Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24052
標題: 位於相鄰XpsE分子界面之胺基酸突變對其生化特性之影響
Effect of mutation in XpsE at residues near intermolecular interface on its biochemical properties.
作者: 陳玟秀
Chen, Wen-Shiou
關鍵字: XpsE
十字花科黑腐病菌
intermolecular interface
biochemical properties
分子界面胺基酸
生化特性
出版社: 生物化學研究所
引用: Abendroth, J., Murphy, P., Sandkvist, M., Bagdasarian, M., and Hol, W.G. (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, 845-855. Bingle, L.E., Bailey, C.M., and Pallen, M.J. (2008). Type VI secretion: a beginner''s guide. Curr Opin Microbiol 11, 3-8. Camberg, J.L., and Sandkvist, M. (2005). Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187, 249-256. Chan, S., Horner, S.R., Fauchet, P.M., and Miller, B.L. (2001). Identification of Gram negative bacteria using nanoscale silicon microcavities. J Am Chem Soc 123, 11797-11798. 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. Chiang W. L. (2009). The N-terminal region of XpsE is located at the interface of protein-protein interaction with itself. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Cornelis, G.R. (2006). The type III secretion injectisome. Nat Rev Microbiol 4, 811-825. Delepelaire, P. (2004). Type I secretion in gram-negative bacteria. Biochim Biophys Acta 1694, 149-161. 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. Fullner, K.J., Lara, J.C., and Nester, E.W. (1996). Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273, 1107-1109. Filloux, A. (2004). The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694, 163-179. Henderson, I.R., Navarro-Garcia, F., Desvaux, M., Fernandez, R.C., and Ala''Aldeen, D. (2004). Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68, 692-744. 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. 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, M.S., Chen, L.Y., Leu, W.M., Shiau, R.J., and Hu, N.T. (2005). Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. J Biol Chem 280, 4585-4591. Neuwald, A.F., Aravind, L., Spouge, J.L., and Koonin, E.V. (1999). AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 9, 27-43. 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. Possot, O., and Pugsley, A.P. (1994). Molecular characterization of PulE, a protein required for pullulanase secretion. Mol Microbiol 12, 287-299. 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., Bagdasarian, M., Howard, S.P., and DiRita, V.J. (1995). Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J 14, 1664-1673. 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. Savvides, S.N., Yeo, H.J., Beck, M.R., Blaesing, F., Lurz, R., Lanka, E., Buhrdorf, R., Fischer, W., Haas, R., and Waksman, G. (2003). VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. EMBO J 22, 1969-1980. Shiue, S.J., Kao, K.M., Leu, W.M., Chen, L.Y., Chan, N.L., and Hu, N.T. (2006). XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25, 1426-1435. Shiue, S.J., Chien, I.L., Chan, N.L., Leu, W.M., and Hu, N.T. (2007). Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Mol Microbiol 65, 401-412. Strom, M.S., Bergman, P., and Lory, S. (1993). Identification of active-site cysteines in the conserved domain of PilD, the bifunctional type IV pilin leader peptidase/N-methyltransferase of Pseudomonas aeruginosa. J Biol Chem 268, 15788-15794. 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. Tsai, K. J. (2009) Analysis of bound nucleotides on XpsE and its ATP hydrolysis activity by HPLC. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Yamagata, A., and Tainer, J.A. (2007). Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J 26, 878-890. Yo, T. T. (2003). Detection of interactions between XpsF and XpsL,or XpsE, in the type II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.
摘要: XpsE是十字花科黑腐病菌第二型分泌機器中唯一的胞內蛋白,可區分為N端和C端兩個獨立區域,其C端有四個具高度保留性的nucleotide binding motifs,呈現ATPase活性。先前in vitro實驗發現,單體 XpsE 蛋白與ATP結合後會形成六聚體,並扮演水解ATP提供分泌所需能量的角色。在已解出與XpsE具同源性的aaPilT蛋白六聚體結構中發現,相鄰兩個subunits間含有三個界面,其中以N2-C1界面涵蓋面積最大且具高度保留性,具高度保留性的R385、D387胺基酸則位於此界面上,推測位於N2-C1區界面的胺基酸彼此間的交互作用力,可能對蛋白六聚體的形成十分重要。本研究希望藉由R385A、D387A突變蛋白與正常XpsE蛋白作生化特性的比較,來了解位於界面上這兩個胺基酸在XpsE行使功能時所扮演的角色。利用分子篩管柱分析的圖譜及西方墨點法的追蹤,發現R385A及D387A突變使蛋白形成的六聚體較野生型XpsE蛋白少;另一方面,XpsE(R385A)及XpsE(D387A) 突變蛋白測得的ATP水解活性,大約是野生型XpsE蛋白的1/10。本研究測定ATPase活性時,為了減少其他污染性ATPase並增加蛋白均質性,將XpsE或其突變蛋白經過親和性管柱純化後,再經過陰離子交換樹脂以收集污染性低且均質性高的單體蛋白,濃縮後再利用本研究發展的非放射性方式進行ATPase活性測定。上述結果說明破壞XpsE N2-C1界面上高度保留性的R385、D387胺基酸會使蛋白形成六聚體能力變差,且會使XpsE蛋白ATP水解活性減弱。
XpsE is the only cytoplasmic protein component of the Xanthomonas campestris pv. campestris type II secretion system. It can be divided into N and C domain, and its C domain contains four conserved nucleotide binding motifs, which exhibit ATPase activity. As suggested from previous in vitro studies, ATP binding to XpsE triggers its hexamerization, and XpsE plays an important part in supplying energy for secretion process by hydrolyzing ATP. In the hexameric structure of aaPilT, an XpsE homologues, three interfaces between two neighboring subunits were revealed. The interface between N2 and C1 covers the largest area and contains most highly conserved residues. It was suggested that the interactive residues located at the N2-C1 interface may be important for protein hexamerization. Sequence alignment indicates that R385 and D387 may be located at the interactive interface of two neighboring XpsE. In this study, we conducted biochemical analysis of the mutant XpsE(R385A) and XpsE(D387A) in parallel with the wild type XpsE to get better understanding of the functional role of R385 and D387 in XpsE. By performing size exclusion chromatography and Western blot, we observed reduction in hexamer content of the mutant XpsE(R385A) and XpsE(D387A) relative to that of the wild type XpsE. Their ATP hydrolysis activity was reduced by 10 fold. In order to minimize possible contaminating ATPase and to raise protein homogeneity, we purified the proteins by performing affinity chromatography followed by anion exchange chromatography, from which homogeneous monomers with little contaminating ATPase were obtained. Following concentration, the monomeric protein was analyzed for ATPase activity by using a non-radioisotopic ATPase assay developed in this study. In summary, the experimental evidences presented in this study suggest mutation of the highly conserved residues possibly located at interactive interface of neighboring XpsE, R385 and D387, to alanine diminishes the ability of XpsE in hexamerization and reduces its ATP hydrolysis activity.
URI: http://hdl.handle.net/11455/24052
其他識別: U0005-2007201010223800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2007201010223800
Appears in Collections:生物化學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館

Show full item record
 
TAIR Related Article
 
Citations:


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.