以結構生物學探討阿拉伯芥FIN219對茉莉酸及AtERF96對乙烯訊息的調控機制

Abstract

FIN219 (far-red insensitive 219)為阿拉伯芥中GH3 (Gretchen Hagen 3)家族的成員，功能上屬於腺苷酸形成酶家族(adenylate-forming family)的酵素。FIN219在由茉莉酸調控的防禦反應中負責合成茉莉酸-異白胺酸(jasmonoyl-isoleucine, JA-Ile)的重要角色，並且與FIP1(FIN219-interacting protein 1)在遠紅光訊息下產生交互作用。過去的研究集中在FIN219及FIP1對阿拉伯芥的光反應之生理功能，對於FIN219與FIP1的結合所產生的分子機制尚不明瞭。此外，乙烯(ethylene)是植物中廣泛影響許多發育過程的植物激素，也是植物面對生物與非生物性逆境的防禦反應之重要調控因子。乙烯反應因子(ethylene response factor, ERF)為AP2/ERF家族的成員，是藉由辨識目標DNA上的順式反應元件(cis-element)，來調控逆境反應基因的轉錄因子，對於乙烯訊息途徑的活化至關重要。前人研究僅展示了AtERF100的AP2/ERF domain之結構，對於ERF其他部分的結構資訊仍不清楚。 本研究利用X光晶體學(X-ray crystallography)探討FIN219–FIP1蛋白質複合體，包括與不同受質結合的複合體晶體結構，以及AtERF96–GCC box蛋白質–DNA複合體晶體結構。我們發現FIN219的結構透過與FIP1的結合產生了構型改變，C端的結構域結合至N端結構域的活性區入口，並且改變了活性區內ATP的結合位置與ATP的構型。活性區的改變使得受質的結合更加緊密，更有利於腺苷酸化反應的進行，這些影響與FIN219結合FIP1後活性上升的結果一致。因此，我們推導出阿拉伯芥中存在兩種FIN219的催化模式，分別對應於FIP1結合與否所產生的結構構型，並延伸為茉莉酸與遠紅光訊息之間交互作用的分子機制。此外，我們也發現AtERF96不僅透過AP2/ERF domain辨識並結合GCC box的核心序列，其N端螺旋結構延伸至GCC box的次溝槽(minor groove)位置與鹼基結合。N端螺旋以及AP2/ERF domain上的保守胺基酸一起作用於模板股的鹼基，使得DNA的模板股結構發生改變，造成局部區域的DNA雙股被打開。EDLL motif區域呈現出連續的螺旋結構並帶有較高的B-factor，意味著其保守序列與介體複合物(mediator complex)結合的重要性。

FIN219 (far-red insensitive 219) is a member of the GH3 (Gretchen Hagen 3) family of proteins in Arabidopsis, and functionally belongs to the adenylate-forming family of enzymes. FIN219 plays an important role in the biosynthesis of jasmonoyl-isoleucine in JA-mediated defense responses and interacts with FIP1 (FIN219-interacting protein 1) under far-red light signaling. The previous study focused on the physiological function of light reactions of FIN219 and FIP1, the molecular mechanism of the interaction between FIN219 and FIP1 remained unclear. In addition, ethylene is a phytohormone widely involved in many developmental processes, and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. ERF (ethylene response factor), a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA and is necessary for the activation of ethylene signaling pathway. The previous study showed only the structure of the AP2/ERF domain of AtERF100, the other structural information of ERFs is still unknown. In this study, we aimed to solve the crystal structures of FIN219–FIP1 protein complex, including the complex structures with various bound substrates, and the AtERF96–GCC box protein–DNA complex by X-ray crystallography. We found out that the FIN219 structure generated a conformational change through interacting with FIP1, the C-terminal domain bound to the entrance of active site of the N-terminal domain, and altered the binding position and conformation of ATP. The alteration of the active site is advantageous to the closer binding of substrate, it is consistent with the result of the increased activity of FIP-bound FIN219. Thus, we proposed that the two modes of catalysis of FIN219 exist in Arabidopsis, corresponding to the structural conformation with FIP1-bound and FIP1-free, and extend to the molecular mechanism of the interplay between jasmonic acid and far-red light signaling. Furthermore, we also found out that AtERF96 not only recognized and bound to the core sequence of GCC box motif through the AP2/ERF domain, the N-terminal helix structure extended to the minor groove of GCC box and bound to bases. The N-terminal helix and the conserved residues of AP2/ERF domain function on the nitrogenous bases of template strand, leading to the opening of DNA double strands of the local region. The EDLL motif region revealed a continuous helical structure and had the higher B-factor, implying its importance of a conserved sequence binds to the mediator complex.