阿拉伯芥PIF3與G-box序列結合的分子機制之研究

Abstract

在光敏素調控的訊息傳導路徑中，PHYTOCHROME INTERACTING FACTOR3 (PIF3)被認為是負調控光訊息反應的一個重要因子。先前的研究也指出PIF3是隸屬於可以結合至G-box序列的bHLH轉錄因子巨家族的成員。在本研究中，旨在探討PIF3與G-box DNA專一性結合之機制。 藉由膠體過濾層析法(gel filtration chromatography)以及分析型超高速離心(analyticalultracentrifugation; AUC)的結果顯示，bHLH是以二聚體形式與G-box(CACGTG)進行結合。經蛋白質序列比對及結構模擬等生物資訊分析方式，發現數個重要的疏水性胺基酸：Met60、Val57、Gln56、Leu53、Leu50、Tyr49、Ile47、Leu27以及Met24，其之間形成六層疏水性作用力介面以穩固蛋白質二聚體的形成。另以螢光基礎電泳遷移滯實驗(fluorescein-based electrophoretic mobility shift assay; fEMSA)測量bHLH與G-box專一性結合的能力，結果顯示bHLH除了可以與G-box結合外，也可以與其他不同E-box(CANNTG)進行結合。從蛋白質結構模擬分析，bHLH中與DNA結合的重要保守性殘基為His9、Glu13以及Arg17，Arg17負責與G-box中第四號鹼基形成氫鍵，由於在fEMSA實驗中發現bHLH在第四號鹼基位置分別置換四種鹼基後皆具結合能力，因此推估Arg17並非扮演DNA辨認的角色，另外也發現E-box上的第三號鹼基突變為空間較大的G後，整體結合能力較G-box下降許多。總結以上研究結果，可以推論一個bHLH與G-box之間可能的結合機制：藉疏水性胺基酸交互作用之二聚體bHLH先以帶正電結合溝槽與雙股DNA進行初步結合，辨認到core region之正股六號鹼基(G)以及反股之二號鹼基(A)，進行氫鍵結合後，進一步靠著中心的正股四號鹼基(G)進行氫鍵結合，形成一氫鍵網絡以穩固蛋白質在DNA上的構型。

It is crucial for plants to sense light signals by phytochromes in development and growth. Among phytochrome signaling pathway, PHYTOCHROME INTERACTING FACTOR3 (PIF3) is believed to act as a key component that negatively regulates several light responses in plants. Previous studies have identified PIF3 as a member of basic helix-loop-helix (bHLH) transcription factor superfamily that can directly bind to a typical G-box (CACGTG) DNA. In this study, we aimed to reveal the binding mechanism between PIF3 and G-box DNA from structural view point. In this study, we examined the oligomeric states of apo form bHLH and bHLH-DNA by gel filtration chromatography and analytical ultracentrifugation. The results indicated that bHLH might form a homodimer to associate with G-box DNA. We also found some hydrophobic residues, including Met60, Val57, Gln56, Leu53, Leu50, Tyr49, Ile47, Leu27 and Met24 by sequence alignment and modellng. These residues formed a six-layer hydrophobic interaction interface that might be critical for bHLH dimer formation. From the fluorescein-based electrophoretic mobility shift assay (fEMSA) results, we found that not only G-box but also E-box (CANNTG) could bind to bHLH protein. In addition, we found some conserved residues His9, Glu13 and Arg17 that might play critical roles in G-box recognition. Combined with fEMSA and modelling results, the Arg17 that formed hydrogen bonds with the 4th base of G-box may not act as a DNA recognition residue. Although the 3th base of G-box did not participate in hydrogen bond formation, it might have steric hindrance that affected the hydrogen bond formation between Arg17 and the 4th base of G-box. In summary, we concluded a possible mechanism of bHLH binding to G-box sequence: The bHLH formed homodimers by the hydrophobic residues first. The positive-charged binding groove of bHLH could stabilize DNA at the groove. Then, the His9 and Glu13 could recognize the 6th base of G-box and the the 2th base of anti-sense G-box. The Arg17 then formed hydrogen bonds with the 4th base of G-box. Finally, they could form a hydrogen bond network to stabilize the protein-DNA structure.