臺灣博碩士論文加值系統

第二型分泌系統 (T2SS) 是十字花科黑腐病菌分泌水解酵素、攻擊寄主的主要機制，此機制也出現在許多不同的致病菌株中，感染動、植物並造成疾病。 在T2SS中，位於cytosol的XpsE是唯一具ATP結合及水解能力的組成蛋白，推測為T2SS運轉提供所須要的能量。 先前研究指出，XpsE ATP水解活性可能受到其N1區域的抑制，而內膜蛋白XpsL可透過與XpsE N1區域結合，移去N1的抑制而達到刺激XpsE ATP水解活性的效果。 XpsE的N2區域位於N1區域及其C區域ATP水解活性中心之間，推測N2區域可能居中參與XpsE蛋白構形變化之訊息傳遞以及ATP水解活性之調控。 XpsE本身可聚合成多聚體，可能以六聚體的形式推動T2SS的運轉。 XpsE同源蛋白的立體結構暗示，N2區域亦可能參與六聚體中分子間的結合。 本研究選擇高保留性的D202以alanine定點突變的方式進行分析。 結構顯示XpsE同源蛋白N2區域的D202同源胺基酸與C區域所結合的ATP靠近。 D202A突變蛋白失去正常in vivo分泌功能，並具有干擾正常XpsE行使功能的特性。 本研究希望藉由D202A突變蛋白和正常XpsE蛋白生化特性的比較，以瞭解胺基酸D202在XpsE行使功能時所扮演的角色。 結果發現D202A突變蛋白的許多特性都與正常XpsE無明顯差異，它能正常結合ATP，沒有喪失形成多聚體的能力，且仍然可以與XpsL結合並受到XpsL刺激ATP水解活性。 但是，D202A突變蛋白單獨的ATP水解活性略低，同時對ADP可能有異常的結合能力。 最後以四個已知XpsE同源蛋白的結構為依據，分析N2區域以及D202保留性胺基酸鄰近的構造，推測胺基酸D202可能主要參與XpsE水解ATP的過程或其後的下游步驟。

T2SS is the mechanism required for Xanthomonas campestris pv. campestris to secret hydrolytic enzymes in the invasion of its host. Widespread in pathogenic bacteria, the secretion system leads to animal and plant diseases. XpsE, a cytosolic protein, is the only component in T2SS that possesses ATP binding and hydrolyzing activities and may work as the molecular motor. Previous studies indicated that the N1 domain of XpsE is inhibitory to its ATPase activity exihibited by its C domain. An inner membrane protein XpsL acts as a positive regulator of the XpsE ATPase, presumably by binding to the XpsE N1 domain and alliviating its inhibition effect. An N2 domain physically located between N1 and C domain of XpsE may coordinate conformational change related ATPase activity regulation. XpsE forms homo-multimer and may function as hexamer in T2SS. Crystal structures of XpsE homologues suggest that the N2 domain may be involved in inter subunit association in hexamer. This study is focused on the analysis of an XpsE mutant that is mutated at the conserved residue D202 in the N2 domain. The XpsE(D202A) is no longer functional in secretion. It also interferes with normal secretion when introduced into the wildtype strain. In crystal structures of XpsE homologue, the D202 equivalent residues are close to ATP bound to the C domain. To get better understanding of functional role of the residue D202 in XpsE, ATP binding and ATP hydrolyzing activities of the mutant were analyzed in parellel with the wildtype XpsE. In most assays, no significant alteration was observed. The mutant remains capable of ATP binding, appears to exihibit normal oligomerization activity and XpsLN binding. However, it exihibits slightly lower intrinsic ATPase activity and deviated ADP binding. By inspecting surroundings of D202 equivalents in the solved crystal structures, we speculated that the D202 residue of XpsE may be required for steps downstream of ATP hydrolysis by XpsE.

摘要 i
Abstract ii
目次 iii
前言 1
實驗方法 8
一、螢光測試 8
(一) 蛋白的表現、純化及處理 8
(二) 利用螢光測試XpsE與TNP-ATP的結合能力 9
二、ATP水解活性的測試 10
(一) 蛋白的表現、純化及處理 10
(二) ATP水解活性測試及分析 11
(三) ATP水解活性的計算方式 12
(四) MBP-XpsLN的表現與純化 13
(五) ADP競爭ATP水解活性測試 13
結果 14
一、D202A突變對XpsE結合ATP的能力無顯著影響 14
二、D202A突變造成XpsE的ATP水解活性略為下降 15
三、D202A突變不影響XpsE的ATP水解活性隨蛋白濃度提高而上升的現象 15
四、XpsE(D202A) 突變蛋白的 ATP水解活性仍受到XpsLN的刺激 16
五、ADP抑制ATP水解活性實驗暗示D202A定點突變蛋白較XpsEwt可能對ADP有較強的結合能力 17
討論 18
D202位於N2區域與相鄰分子C區域的接觸介面 18
D202可能與分子內N2區域的R286形成salt bridge 19
D202在N1、N2區域間訊息傳遞中可能扮演的角色 20
參考文獻 22
圖一 27
圖二 28
圖三 29
圖四 31
圖五 32
圖六 33
圖七 36
表一、菌種 37
表二、表現質體 37
表三、 XpsEwt、XpsE(D202A) 蛋白單獨或添加MBP-XpsLN時的ATP水解活性 39
附錄 40

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