Reposition pathways of GTP in <i>orthoflavivirus</i> NS5-methyltransferase revealed by enhanced molecular dynamics simulations

The NS5-methyltransferase (MTase) domain is highly conserved in the <i>orthoflavivirus</i> genus. This enzyme catalyzes the methylation of the 5'-RNA cap and its adjacent nucleotide, responsible for viral RNA capping that is crucial for the survival and replication of the virus. The catalytic mechanism of this enzymatic domain is yet to be understood. In particular, the experimentally determined cap-binding site on the MTase is outside the catalytic site putatively responsible for <i>N</i>⁷-methylation. Herein we employ GTP as a model of the 5'-RNA cap to investigate the process of repositioning of the cap from the GTP-binding pocket to the putative catalytic center. To overcome the computational challenge in sampling transitions, we deploy a two-step approach in which extensive Gaussian-accelerated molecular dynamics are performed to explore transition paths followed by a path-based umbrella sampling for determining the free energy change during the transition. We find that the GTP substrate interacts with conserved residues along the pathways from the crystallographic pocket to near the methyl donor <i>S</i>-adenosyl-L-methionine (SAM). Interestingly, our observation is in accordance with experimental mutagenesis studies on the <i>N</i>⁷-MTase reaction. Through an energy decomposition analysis along the pathway, we further find that residues E149, R57, H110 and R84 contribute to the placement of the GTP substrate. The complex relationship between MTase global conformational changes and the GTP repositioning process has been revealed which could be relevant to the functional mechanism of this enzyme. This work provides the rationale behind residue contribution and substrate requirement of <i>orthoflavivirus</i> MTase activities and provides invaluable insights for the rational design of MTase-targeted inhibitors.