Stelljes Jampa Tsedön, Klostermeier Dagmar
Institute for Physical Chemistry, University of Muenster, Muenster, Germany.
Institute for Physical Chemistry, University of Muenster, Muenster, Germany.
J Biol Chem. 2025 Jul 18;301(8):110495. doi: 10.1016/j.jbc.2025.110495.
Eukaryotic topoisomerase II (TOP2) catalyzes DNA relaxation and decatenation in an ATP-dependent reaction. TOP2 is a homodimer with three protein-protein interfaces, the N-gate, DNA-gate, and C-gate. Relaxation and decatenation follow a strand-passage mechanism in which one DNA duplex is bound at the DNA-gate in the middle of the enzyme and cleaved, a second DNA duplex is captured by ATP-induced closing of the N-gate through dimerization of the ATPase domains, the DNA-gate opens to enable passage of the second duplex through the gap, then closes for religation of the cleaved duplex, and the C-gate opens to release the second duplex. ATP hydrolysis and re-opening of the N-gate complete the catalytic cycle. Despite their key role in the topoisomerase reaction, gate opening and closing have not been observed directly. Here we use single-molecule FRET and Saccharomyces cerevisiae TOP2 labeled with donor and acceptor fluorophores on opposite sides of the N-gate, the DNA-gate, or the C-gate to investigate the conformational changes of these gates in response to nucleotide, DNA, and the topoisomerase poison etoposide. We show that the N-gate is open in the absence of nucleotides, and closes in response to nucleotide binding. The DNA- and C-gates are in a closed state in the absence of nucleotide and DNA and in the nucleotide-bound state. In the presence of DNA, DNA and nucleotide, or etoposide, conformations with widened or open DNA- or C-gates are observed. Consistent with biochemical data, our results point to a tight coupling between the events at different gates and between the conformational changes of these gates.
真核生物拓扑异构酶II（TOP2）在ATP依赖的反应中催化DNA松弛和解连环。TOP2是一种同型二聚体，具有三个蛋白质-蛋白质界面，即N门、DNA门和C门。松弛和解连环遵循链穿机制，其中一条DNA双链在酶中间的DNA门处结合并被切割，第二条DNA双链通过ATP酶结构域的二聚化由ATP诱导的N门关闭捕获，DNA门打开以使第二条双链通过间隙，然后关闭以重新连接切割的双链，C门打开以释放第二条双链。ATP水解和N门重新打开完成催化循环。尽管它们在拓扑异构酶反应中起关键作用，但尚未直接观察到门的打开和关闭。在这里，我们使用单分子荧光共振能量转移以及在N门、DNA门或C门相对两侧标记有供体和受体荧光团的酿酒酵母TOP2，来研究这些门响应核苷酸、DNA和拓扑异构酶毒药依托泊苷的构象变化。我们表明，N门在没有核苷酸时是开放的，并且响应核苷酸结合而关闭。DNA门和C门在没有核苷酸和DNA以及在核苷酸结合状态下处于关闭状态。在存在DNA、DNA和核苷酸或依托泊苷的情况下，观察到DNA门或C门变宽或开放的构象。与生化数据一致，我们的结果表明不同门处的事件之间以及这些门的构象变化之间存在紧密耦合。
