Cheung Ming-Yan, Li Xiaorong, Ku Yee-Shan, Chen Zhongzhou, Lam Hon-Ming
Center for Soybean Research of the State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
Front Mol Biosci. 2022 Nov 30;9:1061350. doi: 10.3389/fmolb.2022.1061350. eCollection 2022.
AtYchF1 is an unconventional G-protein in that exhibits relaxed nucleotide-binding specificity. The bindings between AtYchF1 and biomolecules including GTP, ATP, and 26S rRNA have been reported. In this study, we demonstrated the binding of AtYchF1 to ppGpp in addition to the above molecules. AtYchF1 is a cytosolic protein previously reported as a negative regulator of both biotic and abiotic stresses while the accumulation of ppGpp in the cytoplasm induces retarded plant growth and development. By co-crystallization, pull-down experiments, and hydrolytic biochemical assays, we demonstrated the binding and hydrolysis of ppGpp by AtYchF1. ppGpp inhibits the binding of AtYchF1 to ATP, GTP, and 26S rRNA. The ppGpp hydrolyzing activity of AtYchF1 failed to be activated by AtGAP1. The AtYchF1-ppGpp co-crystal structure suggests that ppGpp might prevent His from executing nucleotide hydrolysis. In addition, upon the binding of ppGpp, the conformation between the TGS and helical domains of AtYchF1 changes. Such structural changes probably influence the binding between AtYchF1 and other molecules such as 26S rRNA. Since YchF proteins are conserved among different kingdoms of life, the findings advance the knowledge on the role of AtYchF1 in regulating nucleotide signaling as well as hint at the possible involvement of YchF proteins in regulating ppGpp level in other species.
AtYchF1是一种非常规G蛋白，其核苷酸结合特异性较为宽松。此前已有报道AtYchF1与包括GTP、ATP和26S rRNA在内的生物分子之间的结合。在本研究中，我们证明了AtYchF1除了能与上述分子结合外，还能与ppGpp结合。AtYchF1是一种胞质蛋白，此前被报道为生物和非生物胁迫的负调控因子，而细胞质中ppGpp的积累会导致植物生长发育迟缓。通过共结晶、下拉实验和水解生化分析，我们证明了AtYchF1对ppGpp的结合和水解作用。ppGpp会抑制AtYchF1与ATP、GTP和26S rRNA的结合。AtGAP1无法激活AtYchF1的ppGpp水解活性。AtYchF1-ppGpp共晶体结构表明，ppGpp可能会阻止组氨酸进行核苷酸水解。此外，ppGpp结合后，AtYchF1的TGS和螺旋结构域之间的构象会发生变化。这种结构变化可能会影响AtYchF1与其他分子（如26S rRNA）之间的结合。由于YchF蛋白在不同生命王国中是保守的，这些发现增进了我们对AtYchF1在调节核苷酸信号传导中作用的了解，并暗示了YchF蛋白可能参与调节其他物种中ppGpp的水平。
