Conformational Sampling during the Reaction of the SARS-CoV-2 Methyltransferase nsp16

The coronavirus 2'-O-methyltransferase nsp16 catalyzes the methylation of the viral RNA cap structure, playing an essential role in viral RNA immune evasion. Unusually, nsp16 forms a heterodimer with a second viral protein, nsp10, which appears to be essential for activity. Here, we use a combination of density functional theory (DFT) modeling of the nsp16 active site to investigate the methyl transfer reaction and molecular dynamics (MD) simulations to investigate substrate binding and dynamics. The active site cluster models give barrier heights of 76-120 kJ mol^-1, with much of the variation appearing to come from large differences in the relative (de)stabilization of the reactant state. The lower barriers are in good agreement with experiment, suggesting that conformational sampling of nonreactive conformations of the RNA substrate occurs during MD simulations. An analysis of interaction energies shows that nsp10 stabilizes nsp16-RNA interactions, but we see only modest changes in nsp16 structure and dynamics upon removal of nsp10, with these changes centered around the active site loops and the dimer interface. We also observe a considerable conformational sampling of RNA substrates within the active site. The population of potentially reactive substrate configurations is relatively low, and we see no significant effect of nsp10, but differences between RNA substrates 7-methyl-GpppA and 7-methyl-GpppAUU; the larger substrate appears to more frequently sample potentially reactive configurations. This conformational sampling of the RNA substrate is consistent with X-ray crystal structures of substrate (Michaelis) complexes of nsp16 nsp10, where soaking the RNA fragment into a crystal of SAM-bound nsp16 nsp10 can prevent productive sampling of the RNA conformational space within the active site.