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dc.contributor.authorShiue, Sheng-Jieen_US
dc.description.abstract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。zh_TW
dc.description.abstractType 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.en_US
dc.description.tableofcontentsIntroduction 1 Material and methods 7 Bacterial strains, plasmids and antisera 7 Protein purification 7 Gel filtration chromatography 8 MBP-XpsL pull-down assay 8 XpsEC-Strep pull-down assay 9 Subcellular fractionation 9 Assays for α-amylase secretion 9 ATPase assay 10 ATP-binding assay 10 Labeling of XpsE by covalent thiol-reactive fluorescent probes 11 Fluorescence measurements 11 Results 12 XpsE oligomerizes when bound to ATP 12 The isolated AMPPNP-induced oligomeric XpsE is able to associate with XpsL (N-terminal domain of XpsL) in the absence of AMPPNP 13 The double mutant XpsE(K331M, R504A) exhibits reduced ATP binding and defect in AMPPNP-triggered oligomerization 14 The ATP binding mutant XpsE(K331M, R504A) lost the ability of XpsL binding induced by Mg-AMPPNP 15 The XpsE(K331M) is non-functional due to its defect in ATPase activity 16 Subcellular distribution of XpsE correlates with its MBP-XpsLN-binding ability 16 MBP-XpsLN stimulates the ATPase activity of XpsE in vitro 17 The major XpsL binding domain XpsE interacted with MBP-XpsL in an AMPPNP independent matter 17 AMPPNP enhances the interaction between XpsE and XpsE 19 The ATPase activity of XpsEC was reduced by XpsEN 19 The ATPase activity of XpsEC was stimulated slightly by MBP-XpsLN 20 Conformational change in the N-domain of XpsE triggered by AMPPNP 20 ATPase activity of the mutant XpsE(R286A) is higher than wild-type XpsE and similar to that of the XpsEC 21 The interaction between XpsEN and XpsEC was reduced by mutating R286 to alanine 22 XpsE(R286A) remained unchanged in AMPPNP-triggered oligomerization, but lost the XpsL-binding ability 23 The AMPPNP-triggered N terminal conformational change in XpsE was abolished by mutating R286 to alanine 23 Discussion 25 ATP binding to XpsE causes its oligomerization and conformational change, such effects may lead XpsE to interact with XpsL 25 The functional ATPase activity of XpsE may be involved in steps downstream of XpsE-XpsL association 27 The XpsE-XpsL interaction is a two steps matter 28 R286 may be a sensor in GspE 28 GspE as “molecular motor” in T2SS 30 Reference 32 Fig 1 Gel-filtration analysis of XpsE-Strep 37 Fig 2 Gel-filtration analysis of monomeric XpsE with or without preincubating with Mg-AMPPNP 38 Fig 3 Sedimentation coefficient distribution of wild-type XpsE and its variants 39 Fig 4 MBP pull-down assay of association of monomeric XpsE with MBP-XpsLN 40 Fig 5 MBP pull-down assay of oligomeric XpsE and monomeric XpsE for their association with MBP-XpsL in absence of AMPPNP and MgSO4 41 Fig 6 Effect of AMPPNP and MgSO4 on the mutant XpsE oligomerization analyzed upon size exclusion chromatography 42 Fig 7 MBP-XpsL pull-down assay for the effect of AMPPNP and MgSO4 43 Fig 8.zh_TW
dc.subjectATPase 活性zh_TW
dc.titleInteractive relationship among XpsE, ATP and XpsLen_US
dc.typeThesis and Dissertationzh_TW
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item.openairetypeThesis and Dissertation-
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