Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23527
標題: XpsE突變蛋白多聚體穩定性及其ATP結合能力之比較分析
Comparative analysis of XpsE variants for their oligomer stability and ATP-binding activity
作者: 劉仲祐
Liu, Chung-Yu
關鍵字: 第二型分泌系統;T2SS;多聚體;oligomer
出版社: 生物化學研究所
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摘要: 
十字花科黑腐病菌是造成十字花科葉片褐化的病原菌,具有第二型分泌系統(T2SS)。XpsE是T2SS胞內蛋白,可分成N端和C端兩個獨立區域,C端有四個高度保留的motifs,與ATP水解、ATP結合與穩定多聚體有關。單體XpsE與ATP結合後會形成六聚體,並與內膜蛋白XpsL結合,接著水解ATP提供能量進行分泌。XpsE之K331M突變會降低ATP水解活性,K331M R504A突變會降低ATP結合能力。在XpsE同源的PilT、afGspE蛋白六聚體結構中,相鄰兩個subunit間的三個介面中,以N2-C1介面面積最大,其中高保留性R217、D387在此介面形成鹽橋,可能與六聚體穩定性有關。而R385與相鄰subunit中的ADP磷酸根靠近,可能與ATP水解有關。本研究為驗證上述XpsE保留性胺基酸的推論,針對XpsE的R217、K331、R385、D387、E395進行突變,並進行XpsE多聚體穩定性及ATP結合分析。Gel filtration及Western blot分析,顯示XpsE會形成單體與高比例的多聚體,且多聚體所佔比例會隨著蛋白濃度的增加而上升。各種突變蛋白的分析,顯示R217A、K331M、R385A、D387A與K331M R504A突變會減弱多聚體的穩定,以不同程度降低多聚體/單體比例,只有E395Q會提高多聚體/單體比例。K331M與D387A或與R217A D387A對降低多聚體穩定性有加成效應。E395Q提高多聚體效應被K331M R504A降低多聚體效應抵消。利用ATP content determination分析ATP結合,發現XpsE與XpsEK331M突變蛋白再結合ATP的量與原先結合在蛋白中ATP的量相等,XpsEK331M R504A突變蛋白不具有再結合ATP能力。利用Isothermal Titration Calorimetry (ITC)分析ATP結合時,發現XpsEK331M R504A突變蛋白ATP結合係數n值小於1,不具有結合ATP能力,而XpsE與其他突變蛋白ATP結合係數n值均為3,假設XpsE與其他突變蛋白以六聚體形式結合ATP,推測六聚體中僅3個subunits可與ATP進行結合。分析XpsE及其突變蛋白的動力學平衡常數K1值與熱力學自由能ΔG1值,所顯示的結果皆彼此相符。

Xanthomonas campestris pv. campestris is the causal agent of cruciferous leaf browning. A variety of hydrolytic enzymes are secreted through the type II secretion system (T2SS). XpsE is the only cytoplasmic component of the T2SS. It can be divided into an N-domian and a C-domian. Four highly conserved motifs in the C-domain of XpsE are involved in its ATP-binding, ATP hydrolysis activity and oligomer formation. Previous studies indicated the monomeric XpsE, when binding to ATP forms hexamer and associates with the membrane protein XpsL. Subsequently, ATP hydrolysis by XpsE may provide energy for the secretion process. K331M mutation caused XpsE deficient in ATP hydrolysis, and K331M R504A mutation deficient in ATP-binding. The hexameric structure of XpsE homologues PilT and afGspE revealed three interfaces between two adjacent subunits. The interface between the N2 and C1 domain covers the greatest area. The salt bridge formed between the highly conserved residues equivalent to R217 and D387 of XpsE may be involved in kseeping hexamer stable, R385 is near the presumed g-phosphate group of an ATP bound to the adjacent subunit, and predicted to be related to ATP hydrolysis. To determine significance of the conserved residues, including R217, K331, R385, D387 and E395 of XpsE in its function, I examined oligomer abundance and ATP-binding ability of the XpsE variants with mutation at these residues. Gel filtration followed by Western blot analysis of the wild-type XpsE indicated it forms monomer and a great proportion of oligomer of varying sizes. The proportion of oligomer increased as XpsE protein concentration increased. Of all XpsE mutants analyzed, R217A, K331M, R385A, D387A and K331M R504A mutations appeared to cause reduction in the abundance of oligomer, to varying degrees. Only the E395Q appeared to make the oligomer abundance increase. K331M in combination with D387A or R217A D387A exerted additive effect in reducing the abundance of oligomer. K331M R504A mutation is epistatic to E395Q mutation making ologomer abundance drop. To analyze ATP-binding activity of XpsE and its varients, I performed ATP content determination assay. The protein-bound ATP for XpsE and XpsEK331M is equal to additional ATP bound upon incubation with ATP after protein purification. XpsEK331M R504A appeared to lack ATP-binding activity. In agreement, Isothermal Titration Calorimetry (ITC) analysis revealed, the ATP-binding coefficient of XpsEK331M R504A is less than 1, suggesting nil ATP binding capacity. ATP-binding coefficient for XpsE and the rest of XpsE variants analyzed are 3. If the XpsE analyzed is mainly in hexameric form, the observations made here would agree with the proposal that each XpsE hexamer may bind only three ATPs. ITC analysis of XpsE and its variants that indicated kinetic equilibrium constant K1 value and thermodynamic free energy ΔG1 value their are consistent with each other.
URI: http://hdl.handle.net/11455/23527
其他識別: U0005-2706201215130900
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