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標題: 十字花科黑腐病菌之XpsE蛋白與XpsF蛋白N端區域之純化與結晶
Purification and crystallization of the XpsE and XpsF N-terminal domains of the Xanthomonas campestris type II secretion system
作者: 詹朝翔
Chan, Chao-Hsiang
關鍵字: 第二型分泌機制;XpsE
出版社: 生物化學研究所
引用: 參考文獻 Bally, M., Filloux, A., Akrim, M., Ball, G., Lazdunski, A. and Tommassen, J. (1992) Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol, 6, 1121-1131. Chen, L.Y., Chen, D.Y., Miaw, J. and Hu, N.T. (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem, 271, 2703-2708. Chen, Y., Shiue, S.J., Huang, C.W., Chang, J.L., Chien, Y.L., Hu, N.T. and Chan, N.L. (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem, 280, 42356-42363. Drenth, J. (1999) Principle of Protein X-Ray Crystallography. Dums, F., Dow, J.M. and Daniels, M.J. (1991) Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet, 229, 357-364. Hu, N.T., Hung, M.N., Chiou, S.J., Tang, F., Chiang, D.C., Huang, H.Y. and Wu, C.Y. (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol, 174, 2679-2687. Hu, N.T., Leu, W.M., Lee, M.S., Chen, A., Chen, S.C., Song, Y.L. and Chen, L.Y. (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J, 365, 205-211. Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A, 47 (Pt 2), 110-119. Kostakioti, M., Newman, C.L., Thanassi, D.G. and Stathopoulos, C. (2005) Mechanisms of protein export across the bacterial outer membrane. J Bacteriol, 187, 4306-4314. Lee, H.M., Tyan, S.W., Leu, W.M., Chen, L.Y., Chen, D.C. and 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. Lee, H.M., Wang, K.C., Liu, Y.L., Yew, H.Y., Chen, L.Y., Leu, W.M., Chen, D.C. and Hu, N.T. (2000) Association of the cytoplasmic membrane protein XpsN with the outer membrane protein XpsD in the type II protein secretion apparatus of Xanthomonas campestris pv. campestris. J Bacteriol, 182, 1549-1557. Matthews, B.W. (1968) Solvent content of protein crystals. J Mol Biol, 33, 491-497. Nunn, D.N. and Lory, S. (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol, 175, 4375-4382. Planet, P.J., Kachlany, S.C., DeSalle, R. and Figurski, D.H. (2001) Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci U S A, 98, 2503-2508. Py, B., Loiseau, L. and Barras, F. (2001) An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep, 2, 244-248. Robien, M.A., Krumm, B.E., Sandkvist, M. and Hol, W.G. (2003) Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol, 333, 657-674. Sandkvist, M., Hough, L.P., Bagdasarian, M.M. and Bagdasarian, M. (1999) Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae. J Bacteriol, 181, 3129-3135. Tsai, R.T., Leu, W.M., Chen, L.Y. and Hu, N.T. (2002) A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J, 367, 865-871. Walker, J.E., Saraste, M., Runswick, M.J. and Gay, N.J. (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. Embo J, 1, 945-951.

十字花科黑腐病菌(Xanthomonas campestris pv. campestris)係利用第二型蛋白分泌機制(type II secretion pathway)將各種水解酵素運送至胞外。由已知研究得知此分泌機器運作至少需要12個蛋白,其中XpsE是此機制中唯一的胞內蛋白,由於XpsE蛋白序列中含有4個與nucleotide binding有關的motifs,因而被預測為一個可提供能量的ATPase。若與第四型分泌機制之ATPase VirB11比較,XpsE蛋白可被區分為32 kDa的N端區域(1-291)及31 kDa的C端區域( 292-567 ),其中N端區域包含與XpsL結合的subregion (1-153),而nucleotide binding motifs則位於XpsE的C端區域內。由文獻得知,XpsE N端之subregion (1-153)可形成兩種不同的構形,此外由生化分析亦顯示XpsE蛋白必須在ATP存在時才能與XpsL蛋白結合,且ATP能藉由N端區域之subregion(154-291)的參與引發subregion(1-153)之構形變化,為了進一步了解ATP與此構形改變的關連性及其對第二型分泌機制的影響,因此本實驗室曾經嘗試利用X-ray繞射法分析XpsE蛋白N端區域(1-291)的結構,先了解XpsE蛋白N端兩個subregions之間的交互作用方式。然而由於此蛋白晶體易於胺基酸R176及D177間斷裂,因此本研究構築了一個包含R176S、D177A雙突變的XpsEN (1-291)(在本研究中皆以XpsEN(1-291)DM稱之)以其獲此二subregions間交互作用的訊息。經過大量表現、管柱層析純化及晶體培養順利得到繞射解析度達2.3 Å、空間群為I4122 tetragonal晶系的蛋白質晶體,而經由與XpsEN(1-153)蛋白晶體結構之比較,我們發現這兩個結構不論是在單位晶格或是空間群、還是重疊後之立體結構上,皆高度相似,因此推斷經由XpsEN (1-291)DM蛋白所獲得之蛋白之立體結構與XpsEN(1-153)蛋白是相同的。此外由胡念台老師實驗室研究之結果顯示,當ATP存在,XpsE中XpsEN(1-152)與XpsEN(153-291)兩個蛋白片段間可能存在較強之交互作用,因此在XpsEN(1-291)兩個subregions之間交互作用微弱的情況下,蛋白晶體可能較不易形成。未來若想要進一步研究XpsE蛋白之結構,我們可能需要全長的XpsE蛋白,並嘗試在ATP存在的情況下獲得其晶體。此外胡念台老師實驗室的初步結果顯示XpsF可能與XpsL以及XpsE具有交互作用,因此我們亦期望能藉由 XpsF N端區域(1-167)晶體結構的解析,使我們更了解第二型分泌機制。

Xanthomonas campestris pv. campestris transports a number of hydrolytic enzymes across bacterial outer membrane by the type Ⅱ secretion pathway. At least 12 Xps protein components are involved in the assembly of the typeⅡsecretion apparatus. XpsE is the the only cytosolic protein without any membrane spanning sequence among the 12 components. Due to the presence of four conserved nucleotide binding motifs, XpsE has been predicted to function as an ATPase. By comparing the protein sequence of XpsE with that of the typeⅣATPase VirB11 with know three-dimensional structure, the XpsE sequence can be divided into the N-terminal domain (XpsE (1-291)) which has been demonstrated to mediate its association with the cytoplasmic membrane protein XpsL and C-terminal domain (XpsE (292- 567)) which includes nucleotide binding motifs. Pull-down assays have shown that XpsE can interact with XpsL in an ATP-dependent manner. Fluorescent spectroscopy measurement further demonstrated that ATP-binding may induce conformation change in the N-terminal region of XpsE. And the highly conserved amino acid residue R286 plays an essential role in the ATP-induced conformation change. To understand the structural basis of this conformation change, our lab had proviously attempted to determine the structure of
XpsE(1-291).However, this protein was found to be cleaved in between R176 and D177 in the crystal. To solve this problem, I construced an expression plasmid for XpsE(1- 291) which includes R176A and D177S mutations (referred as XpsEN(1- 291)DM in this study).
The XpsEN(1-291)DM was expressed, purified, and crystallized in space group I4122. Unfortunately, after solving the structure, it was found that again the XpsEN(1-291)DM was likely cleaved, giving no structure information after residue 149. Because subregions XpsE(1-153) and XpsE(154-291) only interact in the presence of ATP, so the two subregions may interact very weakly, and the flexible region (residues 154-291) remains susceptible to protease cleavage. Therefore, instead of working on fragments of XpsE, one should probably try to crystallize full-length XpsE in the presence of ATP if enough material can be obtained. In addition, we also tried to crystallize XpsF(1-167), which may interact and regulate the ATPase activity of XpsE.
其他識別: U0005-2507200715004700
Appears in Collections:生物化學研究所

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