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標題: | 藉由結構生物學探討第二型拓樸異構酶其DNA閘門區域之構型變化及其運作之分子機制 Structural insights into the assembly, opening and gating of DNA passage through the Topoisomerase II DNA-gate |
作者: | 陳星甫 Shin-Fu Chen |
指導教授: | 詹迺立 Nei-Li Chan |
關鍵字: | 第二型拓樸異構?,X-射線晶體學,晶體結構,DNA閘門,蛋白DNA複合體, Type II DNA topoisomerases,crystal structure,DNA-gate,T-segment-conducting path,steered molecular dynamics, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 第二型拓樸異構酶(Top2)是一類普遍存在於古生菌、細菌及真核生物中的重要蛋白。Top2可以利用ATP分子之結合與水解所帶動的蛋白構型變化來改變兩條雙股DNA的交會方式(handedness of DNA crossovers)。透過其獨特的酵素活性,Top2可以操控DNA拓樸構型並移除細胞中可能阻礙DNA運作的結構(包括超螺旋、扭結或套鎖),使DNA代謝作用得以順利進行。在作用機制上,這類具有二維對稱構型的酵素首先會與一條稱為G-segment的雙股DNA結合,接著兩個具備催化轉酯反應能力的酵素活性中心會以酪胺酸作為親核基攻擊G-segment DNA磷酸雙酯骨架,並與5′端的磷酸根形成共價的磷酸酪胺基鍵(phosphotyrosyl bond),造成暫時性的DNA雙股斷裂。再透過ATP分子結合與水解所誘發的酵素四級構型變化促使另一條被稱為T-segment的雙股DNA通過由蛋白與G-segment共同形成的閘門(DNA-gate)以達成DNA拓樸構型的改變。然而,儘管DNA-gate的形成及開闔方式對於Top2是否可以順利完成其DNA搬運的功能扮演極為重要的關鍵角色,過去對此區域的結構研究僅侷限於關閉構型。
在本論文中,我們利用了X-射線晶體學成功解析了第一個以開啟狀態呈現的DNA-gate結構。透過對此新構型的分析,我們闡明了過去因為缺乏相關結構資訊而尚未解決的問題,包括: Top2 是如何開啟其DNA-gate區域? 與酵素共價連接的DNA ends在DNA-gate開啟後會產生何種構型變化? 以及預期會讓T-segment穿越的通道(T-segment conducting channel) 在DNA-gate開啟後會呈現出什麼樣的結構特性? 除了這些議題,我們也從T-segment conducting channel的走向了解到第二型拓樸異構酶為何對於正超螺旋DNA有較好的催化效果。由於此構型係對應至T-segment穿越缺口的早期型態,我們團隊也透過電腦的計算,成功模擬出Top2可能會以類似ABC transproter運輸小分子的形變方式(rocker-switch-type movement)協助T-segment DNA穿越DNA-gate。鑒於Top2是臨床許多抗生素與抗癌小分子的標靶,我們認為此新構型可以做為發展新式Top2標的藥物的平台,有機會解決目前難以克服關於抗藥性的問題。另一方面,除了針對DNA-gate 開闔所衍伸出的議題進行探討外,本研究亦藉由解析人類Top2 DNA-gate片段的原態晶體結構,並透過一系列生化實驗及結構比對分析,提出了第二型拓樸異構酶是如何從其原態構型組裝成具有切割活性的蛋白DNA複合體之模型。 Type II DNA topoisomerases (Top2s) exploit protein conformational changes driven by ATP binding and hydrolysis to direct the passage of one DNA duplex through another, leading to a topological transformation of the DNA. This hallmark DNA passage activity makes Top2s particularly suitable for resolving DNA entanglements arising from cellular DNA transactions, including replication, transcription, chromosome segregation and recombination. To change the topology of DNA, Top2 first introduces a transient double-strand break in a stretch of duplex DNA called the G-segment, which is bound to the enzyme. Another stretch of duplex DNA, called the T-segment, passes through this break and then through the part of the enzyme holding the G-segment, which is called the DNA-gate. The structural basis governing the assembly, opening and closure of the DNA-gate is arguably the most important question to be addressed for a complete mechanistic understanding of Top2 function, as it is this step which allows DNA passage and hence the change in topology. Because directional transport of the T-segment is achieved via coordinated opening and closure of the N-gate, DNA-gate, and C-gate, a full understanding of the catalytic mechanism of Top2 requires structural characterization of these three gates in distinct conformational states. Previous crystallographic studies have revealed the structural details of the N-gate in its open and nucleotide-bound, closed forms. The C-gate has also been resolved in both open and closed conformation. In contrast, whereas multiple crystal structures are available for the closed DNA-gate, opening of the DNA-gate has never been directly visualized. Consequently, outstanding issues regarding the operation of the DNA-gate remain poorly defined, including the architectural and surface features of the T-segment-conducting path, the tertiary and quaternary structural changes associated with gate-opening, and the structural changes of the enzyme-linked DNA cohesive ends upon their detachment from one another. Thus, a step-by-step description of how the T-segment is transported through the DNA-gate is not yet available. In addition, various clinically active anticancer drugs and antibacterials act by targeting the Top2-mediated DNA-gate to produce cytotoxic DNA lesions. Obtaining a more complete picture of the conformational landscape of the DNA-gate will contribute to the development of new Top2-targeting agents. We report herein the first high-resolution view of the opening of the Top2 DNA-gate, which directly mediates the resolution of topological strand crossings. This new structure not only reveals the formation and structural features of the T-segment-conducting path, but also uncovers unexpected, functionally relevant, conformational changes that accompany the closed-to-open transition. Moreover, using this new structure as the starting point, we further simulated the passage of the T-segment through the DNA-gate by steered molecular dynamics (MD) simulations, providing crucial insights into this central yet elusive step in Top2 catalysis. We have also determined the structures of human Top2α and Top2β DNA-gate in their apo, DNA-free state at 2.67 Å and 2.8 Å, respectively. To our knowledge, these structures provide the first view of eukaryotic DNA-gate in a conformation suitable for engaging in G-segment binding. Structural comparisons between the newly determined apo form and Top2-DNA complex reveal that both prokaryotic and eukaryotic Top2 may undergo a similar G-segment-induced rearrangement of its DNA-binding groove, suggesting an unified mechanism of DNA-gate assembly. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79144 |
DOI: | 10.6342/NTU201802353 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-09 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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