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|標題:||Analysis of comformation change at the N domain of XpsE protein by utilizing fluorescence probes
利用螢光探針追蹤 XpsE 蛋白N端區域之結構變化
|關鍵字:||XpsE;第二型分泌系統;Xanthomonas campestris;type II secretion system;fluorogenic probes;螢光探針||出版社:||生物化學研究所||引用:||J. Abendroth, P. Murphy, M. Sandkvist, M. Bagdasarian and W. G. J. Hol (2005) The X-ray Structure of the Type II Secretion System Complex Formed by the N-terminal Domain of EspE and the Cytoplasmic Domain of EspL of Vibrio Cholerae. J. Mol. Biol. 348:845-855 D. M. Anderson and O. Schneewind (1999) Type III machines of Gram- negative pathogens: injecting virulence factors into host cells and more. Curr. Opin. Microbiol. 2:18-24 W. Bitter, M. Koster, M. Latijnhouwers, H. de Cock and J. Tommassen (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 S. Bleves, R. Voulhoux, G. Michel, A. Lazdunski, J. Tommassen, and A. Filloux (1998) The secretion apparatus of Pseudomonas aeruginosa : identification of a fifth pseudopilin, XcpX (GspK family). 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DiRita (1997) General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae. J Bacteriol 179:6994-7003 S. J. Shiue, K. M. Kao, W. M. Leu, L. Y. Chen, N. L. Chen and N. T. Hu (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J. 25:1426-1435 R. Silverstein, C. C. Lin and A. B. Rawitch (1980) Evidence for an Essential Hydrophobic Domain in the Maintenance of Phosphoenol- pyruvate Carboxykinase Activity. J. Biol. Chem. 256:1374-1379 T. Toyo'oka and K. Imai (1984) New Fluorogenic Reagent Having Halogenobenzofurazen Structure fo Thiols: 4-(Aminosulfonyl)-7-fluoro -2,1,3-benzoxadiazole. Anal. Biochem. 56:2461-2464 R. T. Tsai, W. M. Leu, L. Y. Chen and N. T. Hu (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 Y. N. Tseng (2006) Significance of the N-terminal domain of XpsE in its interaction with XpsL analyzed by site-directed mutation. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R.O.C. M. J. Treuheit and T. L. Kirley (1993) Reversibility of Cysteine Labeling by 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Anal. Biochem. 212: 138-142 D. C. Turner and L. Brand (1968) Quantitative Estimation of Protein Binding Site Polarity Fluorescence of N-Arylaminonaphthalenesulfonates. Biochem. J. 7:3381-3390 J. E. Walker, M. Saraste, M. J. Runswick and N. J. Gay (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinase, and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945-951 C. Wandersman and P. Delepelaire (1990) TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 87:4776-4780 Y. Watanabe, M. Takano, and M. Yoshida (2005) ATP Binding to Nucleotide Binding Domain (NBD)1 of the ClpB Chaperone Induces Motion of the Long Coiled-coil, Stabilizes the Hexamer, and Activates NBD2. J. Biol. Chem. 280:24562-24567 T. T. Yo (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 Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R.O.C. X. R. Zhou and P. J. Christie (1997) Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase and the effect of ATP-binding cassette mutations on assembly and function of the T-DNA transporter. Mol. Microbiol. 32:1239-1253 Z. L. Zhu (2006) Type II Secretion Apparatus of Xanthomonas campestris: Expression and Functional Characterization of the Cytoplasmic Domain of XpsF. Master thesis. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R.O.C.||摘要:||
XpsE is the only cytoplasmic protein component of the type II secretion system of Xanthomonas campestris pv. campestris. XpsE interacts with XpsL via tis N-terminal domain, whereas its C-terminal domain could be subdivided into an N2 and an ATP-binding domain. Triggered by ATP-binding, XpsE oligomerises leading to its interaction with XpsL. X-ray crystallography of XpsEN (the N-terminal domain) revealed an open and a closed form that differ at their N-termini. A hydrophobic patch surrounded by residues 11, 15, 25 and 39 was detected only in the closed form, not in the open form. The hydrophobic patch was implicated to be involved in normal function of XpsE as suggested from mutant analysis. Trypsin limited digestion implied ATP-binding to XpsE causes conformational change near its N-terminal domain. To gain further understanding on the conformational change at the N-terminal domain of XpsE triggered by ATP-binding, I used AMPPNP for mimicking ATP-binding state and two fluorogenic probes for monitoring XpsE conformational changes. Both are hydrophobic probes, but bind differently to the target protein. ANS binds to the protein neither specifically nor covalently, whereas ABD-F binds covalently to the cysteine residue. Results from both probes suggested changes in XpsE conformation was caused by addition of AMPPNP. Since ABD is bound specifically to the 39th residue of XpsE, conformational change in the surroundings of the 39th residue was implied from the observation that Cys-ABD fluorescence intensity varied upon ATP-binding. Such prediction was confirmed with similar experiment using a mutant with weakened ATP-binding ability. In addition, I observed that mutation of a residue in the N2 domain R286 to alanine made XpsE lose the property of changing Cys-ABD fluorescence intensity induced by ATP-binding. The mutant R286A was shown in previous studies to remain normal in ATP-binding but have lost its ability to interact with XpsL. To summarize, I observed in the study that ATP-binding at the C-terminal domain of XpsE caused conformational changes in the surroundings of the 39th residue near its N-terminus that might be directly involved in interaction of XpsE with XpsL. In the transmission of the “signal” initiated by ATP-binding at the C-terminal domain of XpsE to its N-terminus, the R286 residue located within the N2 domain appears to play a key role.
XpsE為Xanthomonas campestris pv. campestris中參與第二型分泌機制 (type II secretion system) 組成的唯一胞內蛋白，其N端區域可能參與與內膜蛋白XpsL的結合；其C端序列可再被細分為一N2 domain及一ATP-binding domain，當ATP與XpsE結合時，會促使XpsE形成六聚體，進而與XpsL 結合。XpsEN晶體結構呈現兩種構造：open form與closed form，在closed form中第11、15、25、39號胺基酸形成一個hydrophobic patch，而在open form中此結構則不存在，突變實驗的結果暗示，具有功能的XpsE其N端可能需要形成類似XpsEN closed form中的hydrophobic patch。Trypsin limited digestion分析結果亦暗示ATP 結合會使XpsE 發生構形變化，而此變化接近N端。為進一步探討ATP 結合對XpsE蛋白結構變化的影響，本研究利用AMPPNP來模擬ATP結合狀態，並選用兩種螢光探針：疏水性探針ANS及可和cysteine形成共價鍵的疏水性探針ABD-F分析ATP binding對XpsE構形的影響，兩種實驗結果皆顯示AMPPNP的添加會使XpsE蛋白發生構形上的變化。此外，Cys-ABD呈現的螢光訊號反映的是XpsE第39號胺基酸的環境變化，暗示ATP binding引起的構形改變及於第39號胺基酸附近。此推論更進一步經由失去ATP binding能力的突變蛋白實驗結果得到印證。最後，我並發現位於N2 domain的R286A突變使得XpsE失去因添加AMPPNP而改變螢光強度的性質。綜上所述，當XpsE C端與ATP結合時，可能會導致第39號胺基酸附近的結構改變，而位於N2 domain的R286，在此過程中扮演關鍵性的角色。
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