Catalysis Tuned for Metabolic Sensing: Dynamics and Mechanism of the RNA Methyltransferase METTL16

The methyltransferase-like protein 16 (METTL16) is the writer enzyme that methylates adenosine to form N6-methyladenosine (m6A) on U6 spliceosomal RNA and MAT2A mRNA. The latter encodes the enzyme responsible for the synthesis of S-adenosyl methionine (SAM). Since methylation influences the stability of MAT2A mRNA, METTL16 is the sensor of SAM in the cell. Here, we investigate the (co)substrate recognition and catalytic mechanism of METTL16 by a combination of in vitro and simulation methods. We first solved the crystal structures of METTL16 in complex with bisubstrate analogs (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. The METTL16/BA crystal structures provide an atomistic picture of a transition-state mimic of methyl transfer. Molecular dynamics (MD) simulations started from complexes with oligonucleotide-RNA modeled on the METTL16/BAs structures show rapid unbinding of SAM. The presence of the MAT2A hairpin 1 RNA stabilizes SAM in its recognition pocket, and substantially reduces the intrinsic flexibility of the R-loop and N-terminal segment of METTL16. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations provide evidence that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the simulations indicate that electrostatic interactions between the charged groups of SAM and the catalytic pocket are less pronounced for METTL16 than for METTL3-14, which is the main m6A-RNA writer enzyme. The multidisciplinary approach used here sheds light on the intrinsic plasticity of METTL16, the rigidification upon RNA-substrate binding, the catalytic mechanism, and (co)product release. Overall, our study suggests that the latter step is rate-limiting.