Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92218
標題: Xanthomonas oryzae pv. oryzae的FimX domains與PilZ蛋白的交互作用與結構分析
Structure and interaction of Xanthomonas oryzae pv. oryzae FimX domains with PilZ protein
作者: 徐偉哲
Wei-Jhe Hsu
關鍵字: 二級訊息傳訊子;纖毛;c-di-GMP;FimX;PilZ
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'A novel tetrameric PilZ domain structure from Xanthomonads.' PLoS ONE 6: e22036. Li, T.-N., et al. (2009). 'XC1028 from Xanthomonas campestris adopts a PilZ domain-like structure without a c-di-GMP switch.' Proteins: Structure, Function and Bioinformatics 75: 282-288. Liao, Y.-T., et al. (2012). 'On the crystallization and preliminary X-ray diffraction characterization of FimXEAL-c-di-GMP and FimXEAL-c-di-GMP-PilZ complexes from Xanthomonas campestris.' Acta Crystallogr. F68: 301-305. Mattick, J. S. (2002). 'Type IV pili and twitching motility.' Annu Rev Microbiol. 56: 289-314. Navarro, M. V. A. S., et al. (2009). 'Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX.' Structure 17: 1104-1116. Navarro, M. V. A. S., et al. (2011). 'Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis.' PLoS Biology 9: e1000588. Paul, K., et al. (2010). 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摘要: 
第四型纖毛為控制細菌運動性,生物膜形成,和致病性的重要細胞結構。而此第四型纖毛的形成主要是經由FimX 蛋白調控。最近,有研究指出在植物病原菌Xanthomonas axonopodis pv citri (Xac)中的第二型PilZ輔助蛋白和二級傳訊者c-di-GMP會共同協調FimX蛋白與PilB蛋白之間的相互作用,以組裝纖毛結構。其中兩個分別在Xac 跟 Xanthomonas campestris pv. campestris (Xcc)植物病原菌中有關FimXEAL跟PilZ蛋白交互作用並受c-di-GMP的調控研究已被報導出來。由於FimX蛋白包含著REC-PAS-GGDEF-EAL四個結構域,因此跟PilZ蛋白之間的交互作用可能並不完全由FimXEAL此一domain所扮演。事實上,相關的研究也指出c-di-GMP跟FimX蛋白的EAL domain的結合有很大的可能會受到REC domain尾端區域的影響。為了更進一步了解FimX-PilZ蛋白之間的交互作用,構築較長片段的FimX蛋白與PilZ蛋白作用與解析出此種複合體的結構極為重要。為了實現這一個目標,本論文我們利用了Xanthomonas oryzae pv. oryzae (Xoo)當中的FimX蛋白,得到截斷REC-PAS domains保留後端GGDEF-EAL domains與截斷REC domain 而保留後端PAS-GGDEF-EAL domains較長片段的FimX蛋白。與c-di-GMP和XccPilZ蛋白進行共結晶實驗,並得到了兩種不同空間構型的晶體結構。它們屬於三斜晶系的空間構型,分別是P222與R32的兩個不同的晶胞尺寸,最終得到的解析度分別是2.85 A跟3.1 A。但是由於複合體結構包含多個domain蛋白,因此蛋白是較具動態的,XooFimXGGDEF domain其電子密度圖較不清楚,要成功解析出結構,可能需要手動微調得到解析度更高的晶體。

Type IV pili (T4P) are important cellular structure that controls bacterial motility, biofilm formation, and pathogenicity. The formation of T4P is believed to be triggered by the action of FimX protein. Recently, it was also reported that a type II PilZ accessory protein and the novel second messenger molecule c-di-GMP are required to mediate the interaction between FimX and PilB to assemble the pilus structure in the plant pathogen Xanthomonas axonopodis pv citri (Xac). Two studies about interaction between the FimXEAL domain and the PilZ protein interaction from Xac and Xanthomonas campestris pv. campestris (Xcc) in the presence of c-di-GMP have been reported. Since FimX comprises the REC-PAS-GGDEF-EAL domains, its interaction with the PilZ protein may not be entirely triggered by the FimXEAL domain alone. In fact, it has also been reported that binding of c-di-GMP with the EAL domain can exhibit considerable conformational change on the far-end REC domain of FimX. To better understand the FimX-PilZ interaction, it is necessary to determine the complex structures of PilZ with longer FimX protein. Toward this goal, we report in the current manuscript the successful cloning, and purification of REC-PAS-truncated FimXGGDEF-EAL and REC-truncated FimXPAS-GGDEF-EAL from Xanthomonas oryzae pv. oryzae (Xoo) and its cocrystallization with c-di-GMP and XccPilZ. The XooFimXGGDEF-EAL/c-di-GMP/XccPilZ complexes cocrystallize into two different space groups belonging to the triclinic space group P222 and R32 with different unit-cell dimensions, and are diffracted to a resolution of 2.85 A and 3.1 A , respectively. It may be necessary to get better quality of crystal to determine the complex structures.
URI: http://hdl.handle.net/11455/92218
Rights: 同意授權瀏覽/列印電子全文服務,2017-10-21起公開。
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