Cabral Oliveira Celio, Bassi Simoni Eduardo, Abrahão Bueno Morais Mariana, Pacheco Batista Fontes Elizabeth, Braga Dos Reis Pedro A, Urano Daisuke, Jones Alan M
Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, SP 13083-970, Brazil.
Department of Biology, University of North Carolina, Chapel Hill, NC 27899, USA.
Comput Struct Biotechnol J. 2025 Apr 11;27:1529-1537. doi: 10.1016/j.csbj.2025.04.013. eCollection 2025.
Protein phosphorylation regulates G protein signaling in plants. AtRGS1 primarily modulates AtGPA1, the canonical Gα subunit in the heterotrimeric G protein complex. AtRGS1 possesses both a seven-transmembrane (7TM) domain connected to a cytoplasmic Regulator of G Protein Signaling domain (RGS box domain) by a flexible linker region. This study presents the novel function of a highly conserved, known phosphorylation site, Ser278, within this linker region utilizing molecular dynamics (MD) simulations with experimental validation. We show that phosphorylation at Ser278 is crucial for establishing specific AtRGS1 interactions with AtGPA1, primarily by stabilizing the positioning and orientation of the RGS domain within the membrane. Phosphorylation at Ser278 enhances the formation of stable hydrogen bonds between phosphorylated Ser278 and conserved residues within the RGS box domain, influencing the flexibility of RGS domain mobility and thus modulating its interface to AtGPA1. Consistent with the MD simulations, assays demonstrated that this phosphorylation reduced the binding of AtRGS1 to AtGPA1 and conferred changes in physiology. Specifically, the non-phosphorylation mutation of Ser278 decreased both plant immune responses and AtRGS1 endocytosis evoked by the bacterial effector, flg22. MD simulations and sequence analysis of diverse plant 7TM-RGS proteins suggest conservation of this mechanism across land plants, emphasizing the critical role of this previously overlooked linker region.
蛋白质磷酸化调节植物中的G蛋白信号传导。AtRGS1主要调节AtGPA1，即异源三聚体G蛋白复合物中的典型Gα亚基。AtRGS1具有一个七跨膜（7TM）结构域，该结构域通过一个柔性连接区与细胞质G蛋白信号调节结构域（RGS盒结构域）相连。本研究利用分子动力学（MD）模拟并结合实验验证，揭示了该连接区内一个高度保守的已知磷酸化位点Ser278的新功能。我们发现，Ser278位点的磷酸化对于建立AtRGS1与AtGPA1之间的特异性相互作用至关重要，主要是通过稳定RGS结构域在膜内的定位和方向。Ser278位点的磷酸化增强了磷酸化的Ser278与RGS盒结构域内保守残基之间稳定氢键的形成，影响了RGS结构域移动的灵活性，从而调节其与AtGPA1的界面。与MD模拟结果一致，实验分析表明这种磷酸化降低了AtRGS1与AtGPA1的结合，并导致了生理变化。具体而言，Ser278的非磷酸化突变降低了植物免疫反应以及细菌效应物flg22引发的AtRGS1内吞作用。对多种植物7TM-RGS蛋白的MD模拟和序列分析表明，这种机制在陆地植物中具有保守性，强调了这个之前被忽视的连接区的关键作用。
