嗜酸熱硫化葉菌中DNA結合蛋白Sac10b之結構分析及其與DNA相互作用;Crystal structure of DNA binding protein Sac10b from Sulfolobus acidocaldarius and its interaction with DNA

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题名:	嗜酸熱硫化葉菌中DNA結合蛋白Sac10b之結構分析及其與DNA相互作用;Crystal structure of DNA binding protein Sac10b from Sulfolobus acidocaldarius and its interaction with DNA
作者:	黃正宏;Huang, Cheng-Hung
贡献者:	生命科學系
关键词:	嗜酸熱硫化葉菌;DNA結合蛋白;X光繞射;蛋白質結晶學;Sulfolobus acidocaldarius;DNA-binding protein;X-ray crystallography;crystal structure
日期:	2020-07-24
上传时间:	2020-09-02 15:27:05 (UTC+8)
出版者:	國立中央大學
摘要:	中文摘要在生物體中有一負責結合與調節遺傳物質的重要蛋白，稱之為DNA結合蛋白，顧名思義，它會透過不同方式與DNA結合，像是橋接、彎曲或纏繞。組蛋白是真核生物中常見的DNA結合蛋白，它以纏繞的方式包裹著DNA使DNA組織成緊密結實的染色質構造；與真核生物不同，古生菌沒有組蛋白而是利用各種小的類核蛋白，通過橋接或彎曲DNA分子的方式來組織基因組，Sac10b就是在古生菌中高度保守的核酸結合蛋白。本實驗中，使用各種方式更深入研究Sac10b蛋白質，從分析型超高速離心和圓二色譜分析結果顯示，兩種蛋白質Sac10b1、Sac10b2在溶液中均以熱穩定和耐酸的二聚體形式存在；從得到2.05-Å和1.70-Å解析度的Sac10b1、Sac10b2晶體結構中，分析出Sac10b1二聚體是以R58和F59為中心與另二聚體產生交互作用，再透過電子顯微鏡觀察，Sac10b1與DNA產生了協同結合形成一個完全橋接複合物，而Sac10b2卻無法和DNA形成完全橋接，可能就是缺少F59胺基酸。為了進一步證實R58和F59胺基酸的作用，從電子顯微鏡影像來看三個Sac10b1突變R58A、F59A和R58A/F59A與DNA作用，結果與Sac10b2相似，沒有觀察到與Sac10b1相同的協同結合完全橋接的行為。因此可以證實Sac10b透過橋接作用來組織DNA，R58和F59更是扮演關鍵角色的胺基酸。;Abstract DNA-binding proteins that compact and regulate genetic materials are crucial in all organisms. Eukaryotic nuclear DNA is generally wrapped by histones into nucleosomes. Likewise, thermoacidophilic archaea utilize a variety of small nucleoid-associated proteins to organize the genome predominantly by bridging or bending the DNA molecule. Sac10b is a highly conserved nucleic acid-binding protein in archaea. Here we present two crystal structures of Sac10b1 and Sac10b2 from Sulfolobus acidocaldarius at 2.05-Å and 1.70-Å resolution, respectively. Each protein adopts a mixed α/β-fold with an extended β3-β4 hairpin. Analytical ultracentrifuge and circular dichroism results show that both proteins exist as thermal stable and acid tolerant dimers in solution. Electron microscopic images indicate that Sac10b1 and Sac10b2 can form condensed protein-DNA complexes by multiple bridged patches to different extents. Crystal packing analysis suggests that the dimer-dimer interactions, which involve the conservation of the interface residues, centered on R58 and F59 of Sac10b1, may account for the cooperative binding to DNA. The result is a completely bridged Sac10b1-DNA complex, which does not occur with the Sac10b2 homodimer due to the lack of an F59-equivalent residue. To further confirm the role of R58 and F59 residues, the EM images show that three Sac10b1 mutants R58A, F59A and R58A/F59A, similar to Sac10b2, result in the formation of intra-molecular bridging complexes but do not exhibit the cooperative binding behavior as Sac10b1, as no completely bridged DNA molecules were observed. A functional role for Sac10b in the organization and stabilization of chromosomal DNA through bridging interactions is suggested.
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