PYL Interacting Proteins Capture Via Photo-Cross-Linking

Protein-protein interactions play an important role in various biological processes.Probing the complex PPIs provides new insight into the biological system and paves the way for the subsequent development of therapeutics.The discovery and elucidation of PPIs,as well as the development of methods for PPIs detection and analysis,have always been the research priorities of biology and chemical biology.Among the numerous techniques,photo-crosslinking has been increasingly favored in recent years due to its unbiased covalent capture.Herein,we introduce a photo-crosslinking method coupled with HPLC-MS/MS analysis strategy for PYL(Pyrabactin Resistance 1-like)new interacting partners capture,and developed efficient photo-crosslinkers for protein crosslinking.ABA plays essential roles in plant growth and development.The function of ABA is regulated by its receptors PYR/PYL/RCAR(hereinafter referred to as PYL)through PYL-PP2C-Sn RK2 interaction,which is the core of the abscisic acid signal transduction network.Revealing the new interacting partners of PYL would advance our understanding of the ABA signaling network.In this study,we present a strategy combing photo-crosslinking with mass spectrometry-based label-free quantification(LFQ)proteomics,which enabled us to trap PYL5-interacting partners in Arabidopsis lysates.Fortunately,the known interacting partner HAB1 and three new PYL5-interacting partners,RAD23 C,CSN1 and CAP1,along with their crosslinked sites involved in PYL5-partners interacting surface were identified using further HPLC-MS/MS analysis and site directed mutagenesis.Among these proteins,CAP1 was verified to interact with PYL5 both in vitro and in vivo.The discovery of new PYL interacting proteins not only help us to further understand the ABA signaling pathway,but also show the potential of the photo-crosslinking coupled with mass spectrometry-based proteomics quantification strategy in protein-protein interactions capture.Photo-crosslinkers are vital in trapping protein-protein interactions by photo-crosslinking.Herein,we present a new class of photo-crosslinkers and the strategy for protein crosslinking using palladium oxidative addition complexes.The biarylphosphine-based palladium reagents are used to transfer a series of photoaffinity groups into peptides or proteins by the arylation modification of cysteine residue.Next,the photoaffinity groups are activated by UV irradiation and produce reactive species to capture adjacent proteins for protein cross-linking.We synthesized four photoaffinity palladium reagents that containing three types of common-used photoaffinity groups(alkyl diazirine,trifluoromethylaryl diazirine and benzophenon),which indicates high activity and high selectivity in peptide and protein modifications,and successfully applied to capture the PYR1-ABA-ABI1 interaction.The results demonstrated that palladium oxidative addition complexes which selectively modify natural amino acids can be used as potential platform for the development of multifunctional crosslinkers in the study of protein-protein/peptide crosslinking.