Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92187
標題: c-di-GMP如何增進LtmA與其同源DNA結合的結構研究
How c-di-GMP enhances the binding of LtmA toward its cognate DNA sequences
作者: 胡惠喻
Hui-Yu Hu
關鍵字: 二級訊息傳遞分子
轉錄因子
c-di-GMP
transcription factor
secondary messenger
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摘要: 二級訊息傳遞分子cyclic-di-GMP (CDG)廣泛存在於細菌中,並參與調控許多重要的生物功能,包括致病性因子的產生、生物膜(biofilm)的合成以及細菌的移動。透過調控轉錄因子的活性,細菌能夠一次活化或抑制一群基因的表現。前人研究發現,許多調控細菌中重要生理活性的轉錄因子為CDG的受體蛋白,而CDG的結合會活化或抑制這些轉錄因子的活性。然而,目前對CDG是如何調控這些轉錄因子的詳細分子機制仍然不是很清楚。在2012年的文獻中指出恥垢分枝桿菌(Mycobacterium smegmatis)中的轉錄因子LtmA為CDG受體,此蛋白能辨識特定幾種脂質運輸和代謝基因的啟動子,當CDG結合到LtmA時,可增強此蛋白結合到其目標基因的能力。 為了瞭解CDG是如何影響LtmA的構形並增強其結合DNA的能力,本研究針對LtmA蛋白進行不同長度片段的構築及大量表現,並利用ITC及EMSA assay來研究,同時我們利用X-ray晶體繞射實驗來解析蛋白質結構,在養晶過程中加入CDG和/或DNA進行共結晶條件的篩選。最初有獲得LtmA9-184晶體且收到解析度為3.0 Å的X-ray繞射數據。目前已獲得native和SeMet-labeled LtmA9-184-CDG的晶體,且分別收到解析度為2.3和2.1 Å的X-ray繞射數據。接著利用異常散射(anomalous diffraction)解決相位角的問題,並成功解析出結構。從結構中得知,LtmA包含N端的DNA結合域(DNA-binding domain)和C端的配體結合域(ligand-binding domain),屬於類TetR家族的一員。然而,電子雲密度圖中卻無法偵測到CDG的存在。此外, 在LtmA與DNA的複合體結晶中,也收到解析度為2.6-Å的繞射數據,並順利解出結構,但也無法偵測到DNA的電子密度。未來將利用浸泡(soaking)或種晶(seeding)的方式嘗試解決配體問題,並且繼續篩選不同長度的DNA以期能獲得更高解析度的繞射數據。
Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP, CDG) is an important bacterial secondary messenger that is involved in the regulation of many critical processes including motility, biofilm formation and virulence. CDG exerts its regulatory function by directly binding to several types of receptor. A genetic screen in the non-pathogenic microorganism Mycobacterium smegmatis identified LtmA as a new CDG-responsive regulator, which is involved in lipid transport and metabolism. CDG could directly bind to LtmA and enhance its binding ability to its target gene. However, the reason for this enhancement is unclear. In addition, LtmA seems to lack the previously reported CDG binding motifs. To elucidate how CDG binds to LtmA and enhance its DNA-binding ability, several structural and functional assays have been conducted. ITC and EMSA assay were carried out to detect binding between CDG-LtmA and DNA-LtmA. Initially we obtained the apo-form LtmA9-184 crystal and collected its X-ray diffraction data, which reached to a resolution of 3.0 Å. Currently, we have also obtained the native and SeMet-labeled co-crystals of LtmA with CDG. The X-ray diffraction data of native and SeMet-labeled co-crystals were collected to a resolution of 2.3 and 2.1 Å , respectively, and the initial structure of LtmA has been solved by SAD method (single wavelength anomalous dispersion). The results showed that LtmA contains a N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which are conserved in the TetR superfamily. However, we were unable to detect the presence of CDG in the electron density map. In addition, although we could obtain the crystals of LtmA in complex with DNA/CDG and collected their diffraction data to a 2.6 Å, we were unable to detect the presence of DNA/CDG in the electron density map. In the future, we will use soaking method at a higher ligand/protein ratio to obtain the LtmA-CDG co-crystal, and continue to screening DNA targets of varying-length for obtaining the LtmA-DNA-CDG ternary complex co-crystal of higher quality.
URI: http://hdl.handle.net/11455/92187
文章公開時間: 2018-08-21
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