The structral aspects of cyclic GMP and cyclic di-GMP-dependent bacterial signaling pathways

二級訊息傳導物質Cyclic guanosine 3',5'-monophosphate (cyclic GMP) 在細菌訊號傳遞中所扮演的功能在過去並非相當明確。然而在植物病原菌Xanthomonas campestris pv campestris (Xcc)的基因篩選實驗中發現，編碼XC_0250會轉譯出第三型nucleotidyl 環化酶，並合成cyclic GMP。在in vitro的實驗也證實純化出來的XC_0250具有合成cyclic GMP的能力。而其相連的基因XC_0249，被發現是一個帶有cyclic mononucleotide-binding (cNMP) domain以及 GGDEF diguanylate cyclase domain的蛋白。實驗發現，在cyclic GMP存在的條件下會提升XC_0249的cyclic di-GMP 合成能力。本篇研究我們解析了XC_0249 cNMP domain和cyclic GMP的複合體結構，並從結構的角度探討cyclic GMP和cyclic di-GMP是如何互相影響進而調控下游的生物生理機轉。而突變XC_0250或XC_0249的Xcc都將降低對植物的毒性以及降低生物膜的生合成。這樣的發現也因此連接了cGMP藉由影響cyclic di-GMP的合成調控整個訊息傳導路徑。除此之外，我們也嘗試著解析會和XC_0249的同源蛋白SM_0233交互作用的蛋白SM_RpfGD81E以及SM_RpfGD81E和SM_0233 GGDEF domain以及其cyclic di-GMP binding form的複合體結構，目前已初步得到SM_RpfGD81E-cyclic di-GMP的粗略結構。Cyclic guanosine 3',5'-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signaling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified XC_0250 as a protein with a class III nucleotidyl cyclase domain, which is required for cyclic GMP synthesis. Purified XC_0250 is active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 contains a cyclic GMP binding site and a structure–function analysis, directed by determination of the crystal structure of the holo-complex and enzymatic assay confirm the cyclic GMP binding residues, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector XC_0249 that directly links cyclic GMP and cyclic di-GMP signaling. Besides, we also tried to determine the structure of SM_RpfGD81E which can interact with GGDEF domain of SM_0233, a XC_0249 homologous protein. We have obtained the crude full length SM_RpfGD81E crystal structure in complex with cyclic di-GMP. Further refinement is necessary to truly reveal its c-di-GMP binding mode.目錄 致謝....................................................................................................................................i 中文摘要...........................................................................................................................ii Abstract............................................................................................................................iii 目錄..................................................................................................................................iv 表目錄.............................................................................................................................vii 圖目錄............................................................................................................................viii 縮寫檢索表.......................................................................................................................x 第一章、 前言................................................................................................................1 第二章、 材料與方法....................................................................................................5 一、 轉位子突變與基因篩選........................................................................5 二、 辨識轉位子突變的插入點....................................................................5 三、 表現載體之構築....................................................................................5 (一)、本實驗室染色體 DNA 之取得...................................................5 (二)、目標引子的設計............................................................................6 (三)、聚合酶鏈鎖反應............................................................................7 (四)、膠體電泳........................................................................................8 (五)、DNA 之純化與萃取.....................................................................8 (六)、Vector及Insert的製備..................................................................8 (七)、Ligation-Independent cloning......................................................11 (八)、E. coli 勝任細胞之製備.............................................................12 (九)、轉殖作用......................................................................................12 (十)、質體DNA之抽取........................................................................13 (十一)、PCR check 與DNA 定序. .....................................................13 四、 環核苷酸環化酶實驗..........................................................................14 五、 RNA萃取與製備.................................................................................14 六、 蛋白質之大量表現與純化..................................................................14 (一)、蛋白質之大量表現......................................................................15 (二)、以親和性管柱進行目標蛋白的初步純化..................................15 (三)、TEV 蛋白水解酶作用................................................................16 (四)、使用凝膠過濾法純化目標蛋白..................................................16 七、利用X-ray 晶體繞射技術解析蛋白質之結構...................................17 (一)、蛋白質結晶條件篩選...............................................................17 (二)、protein 與ligand 的共結晶.......................................................18 (三)、protein 與protein 的共結晶.....................................................18 (四)、繞射數據的收集.........................................................................19 (五)、決定相位角.................................................................................19 (六)、結構的建立與精算.....................................................................19 八、點突變.................................................................................................20 九、利用區分熱穩定度來篩選適當的蛋白緩衝溶液.............................22 十、恆溫滴定微卡計實驗.........................................................................22 十一、熱示差掃描實驗.............................................................................23 十二、XC_0249酵素活性測試.................................................................23 第三章、 結果..............................................................................................................25 一、 基因篩檢證實在Xanthomonas中XC_0250是參與cyclic GMP生合成..........................................................................................................25 二、 XC_0250的cyclase domain具有合成cyclic GMP活性.....................25 三、 XC_0250對於最大致病性和生物膜形成是很重要的......................26 四、 XC_0249是一個會結合cyclic GMP的蛋白......................................27 五、 XC_0249的調節特性和XC_0250是有重疊的..................................28 六、 Cyclic GMP透過結合XC_0249的cyclic NMP-binding domain去調節cyclic di-GMP的訊息傳遞..............................................................28 七、 XC_0249所調控的生物膜合成與基因轉錄是仰賴它自身的DGC活性..........................................................................................................29 八、 SM_RpfGD81E具有能和cyclic di-GMP結合的能力...........................29 九、 SM_RpfGD81E結構探討.......................................................................30 十、 SM_0233的GGDEF domain與SM_RpfG之間的蛋白交互作用..31 十一、 根據熱穩定度篩選更適合的XC_0249 full-length以及SM_RpfGD81E緩衝液..................................................................................................32 第四章、 討論..............................................................................................................33 參考文獻.........................................................................................................................36 表目錄 表一、本研究各個蛋白結晶條件與晶體照片...............................................................42 表二、基因表現在XC_0249及XC_0250突變株中跟wild-type有差異的基因..........43 表三、利用ITC分析XC_0249WT以及其突變株和核苷酸的結合能力.......................51 表四、XC_0249 cNMP domain和cyclic GMP複合體的繞射數據與修正...................52 表五、SM_RpfGD81E 和 cyclic di-GMP 複合體繞射數據...........................................54 圖目錄 圖一、透過轉位子突變鑑定推測的鳥糞嘌呤環化酶...................................................55 圖二、XC_0250 cyclase domain和其他物種的胺基酸序列比對..................................56 圖三、單獨的XC_0250 CYC domain具有鳥糞嘌呤環化酶的活性.............................57 圖四、XC_0250酵素活性對於cyclic GMP生合成與生物膜形成的影響...................58 圖五、XC_0250突變株對於Xcc致病性和生物膜合成的影響....................................59 圖六、XC_0249蛋白的組成以及結合cyclic GMP能力的探討...................................60 圖七、ITC以及晶體結構分析顯示XC_0249是一個cyclic GMP結合蛋白................61 圖八、XC_0249 cNMP domain與cyclic GMP的結構圖...............................................62 圖九、XC_0249和E. coli CRP (EcCRP)的胺基酸序列比對.........................................63 圖十、利用ITC檢測XC_0249不同突變株和cyclic GMP的結合能力.......................64 圖十一、比較XC_0249以及XC_0250在轉錄組和表現型的影響..............................65 圖十二、Cyclic GMP濃度存在多寡與XC_0249酵素活性之關係..............................66 圖十三、Cyclic GMP的結合會影響XC_0249蛋白的雙鳥糞嘌呤環化酶活性，並且影響其他基因的表現.....................................................................................................67 圖十四、XC_0249的DGC活性會因為加入cyclic GMP而提升..................................68 圖十五、XC_0249突變株 (F73A, E90A)生物膜合成的能力比wild type來的差.......69 圖十六、SM_RpfGD81E和XC_RpfGD80E對於cyclic di-GMP的結合能力測試............70 圖十七、SM_RpfGD81E-cyclic di-GMP的初步結構.......................................................71 圖十八、SM_RpfGD81E和PmGH 的HD-GYP domain的疊合比較..............................72 圖十九、SM_RpfGD81E和PmGH HD-GYP domain binding site比較............................73 圖二十、SM_RpfGD81E HD-GYP domain binding site的疏水性鍵結............................74 圖二十一、SM_0233GGDEF+SM_RpfGWT 的膠體過濾法層析圖與SDS-PAGE圖......75 圖二十二、SM_RpfGD81E +SM_0233GGDEF 的膠體過濾法層析圖與SDS-PAGE圖.....................................................................................................................................76 圖二十三、SM_RpfGD81E +SM_0233GGDEF+cyclic di-GMP的晶體..............................77 圖二十四、SM_RpfGD81E 不同緩衝液的熱穩定度測試..............................................78 圖二十五、XC_0249 full length不同緩衝液的熱穩定度測試............................................79 圖二十六、Xcc中針對cyclic GMP和cyclic di-GMP所調控的生物膜合成以及致病性的模擬示意圖.............................................................................................................80 圖二十七、XC_0249 cNMP domain (紅色) 和 E. coli CRP (1G6N, 藍色) 的疊合比較.....................................................................................................................................8