利用核磁共振光譜研究趨化素CXCL9的結構特性及與CXCR3受體胞外區結合能力

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研究生：	羅巧玲 Lo, Chiao-Ling
論文名稱：	利用核磁共振光譜研究趨化素CXCL9的結構特性及與CXCR3受體胞外區結合能力 NMR study on CXCL9 structure and the interaction with the extracellular regions of CXCR3 receptor
指導教授：	蘇士哲 Sue, Shih-Che
口試委員:	徐駿森 Hsu, Chun-Hua 蕭育源 Hsiao, Yu-Yuan
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	56
中文關鍵詞：	CXCL9 、趨化因子、核磁共振 (NMR) 、CXCR3 、胞外區域
外文關鍵詞：	CXCL9, chemokine, NMR, CXCR3, extracellular regions
相關次數：	點閱：2 下載：0
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趨化因子 (chemokine) 藉由結合細胞表面的G蛋白偶聯受體 (GPCR) 來趨化細胞。CXCL9屬於CXC型趨化因子家族，由干擾素-γ (IFN-γ) 誘導，並且與其他兩類趨化因子CXCL10和CXCL11密切相關。因為這三種趨化因子會結合到相同的GPCR受體CXCR3。彼此間的相互作用促進了免疫細胞的遷移與活化以及與腫瘤抑制有關。特別是CXCL9主要以吸引T淋巴細胞為主，因此參與了許多生理疾病的發病機制。CXCL9在結構方面資訊缺乏，如何與CXCR3專注結合的資訊也有限，本研究希望了解CXCL9的結構特性，並探討CXCR3和CXCL9之間的結合特異性。我們發現在生理環境下，CXCL9以單體形式存在，這樣的形式使其應該能直接與CXCR3作用。而我們也完成了CXCL9骨架胺基酸序列判定 (backbone assignment) 並從骨架胺基酸之化學位移評估了CXCL9二級結構。CXCL9擁有典型的趨化因子結構，折疊成三個反向平行β折板與一個α螺旋。不同的是，CXCL9 具有一個延伸的C末端，其中的序列包含許多正電荷殘基。經由骨架胺基酸的化學位移和圓二色光譜實驗發現延伸區域不具二級結構特性 (random coil)，而C端延伸似乎與CXCL9核心結構有短暫的交互作用，我們也證實CXCL9對pH敏感的特性。最後，我們測試了CXCL9與CXCR3不同的胞外區間之結合，發現CXCL9與CXCR3 N端結合作用最強，因此我們認為CXCR3 N端扮演吸引CXCL9與CXCR3結合的角色。

Chemokines induce chemotaxis through binding G protein-coupled receptors (GPCRs) on cell surface. CXCL9 belongs to CXC-type chemokine family, induced by interferon-γ (IFN-γ), and closely related to CXCL10 and CXCL11. The three chemokines bind the same GPCR target, CXCR3, and the interactions further contribute the immune cell migration, activation, and tumor suppression. Particularly, CXCL9 attracts T lymphocytes, therefore, involved in the pathogenesis of physiologic diseases. There is limited information of CXCL9 structure and binding behavior with CXCR3. The study intends to understand the structural property of CXCL9, and subsequently, investigate the binding specificity between CXCR3 and CXCL9. Under a physiological condition, we characterized CXCL9 as a monomer, allowing to directly interact with CXCR3. We finished the NMR protein backbone assignment and evaluated its secondary structure from backbone chemical shifts. CXCL9 core adopts a canonical chemokine structural fold that is a three-stranded antiparallel β-sheet followed by an α-helix. Differently, CXCL9 further consists a highly basic C-terminal extension where the sequence contains many positively charged residues. The backbone chemical shifts together with circular dichroism (CD) spectrum reported the C-terminal extension to have a random coil-like structure and a transient interaction with the CXCL9 core. In additional, a pH-sensitive property of CXCL9 was charaertized. In final, we tested the binding between CXCL9 and different CXCR3 extracellular regions. The CXCR3 N-terminal peptide has the most effect in binding, therefore, establishing its potent role in recruiting CXCL9 to recognize CXCR3.

Abstract    2
中文摘要    3
第一章、前言    7
1 趨化因子 (Chemokines)    7
1.1 CXCL9    7
2 趨化因子與G蛋白偶聯受體 (G-protein coupled receptor, GPCR) 相互作用    8
2.1 GPCR    8
2.2 CXCR3    9
3 研究目的    10
第二章、材料與方法    19
1 重組蛋白的表現與純化    19
1.1 CXCL9表現和純化    19
1.2 CXCL91-73表現和純化    19
1.3 同位素標記蛋白的培養    20
2 胜肽設計與合成    20
3 圓二色光譜實驗 (circular dichroism, CD)    20
4 粒徑篩層析-多角度散射分析 (SEC-MALS)    21
5 三維核磁共振骨架光譜實驗    21
6 I-PINE自動判定骨架光譜之胺基酸(NMR automated backbone assignment)    21
7 核磁共振1H-15N-二維光譜滴定實驗    22
8 Phyre2軟體結構預測    23
第三章、實驗結果    24
1 重組蛋白CXCL9表現與純化    24
2 CXCL9在生理環境下之寡聚體狀態    24
3 模擬結構模型    24
4 核磁共振 (Nuclear Magnetic Resonance, NMR) 技術分析水溶性蛋白CXCL9之單體    25
4.1 CXCL9分子骨架在二維光譜HSQC之胺基酸判定 (backbone assignment)    25
4.2 趨化因子CXCL9對酸鹼環境的敏感性    25
5 探討截短C端的CXCL9(CXCL91-73)對結構上的影響    26
5.1 CXCL91-73之純化    26
5.2 CXCL91-73分子骨架在二維光譜HSQC之胺基酸判定 (backbone assignment)    27
5.3 比較CXCL9和CXCL91-73之間的結構變化    27
6 CXCL9和CXCR3胜肽片段之交互作用    28
第四章、討論    50
1 趨化因子CXCL9與CXCR3其他配體結構差異性    50
2 CXCR3與CXCL9間之交互作用    50
參考資料    53


                                

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