Please use this identifier to cite or link to this item:
標題: XpsE Walker B motif中保留性胺基酸重要性之分析
Significance of the conserved residues in Walker B motif of XpsE
作者: 余適伶
Yu, Shi-Ling
關鍵字: oligomer;多聚體
出版社: 生物化學研究所
引用: 1.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. 2.Andersen, C. (2003) Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria. Rev Physiol Biochem Pharmacol, 147, 122-165. 3.Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H. and 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, 209-219. 4.Brok, R., Van Gelder, P., Winterhalter, M., Ziese, U., Koster, A.J., de Cock, H., Koster, M., Tommassen, J. and Bitter, W. (1999) The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity. J Mol Biol, 294, 1169-1179. 5.Buttner, D. and Bonas, U. (2002) Port of entry--the type III secretion translocon. Trends Microbiol, 10, 186-192. 6.Camberg, J.L. and Sandkvist, M. (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol, 187, 249-256. 7.Cascales, E. and Christie, P.J. (2003) The versatile bacterial type IV secretion systems. Nat Rev Microbiol, 1, 137-149. 8.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. 9.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. Keyzer, J., van der Does, C. and Driessen, A.J. (2003) The bacterial translocase: a dynamic protein channel complex. Cell Mol Life Sci, 60, 2034-2052. 11.d''Enfert, C., Ryter, A. and Pugsley, A.P. (1987) Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. Embo J, 6, 3531-3538. 12.Desvaux, M., Parham, N.J. and Henderson, I.R. (2004) The autotransporter secretion system. Res Microbiol, 155, 53-60. 13.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. 14.Genin, S. and Boucher, C.A. (1994) A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mol Gen Genet, 243, 112-118. 15.Hanson, P.I. and Whiteheart, S.W. (2005) AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol, 6, 519-529. 16.Higgins, C.F., Hiles, I.D., Salmond, G.P., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W. and et al. (1986) A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature, 323, 448-450. 17.Hu, N.T., Hung, M.N., Chen, D.C. and Tsai, R.T. (1998) Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris. Microbiology, 144 ( Pt 6), 1479-1486. 18.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. 19.Hu, N.T., Hung, M.N., Liao, C.T. and Lin, M.H. (1995) Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology, 141 ( Pt 6), 1395-1406. 20.Hu, N.T., Lee, P.F. and Chen, C. (1995) The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues. Mol Microbiol, 18, 769-777. 21.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. 22.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. 23.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. 24.Lin. C.C. (2000) Interaction of cytoplasmic membrane protein XpsF with XpsL/M/N and XpsE in Xanthomonas campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 25.Nouwen, N., Ranson, N., Saibil, H., Wolpensinger, B., Engel, A., Ghazi, A. and Pugsley, A.P. (1999) Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci U S A, 96, 8173-8177. 26.Nouwen, N., Stahlberg, H., Pugsley, A.P. and Engel, A. (2000) Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. Embo J, 19, 2229-2236. 27.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. 28.Palmer, T., Sargent, F. and Berks, B.C. (2005) Export of complex cofactor-containing proteins by the bacterial Tat pathway. Trends Microbiol, 13, 175-180. 29.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. 30.Pugsley, A.P. (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev, 57, 50-108. 31.Pukatzki, S., Ma, A.T., Revel, A.T., Sturtevant, D. and Mekalanos, J.J. (2007) Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A, 104, 15508-15513. 32.Py, B., Loiseau, L. and Barras, F. (1999) Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL. J Mol Biol, 289, 659-670. 33.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. 34.Sandkvist, M. (2001) Type II secretion and pathogenesis. Infect Immun, 69, 3523-3535. 35.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. 36.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. 37.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. 38.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. 39.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. 40.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. 41.Tseng, T.T., Tyler, B.M. and Setubal, J.C. (2009) Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol, 9 Suppl 1, S2. 42.Voulhoux, R., Ball, G., Ize, B., Vasil, M.L., Lazdunski, A., Wu, L.F. and Filloux, A. (2001) Involvement of the twin-arginine translocation system in protein secretion via the type II pathway. Embo J, 20, 6735-6741. 43.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. 44.Yo T.T. (2003) Detection of interaction between XpsF and XpsL, or XpsE, in thentype II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.
XpsE是十字花科黑腐病菌第二型分泌系統中唯一的胞內蛋白,且具有微弱的ATPase活性。先前in vitro實驗發現,XpsE與ATP結合後會聚合成六聚體;且形成六聚體的XpsE可能與穿膜蛋白XpsL朝向胞內的N端結合。本研究欲深入探討XpsE與XpsE相互作用的介面。將XpsE與同源性蛋白EpsE△N90二聚體晶體結構比對,發現在C1 domain中三個胺基酸R385、D387、D389可能位於兩個相鄰XpsE介面-C1moleculeA:N2moleculeB,並延伸至與相鄰分子中結合的AMPPNP,而且這三個胺基酸在第二型分泌系統的GspE蛋白中具高度保留性。首先將這三個胺基酸分別進行定點突變成alanine,再利用starch plate分析其於十自花科黑腐病菌中分泌澱粉分解酶的能力,發現這三個胺基酸皆為XpsE正常分泌所必須。其次利用分子篩管柱層析分析由親合性管柱純化的突變蛋白其內生性多聚體含量,發現XpsED387A突變蛋白的多聚體含量較野生型蛋白減少;而XpsED389A則呈現單體含量減少的現象。進一步比較各突變蛋白是否和XpsLN形成穩定的複合體,藉由與MBP-XpsLN在E. coli中共表現,並經由連續兩種不同的親合性管柱純化XpsE與MBP-XpsLN的複合體,相較於野生型蛋白,XpsED387A與MBP-XpsLN的結合呈現較不穩定的現象;而單獨表現時不易形成可溶性的XpsED389A卻在與MBP-XpsLN共表現形成可溶且穩定複合體。接著利用in vitro MBP-XpsLN pull down實驗,結果發現XpsED387A及XpsED389A分別與MBP-XpsLN結合能力減弱。利用陰離子交換樹脂純化單體的野生型XpsE或突變蛋白,並進行ATPase活性的測試,發現XpsER385A及XpsED387A的ATPase活性均低於正常XpsE。由以上結果推測,在維持XpsE多聚體穩定性中扮演關鍵性角色的可能是D387。R385則可能扮演arginine finger參與相鄰分子水解ATP的活性。

XpsE is the only cytoplasmic protein component of the Xanthomonas campestris pv. campestris type II secretion system. It exhibits weak ATPase activity. As suggested from previous in vitro studies, ATP binding to XpsE triggers its hexamerization as well as its association with the cytoplasmic N-terminal domain of the integral membrane protein XpsL. In order to determine the significance of major XpsE-XpsE interactive interface between the C1 domain of molecule A and the N2 domain of molecule B, C1moleculeA: N2moleculeB, three conserved residues were identified by aligning five proteins of the Type II/IV secretion NTPase family. They are R385, D387 and D389. Each was mutated to Ala, transformed into the xpsE-null mutant XC1723 and analyzed for their secretion function on starch plate. All three mutants XpsER385A, XpsED387A and XpsED389A were unable to resume secretion of α-amylase in XC1723. And they all interfered with normal secretion in the wild type strain XC1701. As revealed from size exclusion chromatography, of all three mutants, XpsED387A was the only one that displayed distinctly decreased level of hexamer. Furthermore, weaker association of XpsED387A with MBP-XpsLN was suggested by reduced level of XpsED387A in the XpsE/MBP-XpsLN complex formed by their co-expression in E. coli. In contrast, the MBP-XpsLN pull down assay, showed that both XpsED387A and XpsED389A exhibited XpsLN binding activity slightly weaker than that of the wild-type XpsE. To minimize contaminating ATPase, monomeric XpsE and its variants (except XpsED389A) were purified through affinity chromatography followed by anion exchange chromatography. Results from ATPase assay indicated that the ATP hydrolysis activity of XpsER385A and XpsED387A were reduced by four and five fold, respectively. In summary, D387 of XpsE appears to play significant role in keeping hexamer stable. Its deficiency in ATP hydrolysis may be an indirect effect. As a contrast, R385 may act as an “arginine finger” and be directly required for ATP hydrolysis by the neighboring XpsE molecule.
其他識別: U0005-2807200900291100
Appears in Collections:生物化學研究所

Show full item record

Google ScholarTM


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