硫磺礦硫化葉菌程序性細胞死亡蛋白5晶體結構分析及其與DNA的相互作用;Crystal Structure Analysis of Sulfolobus solfataricus Programmed Cell Death Protein 5 and Its Interaction with DNA

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Title:	硫磺礦硫化葉菌程序性細胞死亡蛋白5晶體結構分析及其與DNA的相互作用;Crystal Structure Analysis of Sulfolobus solfataricus Programmed Cell Death Protein 5 and Its Interaction with DNA
Authors:	蔡孟儒;Tsai, Meng-Ju
Contributors:	生命科學系
Keywords:	硫磺礦硫化葉菌;程序性細胞死亡蛋白5;X光繞射;蛋白質結晶學;DNA結合蛋白;Sulfolobus solfataricus;the programmed cell death protein 5;crystal structure;X-ray crystallography;DNA-binding protein
Date:	2019-07-17
Issue Date:	2019-09-03 14:32:46 (UTC+8)
Publisher:	國立中央大學
Abstract:	中文摘要程序性細胞死亡蛋白5 (PDCD5)在真核生物的細胞凋亡途徑中扮演著重要信號蛋白的角色，在現有的研究中，已知人類PDCD5蛋白中存在著三螺旋束和兩個游離的氮端區域，然而對於古生菌中PDCD5的研究仍尚不清楚。本研究中，我們鑑定了來自硫磺礦硫化葉菌的PDCD5同源蛋白(Sso-PDCD5)，並且以1.55Å的高解析度呈現Sso-PDCD5的晶體結構，Sso-PDCD5為PDCD5同源蛋白中第一個解析出來的晶體結構，此蛋白質具有低柔韌性的緊密核心，氮端區域具有四個α-螺旋，以及具有彈性的碳端尾端。而位於碳端區域的色胺酸(W117)在激發螢光的實驗中發現色胺酸(W117)發散出的螢光可以被20bp的雙股DNA給遮蔽，螢光強度隨DNA濃度的增加而減少，這顯示Sso-PDCD5可以透過碳端尾端與DNA進行相互作用。在等溫滴定量熱的結果顯示碳端截斷蛋白(Sso-PDCD5_CTT，刪除K108-K118序列)顯著降低了與DNA結合的親和力，更進一步證明Sso-PDCD5利用動態的碳端尾端與雙股DNA進行結合。除此之外，電子顯微鏡(EM)的圖像顯示，Sso-PDCD5透過橋接的方式與雙股DNA相互作用結合。總而言之，經過結構以及生化方面的實驗數據表明Sso-PDCD5具有與雙股DNA結合的能力，是一種DNA結合蛋白。關鍵字：硫磺礦硫化葉菌、程序性細胞死亡蛋白5、X光繞射、蛋白質結晶學、DNA結合蛋白 ;Abstract The Programmed Cell Death protein 5 (PDCD5) is an important signal protein of apoptosis pathway in eukaryotes. In previous research, it is known there are triple-helix bundle and two dissociated N-terminal regions in human PDCD5 protein. However, the study of PDCD5 in hyperthermophile archaea remains unclear. Here, we identify a PDCD5 homolog from Sulfolobus solfataricus (Sso_PDCD5) and present the crystal structure of Sso_PDCD5 at a high resolution of 1.55 Å. This is the first crystal structure of a PDCD5 homolog to be solved, showing that the protein has a compact core of low flexibility with four alpha-helices at N-terminal region and a flexible unstructured C-terminal tail. The fluorescence of C-terminal tryptophan (W117) can be quenched by 20 bp double-strand DNA which indicates PDCD5 may interact with DNA by the C-terminal tail. The isothermal titration calorimety (ITC) results show C-terminal truncated protein (PDCD5_CTT, detection of K108-K118) significantly reduced the DNA-binding affinity, further demonstrated that the flexible C-terminus of Sso_PDCD5 involved in binding dsDNA. In addition, Sso_PDCD5 binds dsDNA through bridging interactions as shown in electron microscopy (EM) images. In conclusion, the structural and biochemical data suggest that Sso_PDCD5 may function as a DNA-binding protein. Keywords: Sulfolobus solfataricus, the programmed cell death protein 5, crystal structure, X-ray crystallography, DNA-binding protein
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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