STUDY ON RATIONAL DEVELOPMENT OF NOVEL ANTIBIOTICS TAGRETING DnaA DOMAIN IV

靶向DnaA结构域IV的新型抗生素的合理开发研究

• 批准号: 13558090
• 负责人: KATAYAMA Tsutomu
• 金额: $ 7.17万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13558090/
• 关键词: DNA replication; Cell cycle; DnaA protein; E.coli; DNA polymerase; AAAt motif; ATP; Antibiotics; DNA結合; NMR; X線結晶解析; AAA+ファミリー; 染色体複製制御

To establish rational process for developing novel antibiotics that targets DnaA protein, we analyzed structure-function relationship of the DNA-binding domain of DnaA, DnaA domain IV(DAD-IV). First, we overproduced and purified a truncated DnaA protein that contained only the DAD-IV. We found that DAD-IV sustains specific affinity for DNA and secondary structure by gel-retardation assay and CD analysis. Then we determined the secondary structure in solution of DAD-IV using NMR. Determined structure suggested that DAD-IV contained a three helices-bundled type helix-turn-helix motif. In addition, NMR analysis suggested that interaction sites of DAD-IV with DNA. Finally, we determined crystal structure of a complex of DAD-IV with DNA using X-ray diffraction analysis. Determined structure by this well coincided with the data obtained by NMR and by other biochemical and genetic analysis. Candidates of antibiotics were searched by the method of virtual screening, based on the structure of DAD-IV/DNA complex.

为了建立合理的开发针对DnaA蛋白的新型抗生素的方法，我们分析了DnaA的DNA结合结构域DnaA结构域IV（DnaA domain IV，DAD-IV）的结构与功能关系。首先，我们过量生产并纯化了一种仅含有DAD-IV的截短DnaA蛋白。通过凝胶阻滞试验和CD分析，我们发现DAD-IV对DNA和二级结构具有特异性亲和力。然后，我们确定了二级结构在溶液中的DAD-IV使用NMR。结构分析表明DAD-IV含有一个三螺旋束型的螺旋-转角-螺旋基序。此外，NMR分析表明，DAD-IV与DNA的相互作用位点。最后，我们用X射线衍射分析确定了DAD-Ⅳ与DNA复合物的晶体结构。该井确定的结构与核磁共振及其它生化和成因分析结果相吻合。根据DAD-IV/DNA复合物的结构，采用虚拟筛选的方法筛选抗生素候选物。

项目成果

Nishida, S., Fujimitsu, K., Sekimizu, K., Ohmura, T., Ueda, T., Katayama, T.: "A uncleotide switch in E. coil DnaA protein initiates the chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis and in vivo"Journ

Nishida, S.、Fujimitsu, K.、Sekimizu, K.、Ohmura, T.、Ueda, T.、Katayama, T.：“大肠杆菌 DnaA 蛋白中的叔核苷酸开关启动染色体复制：来自突变 DnaA 的证据

片山 勉(分担執筆): "ゲノムの複製とその制御(松影昭夫・正井久雄編)"シュプリンガーフェアラーク東京. 234 (2002)

Tsutomu Katayama（合著者）：“基因组复制及其控制（由 Akio Matsukage 和 Hisao Masai 编辑）”Springer Verlag Tokyo 234（2002）。

Su'etsugu, M., et al.: "Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli : Interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex"Genes to Cells. (in press). (2004)

Suetsugu, M. 等人：“大肠杆菌中起始蛋白 DNA 复制偶联失活的分子机制：DnaA 与滑动夹加载的 DNA 和滑动夹-Hda 复合物的相互作用”基因到细胞。

Katayama, T. et al.: "Replication system in the E.coli genome(Advances in Genomics and Proteomics(T.Imanaka(ed.))"N.T.S.. (2004)

Katayama, T. 等：“大肠杆菌基因组中的复制系统（基因组学和蛋白质组学进展（T.Imanaka（编辑））”N.T.S.（2004 年）

Katayama, T.: "Regulation of chromosome replication in prokayotes(Replication and Partition of the Genome(A.Matsukage and H.Masai(eds.))"Sringer-Verlag Tokyo. (2003)

Katayama, T.：“原核生物染色体复制的调节（基因组的复制和分区（A.Matsukage 和 H.Masai（编））”Sringer-Verlag Tokyo。（2003 年）