Light influences plant growth and development. Far-red light and red light can synergistically affect seed germination and photomorphogenesis through the phytochrome photoreceptors in plant cells. A previous study demonstrated that a jasmonate synthetase, FIN219 (FAR-RED INSENSITIVE 219), also known as JAR1 (JASMONATE RESISTANT 1), functions to conjugate jasmonate (JA) to amino acids such as Isoleucine (Ile) in the JA signaling pathway. JA-Ile is an active phytohormone required for plant defense, growth, and development. Therefore, far-red light-coupled JA signaling is controlled by FIN219 activity. In Arabidopsis, FIN219 interacts with the protein FIP1 (FIN219-INTERACTING PROTEIN 1) in the far-red light signaling pathway. However, the mechanism and function of this interaction are unknown. Here, we use several methods, such as X-ray crystallography and biophysical and biochemical analyses, to elucidate the molecular characteristics of the FIN219-FIP1 complex.
From the crystal structure of the FIN219-FIP1 complex, we observed that the orientation of the FIN219 C-terminal domain shifted after binding FIP1 and moving near the active site of FIN219; this domain could then stabilize the binding of substrates, such as JA, Ile, and ATP. Based on this important structural information, we further resolved 12 FIN219-FIP1 structures with different combinations of substrates such as ATP, JA, and the amino acids Ile, Leu, Met, and Val. The structures of FIN219-FIP1 complexes bound to these substrates reveal the preference of FIN219 for Ile over other amino acids. This amino acid preference results from differences in ATP orientation in the substrate binding pocket of FIN219. Furthermore, the binding order of the substrates JA, ATP, and Ile was examined using a quartz crystal microbalance (QCM), a biophysical assay machine. This assay showed that FIN219 first binds to JA and then binds to ATP or Ile. The purpose of the interaction between FIN219 and FIP1 was determined by enzyme kinetics and QCM. Compared with FIN219 alone, the FIN219-FIP1 complex showed 2.3-fold increased Kcat and 2-fold increased Vmax, demonstrating that FIN219 enzyme activity was enhanced by FIP1 binding. Finally, we propose a model to explain the changes in conformation and enzyme activity of FIN219 after FIP1 binding. In summary, the detailed mechanism of the interaction of FIN219 with FIP1 provides valuable insights into enzyme catalysis in the GH3 (glycoside hydrolase 3) family and molecular regulation in photomorphogenesis.
Figure 1. Proposed catalysis mechanism of the FIN219-FIP1 complex
Reference
Chun-Yen Chen, Sih-Syun Ho, Tzu-Yen Kuo, Hsu-Liang Hsieh, and Yi-Sheng Cheng*. (2017). Structural basis of jasmonate-amido synthetase FIN219 in complex with glutathione S-transferase FIP1 during the JA signal regulation. Proceedings of the National Academy of Sciences of the United States of America (PNAS). 114(10), E1815-E1824. Published online February 21, 2017. DOI: 10.1073/pnas.1609980114
Associate Professor Yi-Sheng Cheng
Department of Life Science & Institute of Plant Biology
chengys@ntu.edu.tw