Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23996
標題: XpsE, ATP以及XpsL三者間相互作用機制的探討
Interactive relationship among XpsE, ATP and XpsL
作者: 薛聖潔
Shiue, Sheng-Jie
關鍵字: ATPase 活性;XpsE;交互作用;ATP;XpsL
出版社: 生物化學研究所
引用: 1. Arts, J., de Groot, A., Ball, G., Durand, E., El Khattabi, M., Filloux, A, Tommassen, J., and Koster, M. 2007. Microbiology. 153:1582-92. Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF). 2. Bitter, W., Koster, M., Latijnhouwers, M., Cock, H., and Tommassen, J. 1998. Mol Microbiol. 27 (1): 209-19. Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. 3. Camberg, J. L., and Sandkvist, M. 2005. J Bacteriol. 187(1): 249-5. Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. 4. Chen, L. Y., Chen, D. Y., Miaw, J.,and Hu, N. T. 1996. J Biol Chem. 271(5):2703-8. XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. 5. Chen, Y., Shiue, S. J., Huang, C. W., Chang, J. L., Chien, Y. L., Hu, N. T., and Chan, N. L. 2005. J Biol Chem. 280(51):42356-63. Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. 6. Crowther, L. J., Yamagata, A., Craig, L., Tainer, J. A., and Donnenberg, M. S. 2005. J Biol Chem 280: 24839-48. The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. 7. Johnson, A., Yao, N. Y., Bowman, G.D., Kuriyan, J., and O''Donnell, M. 2006. J Biol Chem. 281(46): 35531-43. The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading. 8. Karamanou, S., Vrontou, E., Sianidis, G., Baud, C., Roos, T., Kuhn, A., Politou, A. S., Economou, A. 1999. Mol Microbiol. 34(5):1133-45. A molecular switch in SecA protein couples ATP hydrolysis to protein translocation. 9. Korotkov, K.V., and Hol, W. G. 2008. Nat Struct Mol Biol. 15:462-8. Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. 10. Krause, S., Barcena, M., Pansegrau, W., Lurz, R., Carazo, J. M.,and Lanka, E. 2000. Proc Natl Acad Sci USA 97: 3067-72. Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. 11. Lee, M. S., Chen, L. Y., Leu, W. M., Shiau, R. J.,and Hu, N. T. 2005. J Biol Chem. 280(6):4585-91. 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 12. Linderoth, N. A., Simon, M. N., and Russel, M. 1997 Science 278:1635-38. The Filamentous Phage pIV Multimer Visualized by Scanning Transmission Electron Microscopy. 13. Nunn, D. 1999. Trends Cell Biol. 9: 402-8. Bacterial type II protein export and pilus biogenesis: more than just homologies? 14. Nouwen, N., Stahlberg, H., Pugsley, A. P., and Engel, A. 2000. EMBO J. 19:2229-2236. Domain structure of secretion PulD revealed by limited proteolysis and electron microscopy. 15. Planet, P. J., Kachlany, S. C., DeSalle, R.,and Figurski, D. H. 2001. Proc Natl Acad Sci U S A. 98(5):2503-8. Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. 16. Possot, O., and Pugsley, A. P. 1994. Mol Microbiol 12:287-99. Molecular characterization of PulE, a protein required for pullulanase secretion. 17. Pozidis, C., Chalkiadaki, A., Gomez-Serrano, A., Stahlberg, H., Brown, I., Tampakaki, A. P., Lustig, A., Sianidis, G., Politou, A. S., Engel, A., Panopoulos, N. J., Mansfield, J., Pugsley, A. P., Karamanou, S., and Economou, A. 2003. J Biol Chem. 278(28):25816-24. Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization. 18. Pugsley A. P.,and Possot O. 1993. Mol Microbiol. 10(3):665-74. The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex. 19. Pugsley A. P. 1993. Mol Microbiol. 9(2):295-308. Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca. 20. Rivas, S., Bolland, S., Cabezon, E., Goni, F. M., and de la Cruz, F. 1997. J Biol Chem 272:25583-90. TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation. 21. Robien, M. A., Krumm, B. E., Sandkvist, M.,and Hol, W. G. 2003. J Mol Biol. 333(3):657-74. Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholera. 22. Russel, M. 1998 J. Mol. Biol. 279: 485-99. Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. 23. Sandkvist, M., Bagdasarian, M., Howard, S. P., and DiRita, V. J. 1995. EMBO J. 14(8):1664-73. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. 24. Sandkvist, M. 2001. Mol. Microbiol. 40, 271-283. Biology of type II secretion. 25. Sauvonnet, N., Gounon, P., and Pugsley, A. P. 2000. J Bacteriol. 182(3):848-54. PpdD type IV pilin of Escherichia coli K-12 can Be assembled into pili in Pseudomonas aeruginosa. 26. Sauvonnet, N., Vignon, G., Pugsley, A. P., and Gounon, P. 2000. EMBO J. 19(10):2221-8. Pilus formation and protein secretion by the same machinery in Escherichia coli. 27. 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. EMBO J. 22: 1969-80. VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. 28. Scott, M. E., Dossani, Z. Y., and Sandkvist, M. 2001. PNAS 98:13978-83. Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway. 29. Senf, F., Tommassen, J.,and Koster, M. 2008. Microbiology. 154: 3025-32. Polar secretion of proteins via the Xcp type II secretion system in Pseudomonas aeruginosa. 30. Vladimir, E., Shevchik, J. R., and Condemine, G. 1997. EMBO J. 16: 3007-16. Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins. 31. Walker , J. E., Saraste, M., Runswick, M. J., and Gay, N. J. 1982. EMBO J. 1:945-51. 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. 32. Yamagata, A. and Tainer, J. A. 2007. EMBO J. 26: 878-90. Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. 33. Whitchurch, C. B., Hobbs, M., Livingston, S. P., Krishnapillai, V., and Mattick, J. S. 1991. Gene 101:33-44. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. 34. Yeo, H. J., Savvides, S. N., Herr, A. B., Lanka, E., Waksman, G. 2000. Mol Cell 6: 1461-72. Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. 35. Yao, N.Y., Johnson, A., Bowman, G. D., Kuriyan, J., O''Donnell, M. 2006. J Biol Chem. 281(25):17528-39. Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen. 36. Yo, T. T. 2003. Detection of interaction 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: 
革蘭氏陰性細菌的第二型分泌系統 (T2SS) 和第四型纖毛組裝機制及細菌的接合生殖系統的部分組成份子相似,推測機制運作方式可能也非常類似。這些系統中均含有被預測為 NTPase 的蛋白,這些 NTPase 蛋白因為高度的相似性而被歸納為GspE-VirB11 superfamily,並且被推測為各個系統中提供分子運送的能量來源。被推測為第二型分泌機器中的分子馬達蛋白 GspE 含有與核苷酸結合的區域 (NBD),其中包含兩個高度保留的 motifs: Walker A and B boxes,藉由定點突變的分析顯示這兩個 motifs 的完整性對於 GspE 在細胞內行使功能是必要的。GspE 如何將水解 ATP 所獲得的能量用於協助蛋白分泌是本篇論文所要探討的重點。利用不被水解的 ATP 類似物 AMPPNP 取代 ATP 進行分子篩層析管柱及分析型超高速離心實驗,結果顯示 Mg-AMPPNP 會促使單分子的 XpsE 蛋白 (Xanthomonas campestris pv. campestris的 GspE 蛋白) 形成多聚體。此外,在MBP-XpsLN pull-down 實驗中發現 Mg-AMPPNP 會促使 XpsE 結合 XpsLN。由上述觀查推測 ATP 結合可能會促使 XpsE 蛋白聚合並和位於內膜上的 T2SS 組成份子 XpsL 結合。此外,ATP-binding 受損的突變蛋白 XpsE(KMRA) 喪失了上述特性,而另一個只喪失 ATPase 活性的蛋白 XpsE(K331M),AMPPNP 依然會促使其單分子形成多具體並和 XpsL 結合,這些現象均支持先前的假設。進一步的分析發現 XpsL 的結合可能會引起 XpsE 的構形改變而刺激其水解 ATP。在 ATPase 活性測試裡,我們觀察到與 XpsL 結合的主要區域: XpsE 的 N 端蛋白片段會抑制 C 端水解 ATP 的活性,推測 XpsL 與 XpsE 的 N端的結合後可能引起 XpsE 構形改變,使 N端對於 C 端的 ATPase活性抑制效應消失。另一方面,利用定點突變的分析,我們發現位於C端的 N2 domain 的 R286 這個胺基酸相當重要,突變蛋白 XpsE(R286A) 的 ATPase 活性上升至和 C端片段相當,但是此突變蛋白喪失正常功能以及與 XpsL 結合的能力。在此突變蛋白中 N-C domain 的交互作用減弱,並且受 N-C domain 的交互作用而產生的 N1區域構形改變也消失了,暗示 R286 這個胺基酸可能扮演 sensor 的角色。推測當 R286 感受到 C端結合 ATP 後,會促使 N-C domain-domain 的交互作用,防止 XpsE 蛋白水解 ATP 以及引發 N1 區域 的構形改變促進 XpsL 結合。當 XpsE 和 XpsL 結合之後,ATPase 的活性才會被刺激並運用於分泌的進行。由於 R268 這個胺基酸經比對後具高度保留性,推測其他 XpsE 的同源蛋白也可能存在著類似的調控機制。

Type II secretion system (T2SS) is similar to type IV pilus biogenesis and DNA transfer in certain components, and possibly their molecular mechanism. One major component shared by these multiprotein complexes belongs to the GspE-VirB11 superfamily of secretion NTPases that have extensive sequence similarity and probably similar functions as the energizer for macromolecular transport. The GspE protein with characteristic nucleotide-binding domain (NBD) comprising Walker A and B box is predicted to be the molecular motor in T2SS. Mutagenesis analysis clearly showed that intactness of NBD in GspE is essential for its normal function in T2SS. However, it is still unclear how GspE couples energy from ATP hydrolysis to exoprotein secretion through the secretion pore. In this study, we observed that the monomeric XpsE, the GspE protein in Xanthomonas campestris pv. campestris, could form oligomer, analyzed in size exclusion chromatography and analytical ultracentrifugation, when incubated with the non-hydrolyzable ATP analogue Mg-AMPPNP. In addition, preincubation of XpsE with Mg-AMPPNP made XpsE competent in XpsLN binding, as revealed in MBP-XpsLN pull-down assay. These observations suggested that ATP-binding to XpsE triggers it to form oligomer and to interact with XpsL. Such a prediction was confirmed with mutant analysis. A mutant XpsE deficient in ATP binding was blocked in AMPPNP-induced oligomerization and XpsLN binding. As a consequence of interaction with XpsLN, the XpsE ATPase activity was stimulated. Further analysis implicated that the stimulation may be related to XpsLN-induced conformational change in XpsE, which leads to separation of N- and C-domain relieving the suppressive effect exerted by N-domain on the ATP hydrolyzing activity exhibited by C-domain. The mutant XpsE(R286A), albeit exhibited raised ATPase activity, was not functional nor membrane-bound in vivo. Furthermore, interaction of the mutated C-domain with N-domain was weakened. So was the conformational change at N1 domain of XpsE triggered by ATP binding demolished, implicating an ATP-sensing and information-transmitting role for R286 in XpsE and the equivalent residues in its homologues.
URI: http://hdl.handle.net/11455/23996
其他識別: U0005-2201200911080900
Appears in Collections:生物化學研究所

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