中文摘要
細胞結合因子(HpCBF2)為胃幽門螺旋桿菌的一個毒素因子，能夠透過第四型類鐸受體(Toll-like receptor 4)轉活化胃上皮細胞中的上皮生長因子受體進而造成異常的細胞增生。此外，HpCBF2也參與蛋白質的折疊，影響蛋白質折疊的速率，在細胞外膜蛋白熟成機制佔有重要的地位。HpCBF2已成功大量表現及純化，其分子量為31,910 Da，在水溶液中以二聚體的聚合型式。HpCBF2以二聚體進行其生物功能，利用胜肽受質 (Suc-AAPF-pNA) 進行的活性測試 (activity assay)，測得HpCBF2對脯胺酸順反異構化的催化效率 (kcat/Km) 為238 ± 7.2 mM-1s-1。我們利用Jeffamine M-600 為沉澱劑培養得到HpCBF2晶體。其晶體的空間群 (space group) 為P3121，晶格參數為a = 61.7 Å, b = 61.7 Å, c = 367.0 Å，每個不對稱單元中含有兩個HpCBF2分子，其中水溶液含量為61%，Vm值為3.16 Å3Da−1。我們利用單波長異常散射方法(single wavelength anomalous dispersion method)計算其相位角，決定HpCBF2 立體結構，解析度為2.6 Å。每個HpCBF2單體具有肽基脯胺酸順反異構酶 (PPIase)及伴隨蛋白(chaperone)兩個功能區。PPIase功能區主要以三條α螺旋及三條β折疊組成，其的受質結合區主要以六個保留性殘基所構成，為His131, Asp169, Leu181, Met189, Phe193, Phe215 及 His217.。 Chaperone功能區以四條α螺旋所組成，而兩個單體會經由一 α螺旋互相纏繞而形成功能區調換(domain swapped)的現象。這些分子結構的資訊提供了胃幽門螺旋桿菌外膜細胞熟成機制的見解及對抗胃幽門螺旋桿菌藥物開發的發想。

The cell binding factor 2 of Helicobacter pylori (HpCBF2) is a virulence factor and transactivates epidermal growth factor receptor (EGFR) through TLR4 in gastric epithelial cells and lead to abnormal cell proliferation. HpCBF2 catalyzed cis-trans isomerization of proline peptide bond on oligopeptide. HpCBF2 also plays an important role in outer membrane protein maturation and may participate in protein folding at the rate-limiting step. HpCBF2 has been overexpressed and isolated with a molecular weight of 31,910 Da. HpCBF2 functions as a dimer. The isomerization activity of HpCBF2 was performed by a coupling enzyme assay using Suc-AAPF-pNA as a substrate analog. The catalytic efficiency (kcat/Km) of HpCBF2 was calculated as 238 ± 7.2 mM−1s−1. HpCBF2 was crystallized by means of Jeffamine M-600 as a precipitant. HpCBF2 crystal belongs to P3121 space group with cell parameters a = 61.7 Å, b = 61.7 Å, c = 367.0 Å, at 2.6 Å resolution. There are two molecules per asymmetric unit with Vm of 3.16 Å3Da−1 and solvent content of 61%. The crystal structure of HpCBF2 was determined by the single anomalous dispersion (SAD) method via the selenium derivative. The overall structure of HpCBF2 contains a PPIase domain and a chaperone domain. The PPIase domain is composed of three β strands and α helices and the substrate-binding pocket consists of six conserved residues, His131, Asp169, Leu181, Met189, Phe193, Phe215 and His217. The chaperone domain is composed of four α helices and two chaperone domains build up a domain-swapped architecture. These structural results provide a understanding of the biological function of HpCBF2 and suggest the possibility of the drug design on HpCBF2 as well as the potential alinic application in H.pylori.

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