嗜熱短桿菌 Lon 蛋白酶 alpha 區塊與去氧核醣核酸結合模式探討

Abstract

Lon 蛋白酶具有 ATPase、chaperone-like 的活性，屬於一種多功能性的酵素，其主要功能在於降解細胞中錯誤摺疊或是受損的蛋白質，並在原核與真核生物的粒線體中均廣泛的被保留下來。先前的研究中證實了嗜熱短桿菌WR-249的 Lon 蛋白酶具有 DNA 結合的能力，然而 Lon 蛋白酶與 DNA 結合的模式與其在生理上的意義目前仍不清楚。 蛋白質序列比對分析顯示 Lon 蛋白酶的 alpha 區塊並不相似於目前已知的DNA結合蛋白，有可能為呈現一新型態的DNA辨認模式。由於此發現與之前所熟悉的 DNA 結合區塊均不相同，本論文將運用蛋白質結晶學與多維核磁共振光譜學的技術，希望能進一步瞭解 Lon 蛋白酶 alpha 區塊對於 DNA 的結合模式。首先，利用了坐式蒸氣擴散法 (sitting drop vapor diffusion) ，我們成功獲得了 alpha 區塊的蛋白質晶體，並於同步輻射光源下收集到晶體數據。其晶體之空間群 (space group) 為 P23，晶胞參數 (cell parameter) 為 a = b = c = 93.8Å ; α = 90°, β = 90°, γ = 90°。之後利用分子置換法，成功的解析了嗜熱短桿菌 Lon 蛋白酶 alpha 區塊的晶體結構。結構顯示 alpha 區塊由四段 α-螺旋 (α-helix) 與一對平行的β-摺板 (β-strand) 所構成，與其他已知不同 Lon 蛋白酶的 alpha 區塊非常相似。而透過核磁共振光譜中化學位移的擾動實驗結果，我們發現有三個區段對於 DNA 的辨識與結合有較重要的影響 : (1) 第四段的 α-helix ; (2) 以及連結第一段 α-helix 與第一段 β-strand 的環狀結構 (loop) ; (3) 以及連結第三段 α-helix 與第二段 β-strand 的環狀結構。最後，我們以定點突變實驗與核磁共振擾動實驗的結果為基礎，透過了 HADDOCK 軟體模擬出一個可能的蛋白-DNA複合物結構。

Lon protease is a multifunctional enzyme, and it’s functions include the degradation of damaged proteins, ATPase, and chaperone-like activities. It is highly conserved in prokaryotes and eukaryotic organelles. Our previous studies demonstrate the DNA binding ability as a novel function for a thermostable Lon protease from Brevibacillus thermoruber WR-249. However, the physiological role and structure of DNA binding by Lon was still poorly understood. Bioinfomatic analysis reveals that the α-domain of Bt-Lon should be a new member of DNA binding domains with an uncharacterized recognition mode. With the aim of elucidating the DNA recognition mode of Bt-Lon, NMR technique and protein crystallography were employed to determine the structures of free and DNA-bound forms of α-domain. The α-domain was crystallized by the sitting drop, vapor diffusion method. X-ray diffraction data was collected at a synchrotron-radiation source and belonged to space group P23 , with unit cell parameters a = b = c = 93.8 Å , α = 90°, β = 90° , γ = 90° . The Bt-Lon α-domain contains four α helices and two parallel β strands and resembles similar domains found in a variety of ATPases. NMR chemical shift perturbation experiments suggest that there are three major sites responsible for DNA binding : (1) α4 ; (2) the loop between α1 and β1 ; (3) and the loop between α3 and β2. The feasible model of protein-DNA complex was docking with HADDOCK software based on the mutation and NMR perturbation data.