The phosphorylated tyrosine as a gatekeeper for topoisomerase catalytic activity: a molecular dynamics simulation study

DNA topoisomerase-IA is an essential enzyme that relaxes supercoiled DNA by introducing transient single-strand breaks through a covalent phosphorylated tyrosine (PTR) intermediate. This cleavage occurs when the active-site tyrosine of dTopo-IA forms a covalent bond with the DNA phosphate backbone, resulting in PTR formation. Although dTopo-IA is believed to mediate strand passage via an enzyme-induced DNA gate, the actual opening of this gate has not been demonstrated experimentally or theoretically. To address this gap, we employed 200-nanosecond (ns) molecular dynamics (MD) simulations using AMBER18 to explore the catalytic mechanism and conformational dynamics of dTopo-IA. Important parameters like RMSD, RMSF, the number of hydrogen bonds, hydrogen bond distances, the radius of gyration (RoG), binding free energy, solvent-accessible surface area (SASA), and per-residue pair-wise decomposition energy were analyzed. Our simulations revealed that the bond between PTR and nucleotide acts as a gatekeeper, regulating the opening and closing of the DNA gate critical for strand passage. MD trajectories clearly demonstrate that gate opening and strand passage occur only after the formation of the covalent bond between PTR and the C5′ atom of the DNA strand. Additionally, we investigated how topoisomerase selectively binds single-stranded DNA in the presence of double-stranded DNA to initiate its catalytic function. The enzymatic roles of residues Gln-223, Arg-533, and Lys-117 were also elucidated in the process. This provides a novel and deeper understanding of the enzyme’s mechanism, which has been challenging to capture through experimental techniques alone, and potentially aids the development of targeted anticancer therapies by disrupting DNA replication in cancer cells.