Modeling Allosteric Mechanisms of Eukaryotic Type II Topoisomerases

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N- and C-terminals of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point towards specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular-level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process. SIGNIFICANCETypeII Topoisomerases (TopoIIs) are essential and ubiquitous for maintaining DNA topology in the nucleus. The mechanisms by which information about the nucleotide-binding state is transmitted from the ATPase domains throughout the TopoII complex remain poorly understood. We used microsecond timescale molecular dynamics simulations to probe the allosteric mechanisms underlying TopoII function. Results indicate remarkable flexibility of the ATPase domains on this timescale which is modulated by nucleotide binding and local and distant amino acid substitutions. Furthermore, we mapped the allosteric networks linking the ATPase and DNA cleavage domains, and connected them to the ATPase domain dynamics. Our findings provide molecular-level insights into how nucleotide binding and hydrolysis regulate the TopoII catalytic cycle and how mutations can disrupt these processes.