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標題: 利用螢光蛋白標定分析位於XpsE分子間交互作用介面胺基酸的重要性
Significance analysis of the residues predicted to be involved in XpsE-XpsE interaction by utilizing green fluorescence protein
作者: 李揚
Lee, Yang
關鍵字: Xanthomonas Campestris;十字花科黑腐病菌;XpsE;R385A;D387A;D389A;第二型分泌機制
出版社: 生物化學研究所
引用: 1.Chen, W. S. (2010). Effect of mutation in XpsE at residues near intermolecular interface on its biochemical properties. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 2.Filloux, A. (2004). The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694, 163-179.Snadkvist, M. (2001). Biology of type II secretion. Mol Microbiol, 40, 271-283 3.Lee, H. M., Tyan, S. W., Leu, W. M., Chen, L. Y., Chen, D. C. & Hu, N. T. (2001). Involvement of the XpsN protein in formation of the XpsL-XpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol 183, 528-535. 4.Korotkov, K. V., and W. G. Hol. (2008). Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nat. Struct. Mol. Biol. 15, 462-468. 5.Rapoport TA. (2007). Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature. 450. 663-669. 6.Robien, M.A., Krumm, B.E., Sandkvist, M. and Hol, W.G.J. (2003). Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333, 567-674 7.Sauvonnet, N., Vignon, G., Pugsley, A.P. and Gounon, P. (2000). Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J 19, 2221-2228 8.Sandkvist, M., J. M. Keith, et al. (2000). "Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae." J Bacteriol 182(3), 742-8. 9.Shiue SJ, Kao KM, Leu WM, Chen LY, Chan NL, Hu NT (2006). XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25, 1426-1435 10.Satyshur, K.A., Worzalla, G.A., Meyer, L.S., Heiniger, E.K., Aukema, K.G., Misic, A.M. and Forest, K.T. (2007). Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure 15, 363-376 11.Yamagata, A. & Tainer, J. A. (2007). Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO Journal 26, 878-890. 12.Yu S. L. (2009). Significance of the conserved residues in Walker B motif of XpsE. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C.
XpsE為十字花科黑腐病菌第二型分泌機制唯一的胞內蛋白,先前研究發現XpsE會形成六聚體且會和內膜蛋白XpsL的N端結合,並能水解ATP。同源性蛋白質比對發現XpsE可分成N1、N2、C1及C2 domain,而C1 domain中含有保留性高的Walker A box、Asp box、Walker B box及His box序列。與同源性蛋白AaPilT立體結構比對,推測Walker B box中的R385、D387與D389位於XpsE分子間交互作用介面。已知R385A、D387A及D389A會使XpsE失去功能,導致第二型分泌機制無法運作。為分析R385A、D387A或D389A在XpsE-XpsE交互作用中的重要性,利用ECFP標定XpsE或其突變蛋白,追蹤XpsE-ECFP在細胞內的分佈。已知在xpsD+菌株表現的XpsE-ECFP均勻分布於細胞質,僅在少數細胞的邊緣呈點狀分布。而xpsD-突變株表現的XpsE-ECFP在細胞邊緣呈現點狀分布的細胞數目明顯增加,推測此點狀分布可能與分泌過程中XpsE形成多聚體而與XpsL交互作用有關,因此在xpsD-突變株中追蹤XpsE-ECFP的點狀分布變化可作為判定XpsE是否與鄰近XpsE交互作用的依據。首先在xpsD-突變株以廣寄主性載體分別表現帶有R385A、D387A及D389A的XpsE-ECFP,結果顯示R385A突變會導致XpsE-ECFP點狀聚集數量些微降低,而XpsED387A-ECFP及XpsED389A-ECFP則無明顯點狀分布。其次在xpsD-, xpsE-雙突變株觀察R385A、D387A及D389A對XpsE-ECFP螢光分布的影響,發現相似結果。最後利用同源重組將xpsD-突變株染色體中的xpsE置換成xpsER385A-ecfp或xpsED387A-ecfp,發現XpsER385A-ECFP仍在細胞邊緣呈點狀分布,而XpsED387A-ECFP則無點狀分布,然而螢光強度隨蛋白含量降低而減弱。為進一步確認前述結果,以質體大量表現α-amylase,發現XpsE-ECFP及XpsER385A-ECFP在細胞邊緣聚集的現象被強化,而XpsED387A-ECFP沒有改變。上述結果說明位於XpsE分子交互作用介面的保留性胺基酸,可能只有D387與D389直接參與分子間相互作用,而R385可能扮演其他不同的角色。

XpsE is the only cytoplasmic component of the Xanthomonas campestris pv. campestris type II secretion system (T2SS). Previous studies showed XpsE has the following features: weak ATPase activity, hexamerization as well as direct interaction with the N-terminal domain of the inner membrane protein XpsL. Sequence alignment with XpsE homologous proteins shows XpsE can be subdivided into N1, N2, C1 and C2 domains. Four highly conserved sequences, namely Walker A box, Asp box, Walker B box and His box, are located in C1 domain. Three-dimensional structure of the XpsE homologue AaPilT revealed that the equivalent residues of R385, D387 and D389 within Walker B box are located at the molecular interaction interface. Mutation of R385, D387 or D389 to alanine was shown to cause XpsE lose its function in T2SS. To analyze the significance of these three conserved residues in XpsE-XpsE interaction, I fused ECFP at the C-terminus of XpsE variants and observed their distribution in Xanthomonas campestris. As suggested from previous studies, XpsE-ECFP produced in xpsD+ strain appeared diffused in cytoplasm and exhibited foci formation foci at cell boundary in only a few cells. When expressed in the xpsD- strain, XpsE-ECFP formed foci at cell boundary in most cells. We propose these focal appearing XpsE represents the oligomerized XpsE that interacts with XpsL at a late stage of the secretion process. Thus, foci formation in the xpsD- strain is used here to determine if these residues are involved in XpsE-XpsE interaction. First, I used broad host-range plasmid for expressing XpsE-ECFP with individual mutations, R385A, D387A or D389A, in the xpsD- strain. The observations indicated the XpsE-ECFP foci formation of the variant with R385A mutation was slightly reduced, but D387A and D389A mutation caused the XpsE-ECFP foci formation undetectable. Next, I introduced the plasmid expressing XpsE-ECFP with each mutated XpsE into the xpsD- xpsE- double mutant strain. Similar results were observed. Finally, by performing homologous recombination, I replaced the chromosomal xpsE gene of the xpsD- strain with xpsER385A-ecfp or xpsED387A-ecfp at xpsD- strain. XpsER385A-ECFP remained detectable as foci at cell boundary, and XpsED387A-ECFP did not. However, both exhibited weakened fluorescence intensity and reduced protein abundance. By introducing a plasmid to overexpress α-amylase (a protein known to be secreted via T2SS), I was able to enhance foci formation by XpsE-ECFP and XpsER385A-ECFP, but not XpsED387A-ECFP. In summary, of the three highly conserved amino acids predicted to be located at the XpsE-XpsE interaction interface, only D387 and D389 may be directly involved in XpsE-XpsE interaction, whereas R385 may be significant by serving different roles.
其他識別: U0005-2507201111343200
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