Fleury F, Ianoul A, Kryukov E, Sukhanova A, Kudelina I, Wynne-Jones A, Bronstein I B, Maizieres M, Berjot M, Dodson G G, Wilkinson A J, Holden J A, Feofanov A V, Alix A J, Jardillier J C, Nabiev I
Institut Fédératif de Recherche "Biomolécules", Université de Reims Champagne-Ardenne, France.
Biochemistry. 1998 Oct 13;37(41):14630-42. doi: 10.1021/bi9806495.
N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.
N端截短的重组68 kDa人拓扑异构酶（topo）I表现出与野生型蛋白相同的DNA驱动活性。在本研究中，采用拉曼光谱和圆二色光谱技术对68 kDa人拓扑异构酶I及其由序列特异性自杀寡核苷酸（solig）结合和切割诱导的转变进行详细的结构表征。结合光谱数据和统计预测技术构建溶液中沿蛋白质一级结构的二级结构分布模型。发现68 kDa拓扑异构酶I约由59%的α-螺旋、24%的β-链和/或片层以及17%的其他结构组成。发现68 kDa拓扑异构酶I的二级结构转变伴随着solig的结合和切割。当与DNA底物形成复合物时，68 kDa拓扑异构酶I近15%的α-螺旋转变为其他结构。拉曼光谱分析还显示68 kDa拓扑异构酶I二硫键的结构旋转异构体重新分布，以及酪氨酸残基氢键和色氨酸侧链微环境/构象的显著变化。光谱技术未检测到DNA底物的结构修饰。所提供的数据首次直接实验证明了在酶与DNA底物结合和切割反应的切割步骤后人拓扑异构酶I的构象转变。这一证据支持了酶功能需要蛋白质构象转变的模型。酶转变最可能的位置是核心和C端保守的68 kDa拓扑异构酶I结构域。相比之下，发现连接域对solig诱导的结构转变的可能性极低。solig结合和共价自杀复合物形成诱导的蛋白质二级结构重新分布模式支持了拓扑异构酶I/DNA在底物结合和切割反应中分子内二分模式的模型。
