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研究生:王永勝
研究生(外文):Yong-Sheng Wang
論文名稱:以調節中東呼吸道症候群冠狀病毒核殼蛋白N端功能區之蛋白質交互作用方式開發抗病毒藥物
論文名稱(外文):Development of antiviral drug by modulating the protein-protein interactions of the N-terminal domain of MERS-CoV nucleocapsid protein
指導教授:侯明宏
口試委員:孫英傑陳俊榮李弘文
口試日期:2016-07-29
學位類別:碩士
校院名稱:國立中興大學
系所名稱:基因體暨生物資訊學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:158
中文關鍵詞:中東呼吸道症候群冠狀病毒核殼蛋白N端功能區
外文關鍵詞:Middle East respiratory syndrome coronavirusnucleocapsid proteinN-terminal domain
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西元2012年,中東呼吸道症候群冠狀病毒 (Middle East respiratory syndrome coronavirus) 在沙烏地阿拉伯首次被發現,隨後在中東地區及南韓爆發大規模感染,造成1768人感染,其中有630死亡,致死率高達35 %以上,至今仍有感染的病例。一般來說,冠狀病毒也會引起人類的一般性感冒,更嚴重的像是嚴重呼吸道症候群 (severe acute respiratory syndrome) 及中東呼吸道症候群,會造成患者在短時間內死亡,會危及各國人民的健康,也會間接地影響到經濟,因此了解冠狀病毒的感染途徑,開發藥物治療冠狀病毒所引起的疾病是刻不容緩的,然而冠狀病毒屬於正向單股的RNA病毒,其基因體的變異性相當高,容易造成病毒蛋白突變,進而導致抗藥性的產生,使藥物開發的難度提高,不過,冠狀病毒的生命週期中會表現特定的結構性蛋白,這些蛋白具有高度的序列保留性,其中核殼蛋白扮演了相當重要的角色。核殼蛋白會包裹並結合病毒的基因體RNA形成長鏈螺旋狀的核糖核蛋白複合體 (ribonucleoprotein complex, RNP),進而調節其轉錄與複製,因此核殼蛋白對於藥物開發與設計是一個很好的標的,核殼蛋白在結構上有兩個獨立的結構功能域,其中N端功能域主要與RNA的結合有關。本篇研究以具有高度序列保留性的核殼蛋白N端功能區為藥物設計之標的,藉由結晶結構及點突變之結構分析,發現中東冠狀病毒核殼蛋白N端功能區二聚體之交界面有一個具有潛力的藥物結合位,並分析其胺基酸組成,這個結合位不同於RNA結合位,針對這個結合位我們進一步地進行電腦模擬藥物篩選 (virtual screening) 實驗,從龐大的小分子資料庫中篩選出三個有潛力和重要結合位結合的化合物 (P1-P3) ,從螢光光譜分析實驗,我們也發現不管是MERS-CoV N-NTD或MERS-CoV核殼蛋白皆具有藍移的現象,我們更進一步解析出藥物與MERS-CoV N-NTD之複合體結構,再分析小分子藥物與核殼蛋白N端功能區複合體之間的結合及作用方式,最後發現P3可以作為一個媒介,結合在MERS-CoV N-NTD二聚體之交界面,進而穩定MERS-CoV N-NTD二聚體的結構,且在小角度X光散射實驗更證實P3可以促進MERS-CoV核殼蛋白的聚合能力,然而P3並沒有顯著地干擾MERS-CoV核殼蛋白結合RNA的能力,因此我們認為P3是藉由異位調控的方式影響MERS-CoV核殼蛋白的功能,本篇的實驗結果也對於未來抗冠狀病毒藥物發展提供一個新方向。

Coronaviruses (CoVs) cause numerous diseases, including Middle East respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. The development of anti-CoV drug is very urgent. Nucleocapsid protein (N protein), an essential RNA-binding protein in coronavirus, is required for the replication and transcription of viral RNA and shows the high conservation. Coronavirus N proteins contain three intrinsically disordered regions (IDRs): N-arm, central linker region (LKR) and C-tail, and two structural domains: N-terminal domain (NTD) and C-terminal domain. In this study, we aim to utilize the MERS-CoV N-NTD as a target for anti-viral drug development. First, we solved the structure of MERS-CoV N-NTD. In this structure, there were 4 protomers which formed homodimer in an asymmetric unit. We also verified the dimer comformation in the solution by chemical cross-linking assay. In the dimer interface, we found that the side chain of N39 (chain A) protrudes to the hydrophobic core of chain C and interacts with aromatic ring of W43. In order to identify the importance role of N39 in the dimer interface, we performed the site-directed mutagenesis to mutate the N39 to Alanine or Glycine. After solving the structure of MERS-CoV N-NTD N39A and N39G, we found that M37 prior to N39 occupied at the hydrophobic pocket. According to this result, we focused on the hydrophobic interface of dimer structure of MERS-CoV N-NTD as target to perform the structure-based virtual screening using the Zinc database to identify 3 compounds (P1-P3). We determined each crystal structure of the N-NTD-drug complex. P3 binds to the dimer interface of N-NTD with more interaction force, compared to P1 and P2. In the fluorescence spectra measurement, the intrinsic fluorescence of N protein and N-NTD showed the blue-shift in the presence of P3. In addition, P3 could enhance the oligomerization of N protein by chemical cross-linking and small angle scattering assay. P3 didn''t affect the binding of N protein to RNA siginificantly by SPR experiment. Although the dimer interface of N-NTD might not be directly involved in RNA binding, the binding of small molecule compounds to this site could still interfere with N protein structure and function, for example through allostery, and could be alternative targets for antiviral drug development. In summary, we found that P3 could bind to the dimer interface and modulate the oligomerization properties of MERS-CoV N protein. These results will be useful for the development of antiviral agents against CoV.

目次
第一章、 前言 1
一、 冠狀病毒(coronavirus) 1
(一)、 冠狀病毒概述 1
(二)、 冠狀病毒分類 2
(三)、 冠狀病毒基因體 (Genome) 3
(四)、 冠狀病毒感染與生命週期 4
(五)、 冠狀病毒的結構性蛋白及其功能 5
(六)、 冠狀病毒核殼蛋白N端功能區之結晶結構 7
二、 研究動機 7
第二章、 材料與方法 9
一、 實驗流程 9
二、 實驗方法 10
(一)、 ordered及disordered區域預測 10
(二)、 序列比對分析 10
(三)、 構築蛋白質表現載體 10
(四)、 蛋白質之大量表現與純化 17
(五)、 利用X-ray晶體繞射技術解析蛋白質之結構 28
(六)、 化學交聯 (chemical cross-linking) 實驗 38
(七)、 定點突變 (site-directed mutagenesis) 41
(八)、 電腦模擬分子對接實驗 43
(九)、 螢光光譜分析法 (Fluorescence spectroscopy) 44
(十)、 蛋白質熱穩定性分析 (Thermostability measurements) 46
(十一)、 蛋白質與藥物共結晶 (co-crystallization) 實驗 48
(十二)、 蛋白質結晶浸泡實驗 50
(十三)、 小角度X光散射 (small-angle X-ray scattering, SAXS) 52
(十四)、 表面電漿共振 (surface plasmon resonance, SPR) 實驗 53
第三章、 結果 56
一、 構築 HCoV-OC43 核殼蛋白N 端功能區之表現載體 56
(一)、 MERS-CoV 核殼蛋白order 與disorder region預測 56
(二)、 MERS-CoV 核殼蛋白N 端功能區截切蛋白的構築 56
二、 MERS-CoV 核殼蛋白N 端功能區截切蛋白大量表現與純化 56
(一)、 MERS-CoV 核殼蛋白N 端功能區截切蛋白之誘導蛋白質大量表現 56
(二)、 MERS-CoV 核殼蛋白N 端功能區截切蛋白的純化 57
(三)、 MERS-CoV 核殼蛋白N 端功能區截切蛋白的濃縮與透析 57
三、 MERS-CoV 核殼蛋白N 端功能區截切蛋白之結晶結構 58
(一)、 MERS-CoV核殼蛋白N 端功能區截切蛋白的結晶條件篩選 58
(二)、 X-ray 繞射數據分析與單位晶格判斷 58
(三)、 利用分子取代法 (molecular replacement) 進行結構解析 59
四、 以化學交聯分析MERS-CoV N-NTD在溶液中的構型 59
五、 定點突變之MERS-CoV 核殼蛋白N 端功能區之表現與純化 60
(一)、 突變株 N39A 60
(二)、 突變株 N39G 60
六、 MERS-CoV 核殼蛋白N 端功能區突變株N39A之結晶結構 61
(一)、 MERS-CoV 核殼蛋白N 端功能區突變株 N39A之結晶 61
(二)、 X-ray 繞射數據分析與單位晶格判斷 61
(三)、 利用分子取代法 (molecular replacement) 進行結構解析 62
七、 MERS-CoV 核殼蛋白N 端功能區突變株N39G之結晶結構 62
(一)、 MERS-CoV 核殼蛋白N 端功能區突變株 N39G之結晶 62
(二)、 X-ray 繞射數據分析與單位晶格判斷 62
(三)、 利用分子取代法 (molecular replacement) 進行結構解析 63
八、 以電腦模擬分子對接尋找有潛力的小分子藥物 64
(一)、 預測可能的藥物結合位 64
(二)、 電腦模擬分子對接 64
九、 以螢光光譜分析MERS-CoV N-NTD與小分子藥物之交互作用 65
十、 小分子藥物對MERS-CoV N-NTD之熱穩定性分析 66
十一、 以化學交聯分析P3對MERS-CoV N-NTD二聚體構型之影響 66
十二、 P1和MERS-CoV N-NTD之複合體結晶結構 67
(一)、 P1和 MERS-CoV N-NTD 之複合體結晶 67
(二)、 X-ray 繞射數據分析與單位晶格判斷 67
(三)、 MERS-CoV N-NTD與P1複合體之結晶結構解析 67
十三、 P2和MERS-CoV N-NTD之複合體結晶結構 68
(一)、 小分子藥物和 MERS-CoV N-NTD 之複合體結晶 68
(二)、 X-ray 繞射數據分析與單位晶格判斷 68
(三)、 MERS-CoV N-NTD與P2複合體之結晶結構解析 69
十四、 P3和MERS-CoV N-NTD之複合體結晶結構 69
(一)、 小分子藥物和 MERS-CoV N-NTD 之複合體結晶 69
(二)、 X-ray 繞射數據分析與單位晶格判斷 69
(三)、 MERS-CoV N-NTD與P3複合體之結晶結構解析 70
十五、 MERS-CoV 核殼蛋白 (野生型全長) 純化 71
十六、 以螢光光譜分析MERS-CoV核殼蛋白與小分子藥物之交互作用 71
十七、 小分子藥物對MERS-CoV核殼蛋白之熱穩定性分析 72
十八、 以小角度X光散射實驗分析P3對MERS-CoV核殼蛋白聚合能力之影響 73
十九、 以表面電漿共振 (surface plasmon resonance, SPR) 實驗探討MERS-CoV核殼蛋白與RNA結合能力 73
第四章、 討論 75
一、 MERS-CoV核殼蛋白order與disorder region及二級結構的預測 75
二、 分析與比較冠狀病毒核殼蛋白N端功能區的結構 75
三、 MERS-CoV N-NTD之定點突變 76
四、 分析與比較MERS-CoV N-NTD野生型與突變株之結構差異 77
五、 分析與比較MERS-CoV核殼蛋白N端功能區結構與複合體結構之差異 78
(一)、 MERS-CoV N-NTD與P1之複合體結晶結構 78
(二)、 MERS-CoV N-NTD與P2之複合體結晶結構 78
(三)、 MERS-CoV N-NTD與P3之複合體結晶結構 79
六、 比較小分子藥物複合體結合位與模擬分子對接結合位之差異 79
七、 P3對MERS-CoV核殼蛋白聚合能力之影響 80
八、 MERS-CoV核殼蛋白與單股RNA序列結合能力分析及探討 80
九、 以冠狀病毒核殼蛋白為標的之藥物開發 81
第五章、 結論 82
第六章、 表次 83
第七章、 圖次 92
第八章、 參考文獻 152



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