Catalytic Mechanism of ATP Hydrolysis in the ATPase Domain of Human DNA Topoisomerase IIα

Abstract

Human DNA topoisomerase IIα is a biological nanomachine that regulates the topological changes of the DNA molecule and is considered a prime target for anticancer drugs. Despite intensive research, many atomic details about its mechanism of action remain unknown. We investigated the ATPase domain, a segment of the human DNA topoisomerase IIα, using all-atom molecular simulations, multiscale quantum mechanics/molecular mechanics (QM/MM) calculations, and a point mutation study. The results suggested that the binding of ATP affects the overall dynamics of the ATPase dimer. Reaction modeling revealed that ATP hydrolysis favors the dissociative substrate-assisted reaction mechanism with the catalytic Glu87 serving to properly position and polarize the lytic water molecule. The point mutation study complemented our computational results, demonstrating that Lys378, part of the important QTK loop, acts as a stabilizing residue. The work aims to pave the way to a deeper understanding of these important molecular motors and to advance the development of new therapeutics.

Modeling Allosteric Mechanisms of Eukaryotic Type II Topoisomerases

“…In a recent simulation study by Ogrizek et al on the isolated human Topo IIα ATPase domains, it was noted that the transducer domains have significant flexibility relative to the GHKL domains on the hundred nanosecond timescale. (52) This is in line with what we observed in the full complex, where this relative motion between the GHKL and transducer domains leads to large-scale motions of the ATPase domains relative to the remainder of the protein, as the base of the transducer act as a hinge for the full N-gate. Chen et al presented complimentary simulations of the human TopoIIβ DNA gate without the ATPase domains in which they induced tDNA strand passage with steered MD simulations.…”

“…We computed pathways between each of the Glu66 residues with each of the catalytically critical Tyr782 residues in the DNA-gate and the Thr1125 residues in the C-gate, for a total of eight pathway analyses per system. Glu66 was chosen as it makes direct contacts with the γ-phosphate of ATP and acts as a general base in TopoII and gyrase enzymes, (50)(51)(52), while Tyr782 cleaves and binds to the G-DNA strand. (11) Although Tyr1125 is not critically crucial for TopoII function, it is located at the end of an α-helix at the base of the coiled-coil domain and forms direct contacts with the opposing monomer, and was therefore chosen as a representative location of the C-gate.…”

Phenotypic Discovery of Thiocarbohydrazone with Anticancer Properties and Catalytic Inhibition of Human DNA Topoisomerase IIα

“…The temporal RMSD plots show that complexes reached equilibrium after about 20–30 ns of simulation. To further pinpoint the main source of flexibility, we performed root-mean-square fluctuation (RMSF) calculations [ 43 ], where the most profound fluctuations were associated with the movement of residues in the transducer domain of topo IIα, comprising residues 265–405 ( Figure 5 A), which is similar to observations in our previous studies [ 5 ]. The protein flexibility induced by the binding of the two catalytic inhibitors is similar, and two conformations of compound 2 revealed by MD ( Figure S4 ) did not influence protein flexibility considerably ( Table S6 ).…”

“…Rapidly dividing cancer cells require the enhanced activity of a family of DNA topoisomerases, efficient biological nanomachines that catalyze formation of either transient single-strand breaks (type I topoisomerases, topo I) or double-strand breaks (type II topoisomerases, topo II) and regulate the topological changes of the DNA molecule. An important member of this family is the human topoisomerase IIα, an ATP-dependent enzyme [ 5 ] that exists in α and β isoforms [ 6 ] and shares approximately 70% sequence similarity but is differentially regulated during cell growth. Topo IIα is elevated in proliferating cells, whereas topo IIβ is present in proliferating as well as postmitotic cells.…”

Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions

“…In other words, the two activities “target” writhe (“writhase” or “crossover invertase”, [ 3 ]) and helical twist (“twistase”), respectively. ATP hydrolysis is essential for the catalytic function of tf1 [ 77 ], but it is unclear whether it would also be required in tf3 .…”