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Influence of mutations at the N domain of XpsE on its structural and functional properties
|關鍵字:||XpsE protein;XpsE蛋白||出版社:||生物化學研究所||引用:||參考文獻 1. Abendroth J, Murphy P, Sandkvist M, Bagdasarian M, Hol WG (2005) The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J Mol Biol 348: 845-55 2. Bitter W, Koster M, Latijnhouwers M, de Cock H, Tommassen J (1998) Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27: 209-19 3. Chien IL (2002) Purification and characterization of the XpsE protein of the type II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 4. Camberg JL, Sandkvist M (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187: 249-56 5. 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十字花科黑腐病菌中的第二型蛋白分泌系統由12個蛋白所組成，能夠將在胞內合成的多種水解酶分泌至胞外，破壞植物細胞的表面構造。XpsE是第二型蛋白分泌系統中唯一的胞內蛋白，具有ATPase活性，在與ATP結合後，會聚合成六倍體且與內膜上的XpsL的N端 (XpsLN) 交互作用，XpsE主要是以N端區域與XpsLN結合。根據XpsE的N端晶體結構得知，XpsEN有open form與closed form兩種構形，推測只存在於closed form構形中的疏水性表面區域，可能參與XpsE與XpsLN的結合。先前藉由三定點突變 (V11A,L25A,L39A) 的實驗發現突變蛋白XpsE(TM11,25,39) 明顯降低與XpsLN結合的能力，並喪失分泌功能。本研究為進一步了解此三定點突變蛋白與正常XpsE的差異，進行三方面的分析。首先，利用疏水性螢光探針ANS對XpsE與XpsE(TM11,25,39) 分別進行疏水性結合的標定，發現XpsE(TM11,25,39) 的螢光訊號明顯較XpsE強，暗示此三定點突變可能導致XpsE結構產生變化，暴露出較多的疏水性區域。其次，利用gel filtration比較由affinity column純化得到的XpsE與 XpsE(TM11,25,39) 其monomer與oligomer的分佈情形，發現 XpsE(TM11,25,39) 相對於XpsE有較高比例的oligomer存在。最後，在ATPase活性方面，發現在高鹽 ( 500 mM NaCl ) 以及含大量ATP的緩衝液清洗的純化方式下，測得XpsE(TM11,25,39) 蛋白的ATPase活性約為野生型XpsE蛋白的四倍。
Type II secretion apparatus of Xanthomonas campestris pv. campestris is constituted of twelve proteins, and mediates the translocation of hydrolytic enzymes from periplasm to the milieu, possibly to destroy the surface component of plant cell. XpsE is the only cytoplasmic protein component in the type II secretion apparatus, and exhibits ATPase activity. ATP binding triggers XpsE to form hexamer as well as its association with the N-terminal domain of the cytoplasmic membrane protein XpsL (XpsLN). XpsE utilizes its N-terminal domain to associate with XpsLN. Two crystal structures of XpsEN were revealed, designated as an open and a closed form that differ at their N-termini. A hydrophobic patch detected only in the closed form was proposed to be involved in the interaction between XpsE and XpsLN. Triple alanine mutations at Val11, Leu25, Leu39 caused XpsE lose secretion function and the ability to bind XpsL. To further characterize the mutant XpsE(TM11, 25, 39), it was analyzed in parallel with wide type XpsE in three aspects. First of all, a fluorogenic probes ANS was utilized for monitoring possible difference in their hydrophobicity. The fluorescence signal of XpsE(TM11, 25, 39) is significantly stronger than that of XpsE, suggesting conformation change in XpsE conformation may have been caused by triple mutation. Secondly, size exclusion chromatography of the affinity column-purified Strep-tagged XpsE and XpsE(TM11, 25, 39) revealed difference in the proportion of their monomer and oligomer. The XpsE(TM11, 25, 39) appeared as oligomeric form at higher proportion than the wild type XpsE. Finally, the ATP activity of the XpsE(TM11, 25, 39) that has been washed with buffer containing high salt and large amount of ATP is about four-times that of the wide-type XpsE.
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