue and adenosine parts of the BA are indicated. Black lightnings highlight residues in METTL3 involved in hydrophobic contacts with the adenosine moiety of the BA. (E) Structure of METTL3-BA4. Figure composition as in (D). BA4 is coloured salmon, its SAM and adenosine moieties are indicated. Note that BA4 is missing the ribose of the substrate adenosine moiety due to lack of electron density in the crystal structure probably due to flexibility of this group. (F) Outline of METTL3/BA4 interactions from a LigPlot+ analysis, as in (D). The missing ribose of the substrate adenosine moiety in the crystal structure is indicated with a lighter colour.

Crystal structures of METTL3-14 with BA2 and BA4 reveal two distinct adenosine binding modes.

(A) Superposition of the structures of SAM (cyan), BA2 (magenta) and BA4 (salmon) bound to METTL3. The ligands and their moieties are indicated. METTL3 in light/dark grey for BA2/BA4, backbone is shown as ribbon with side chains involved in the interactions with the adenosine moiety of the BAs shown as sticks. Waters are shown as red spheres. Black dashes indicate polar contacts common to BA2/BA4. Magenta/salmon dashes indicate hydrogen bonds unique to BA2/BA4. The green dashes indicate a hydrogen bond that does not form with BA2, but is likely to have favourable geometry to form between D395 and adenosine-N6 (i.e., the NH2 group) of the natural RNA substrate. Note that there is no electron density for the side chain of Y406 in the complex with BA2 and for the ribose of the adenosine moiety in the complex with BA4 which is most likely due to flexibility of these groups. (B) Ligplot+ analysis showing key interactions between METTL3, adenosine, and SAM based on the BA2 and BA4 structures. Dashed lines indicate polar contacts as in (A) Small magenta/salmon lightnings highlight residues in METTL3 involved in hydrophobic contacts with the adenosine moiety in the BA2/BA4 conformation. (C) Mutational analysis of the enzymatic activity of METTL3 active site residues involved in adenosine binding. (D) The melting temperature (Tm) and its shift (ΔTm, in red) for METTL3 WT and mutants with DMSO as control (light grey bars) or in the presence of SAH (dark grey bars) measured using differential scanning fluorimetry. The error bars represent standard deviation from triplicate measurements.

Kinetic parameters of ligand dissociation. Mean lifetime (τ) in ns of the analysed (co)substrates or (co)products as calculated from fitting of a single exponential (A) or an exponential with a multiplicative factor (in parentheses) (B).

Flexibility of Y406 is evidenced by MD simulations.

(A) Distance time series of five 500-ns MD trajectories started from the BA2 conformation with substrates (top), products (middle), or apo METTL3-14 (bottom). The distance between the Y406 side chain and the backbone O of W398 (grey trace) reports on the orientation of Y406 and the flexibility of the loop. The data points are coloured black if AMP/m6AMP is bound, and grey if not. Bound AMP is defined by a distance of less than 6 Å between N6 of adenosine and Cγ of D395. (B) The conformations of the flexible METTL3 backbone (ribbon) and Y406 side chain (sticks) are shown at different timepoints between 0-300 ns of the simulation with SAM and AMP, the Y406 side chain is coloured from red/start to blue/end. SAM and AMP are shown as sticks at the start of the simulation.

SAM binding primes the METTL3 active site for adenosine binding.

(A) Structural overlay of the METTL3 apo (grey) and SAM bound holo state (cyan). Black/cyan dashes indicate intramolecular polar contacts in apo/holo METTL3. Residues are shown as sticks and labelled. SAM and METTL3 residues in the holo state are shown as transparent sticks. Waters are shown as red spheres. (B) Structural overlay of the METTL3 apo (grey) and BA2 bound state (magenta). Black dashes indicate intramolecular polar contacts in apo METTL3, magenta dashes indicate polar contacts in BA2 bound METTL3. Residues are shown as sticks and labelled. BA2 is shown in magenta as sticks, its SAM and adenosine moieties are indicated. METTL3 residues in the apo state are shown as transparent sticks. Waters are shown as red spheres.

The crystal structure of the complex of METTL3-14 with the bisubstrate analogue BA2 represents the transition state of catalysis.

(A) Overlap of the crystal structure of the complex of METTL3-14 (grey) with BA2 (magenta) to the complex with SAM (cyan) and the substrate-cosubstrate pairs of METTL4 (green) and METTL16 (orange). The overlap was generated by aligning SAM from each (co)substrate pair to the SAM of METTL3. (B-D) Measurements of distances and angles between the adenosine-N6 and SAM-CH3 groups in the respective (co)substrate pairs shown in (A). (B) The METTL4 (co)substrate pair was generated by aligning the structure of METTL4-AM to METTL4-SAM. (C) The METTL16 (co)substrate pair was generated by aligning the structure of METTL16-MAT2A 3’UTR hairpin 1 to METTL16-SAH. SAM was then generated from SAH using the Chem3D software. (D) The METTL3 overlay was generated by aligning the structure of METTL3-BA2 to METTL3-SAM. The distance was measured between the N6 of BA2 and Cε of SAM, the angle was measured between the N6 of BA2, Cε of SAM, and 5’S of SAM.

Methyl transfer catalysed by METTL3 without prior deprotonation of adenosine is energetically favourable based on DFTB3/MM simulations.

(A) Proposed mechanism of the methyl transfer reaction catalysed by METTL3. (B) Potential of mean force (PMF) along the antisymmetric stretch coordinate that describes the methyl transfer between the N6 in adenosine and the SAM sulphur atom computed using multiple walker metadynamics simulations. The solid curve represents the PMF averaged over three independent runs, and the shaded area represents the standard error of the mean of the three replicas. (C) Snapshots of the active site for the reactant (left panel), transition state (middle panel), and product windows (right panel). Key distances (in Å) involving the reactive groups and the nearby ion-pair (D395-K513) and P396 backbone carbonyl are shown. METTL3 backbone is shown in grey ribbon representation with side chains shown as sticks and labelled. SAM/adenosine and SAH/m6(NH2+)-adenosine are shown as sticks and labelled in the reactant and product window, respectively, together with the transferred methyl group. The methylium group (CH3+) is indicated in the transition state window.

Reaction energetics (in kcal/mol) computed for model methyl transfer reactions that involve SAM and adenosine using different levels of theory.a

Deprotonation of m6NH2+ may occur readily through water wires that connect the N6 position to the protein-solvent interface.

Shown is a snapshot of the product state from DFTB3/MM simulations illustrating that the deprotonation of N6 following the methyl transfer may proceed along multiple water-mediated pathways that lead to the protein/solvent interface. METTL3 backbone is shown in grey ribbon representation with side chains shown as sticks and labelled, water molecules are shown as spheres. SAH and m6(NH2+)-adenosine are shown as sticks and labelled together with the transferred methyl group. Hydrogen bonds are indicated with dotted lines. The movement of protons through water channels is indicated with arrows.

The experimental and computational data elucidate the individual steps of substrate binding, product release, and methyl transfer catalysed by METTL3.

Schematic illustration of the individual steps making up the (co)substrate binding, methyl transfer, and (co)product release mechanism of METTL3; dashed grey and black lines indicate polar contacts that are intramolecular in METTL3 (including water mediated (waters shown as red spheres)) and intermolecular to the substrate adenosine, respectively. The ASL1 loop containing Y406 is either flexible (grey colour) or stabilized by the interaction with the substrate adenosine (black colour). Step 1: binding of SAM and flexibility of Y406 (supporting evidence from crystal structures and MD simulations of apo and SAM bound states); Step 2: substrate recognition (crystal structure of the complex with BA4); Step 3: flip and slip of the substrate adenosine into the catalytic site (crystal structure of the complex with BA2); Steps 4 and 5: methyl transfer and deprotonation of m6NH2+ through water channels (movement of protons indicated by arrows) (QM/MM free energy calculations); Steps 6 and 7: slip and flip of the product m6A out of the catalytic site and subsequent dissociation from METTL3; Step 8: SAH release to re-establish the apo state (MD simulations).