Xiong Bing, Burk David L, Shen Jianhua, Luo Xiaomin, Liu Hong, Shen Jingkang, Berghuis Albert M
Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3A 1A4.
Proteins. 2008 Jun;71(4):1984-94. doi: 10.1002/prot.21876.
Type IA topoisomerases alter the topological state of DNA to relax the supercoils introduced during the DNA replication and transcription process, giving them critical roles in many cellular functions. To manipulate the DNA, type IA topoisomerases first cleave one DNA strand and form a covalent linkage between a catalytic tyrosine residue and the 5'-phosphoryl of the DNA. This is followed by a movement of domain III of the topoisomerase to accommodate the second DNA strand in the center channel of the topoisomerase. Domain III is then closed for religation of the cleaved DNA and subsequently reopened to release the passing strand. Although numerous biophysical and biochemical studies have examined this catalytic cycle, fundamental questions remain such as how domain III opens and closes during this process. We have used computational simulation methods, namely normal mode analysis and molecular dynamics, to investigate the catalytic cycle of Escherichia coli topoisomerase III as a representative of the type IA topoisomerases. It was found that domain II is intrinsically flexible and may empower the enzyme to perform its function by triggering domain III opening and closing. A molecular dynamics simulation and MM-PBSA analysis shows that topoisomerase III alone cannot overcome the large energy barrier of the conformational transition. A detailed examination of the DNA binding sites suggests that the processing DNA cooperates with the topoisomerase to accomplish this dramatic conformational change. These findings will guide future mutagenesis studies of type IA topoisomerases aimed at dissecting the driving forces and conformations in the catalytic cycle.
IA型拓扑异构酶改变DNA的拓扑状态,以松弛DNA复制和转录过程中引入的超螺旋,使其在许多细胞功能中发挥关键作用。为了操纵DNA,IA型拓扑异构酶首先切割一条DNA链,并在催化酪氨酸残基与DNA的5'-磷酸之间形成共价连接。随后,拓扑异构酶的结构域III移动,以便将第二条DNA链容纳在拓扑异构酶的中心通道中。然后关闭结构域III以重新连接切割后的DNA,随后重新打开以释放通过的链。尽管众多生物物理和生化研究已经考察了这个催化循环,但一些基本问题仍然存在,比如在这个过程中结构域III是如何打开和关闭的。我们使用了计算模拟方法,即简正模式分析和分子动力学,来研究作为IA型拓扑异构酶代表的大肠杆菌拓扑异构酶III的催化循环。研究发现,结构域II具有内在的灵活性,可能通过触发结构域III的打开和关闭来使该酶发挥其功能。分子动力学模拟和MM-PBSA分析表明,单独的拓扑异构酶III无法克服构象转变的巨大能量障碍。对DNA结合位点的详细检查表明,正在进行加工的DNA与拓扑异构酶协同作用以完成这种剧烈的构象变化。这些发现将指导未来针对剖析催化循环中驱动力和构象的IA型拓扑异构酶的诱变研究。