Molecular dynamics simulations reveal DNA gate opening mechanisms for M. smegmatis topoisomerase 1A

Type 1A topoisomerases relax torsional strain in DNA via a strand passage mechanism in which a protein-mediated DNA gate must open during the enzyme's catalytic cycle. This gate-open conformational state of the enzyme has been challenging to observe via experimental methods. In this study, we first used equilibrium molecular dynamics simulations to probe the structural properties of the gate-closed state for the DNA-free apo system and a system with an ssDNA bound at the DNA binding site. For both systems, we followed the equilibrium simulations with umbrella sampling simulations. Umbrella sampling allowed us to bias the protein to adopt a gate-open state to study the properties of this conformation, as well as the pathways leading to it. We observed that several electrostatic interactions contribute to the closed-state stability of the protein, which were broken during the gate opening. The gate opening composed of three major domain motions that were determined from simulation trajectories and principal component analysis. Finally, umbrella sampling results combined with the weighted histogram analysis method allowed us to reconstruct the free energy profiles of gate opening for all simulations.