Structural And Biochemical Investigations Of Receptor Kinase FERONIA In Arabidopsis

The survival of plants depends on their ability to adapt to changes in both internal and external environments.Receptor kinases（RKs）located on the cell membrane of plants act as intermediaries between extracellular environmental signaling and intracellular regulation.It is widely accepted that RKs perceive and identify ligand molecules through the extracellular domain（ECD）,triggering the activation of the intracellular kinase domain（KD）via the transmembrane domain（TM）.Subsequently,RKs recruit and phosphorylate specific intracellular substrates,initiating downstream signal transduction processes that ultimately enable plant survival.RK is one of the largest family of plant protein kinases,which previous research has primarily focused on the identification of RK ligands,the composition of signaling complexes,and the recognition of downstream substrates.However,there is limited investigation into the mechanism by which the intracellular KD of RKs adopts an activated conformation and initiates substrate phosphorylation.Arabidopsis FERONIA（FER）,a representative member of the receptor kinase sub-family consisting of 17 members Catharanthus roseus receptor-like kinase 1-like sub-family,plays significant roles in various aspects of plant biology,including growth,development,sexual reproduction,immunity,and responses to abiotic stress.However,the mechanism of how the intracellular domain of FER forms an activation conformation and initiates substrate phosphorylation has not been studied.Hence,this study focused on investigating the intracellular domain of FER in Arabidopsis as the research subject.The crystal structure of FER protein’s kinase domain（FER-KD）in complex with adenosine diphosphate（ADP）was successfully obtained by co-incubation of protein and nucleotide.Structural analysis revealed that FER-KD adopts an activated conformation similar to the activated state of Interleukin-1 receptor-associated kinase 4（IRAK4）,characterized byαC-in,DFG in,and BLA minus conformations.Notably,the FER-KD structure exhibits the presence of C-spine and R-spine,which are distinctive features observed only in the activated conformations of kinases.In addition,the crystal structure showed FER-KD does not undergo phosphorylation.These can be inferred that the fomation of the active conformation of FER-KD is independent of its phosphorylation.The crystal structures of FER-KD^S525A（a weakly active mutant）and FER-KD^K565R（an inactivated mutant）were further analyzed.Structural analysis revealed that these non-phosphorylated mutations were also adopt activation conformations.These results indicate that the activation of FER-KD is independent of its phosphorylation and unaffected by its kinase activity,which is different from traditional view that protein kinases require phosphorylation for activation and the adoption of the activated conformation.This study further studied the molecular mechanism of FER-KD autophosphorylation and substrate phosphorylation（transphosphorylation）.Kinase activity assays and Western bloting demonstrated that FER-KD exhibits robust autophosphorylation capability.The wild-type Trx-FER-KD can phosphorylates the inactivated mutant FER-KD^K565R,suggesting that FER-KD undergoes intermolecular catalysis during autophosphorylation.Analytical ultracentrifugation experiments revealed that autophosphorylation of FER-KD significantly enhances the formation of homodimers.Based on the intermolecular catalysis model,the homodimerization of FER-KD likely enhances its autophosphorylation,creating a positive feedback loop.The autophosphorylation sites of FER-KD were identified using liquid chromatography-tandem mass spectrometry,and site-directed mutagenesis experiments confirmed that T696,S701,and Y704 are critical autophosphorylation sites of FER-KD.Further investigation revealed that autophosphorylation of FER-KD is vital for the transphosphorylation of substrates,such as Glycine-rich RNA-binding protein7（GRP7）and Myelocytomatosis protein 2（MYC2）.This study significantly contributes to our understanding of the regulatory mechanisms of RKs and provides valuable structural biology and biochemical insights into the early processes involved in FER,FER family proteins,and the initiation of RK signaling.