Established The XL-MS And To Study The Interaction Between AHAS Subunits And Ran GTPase Protein

Chemical cross-linking coupled with mass spectrometry(XL-MS)is a new method that combines chemical cross-linking of protein and mass spectrometry to study the structure of protein complexes and protein-protein interactions.It has the advantages of high throughput,sensitivity,and clearly action mechanism.In this study,reaction system of BSA cross-linking in vitro,the protein digestion method of cross-linked proteins and collection methods of mass spectrometry data were optimized systematically.The cross-linked peptides that been indentified are accounted for 68% of the total theoretical cross-linked peptides,which the crosslinked peptides within the distance are accounted for 96% of the total number of identifications.The high credibility and the depth of coverage about cross-linking of data indicates that the XL-MS system has been established successfully.Acetohydroxyacid synthases(AHAS)is a key enzyme in the biosynthesis of branched-chain amino acids.It consists of catalytic subunits and regulatory subunits.The interaction between subunits plays an important role for the regulation of enzyme activity mechanisms.And the interface surface can be used as a potential inhibitor target.This study using XL-MS and site-directed mutation shows the ?-? linker of ilv I with its near ?,? domains and the ACT domain of ilv H is the key interface for interaction;Asp111 of ilv I ? domain,Lys193 and Lys197 of ilv I ? domain,Arg26 of ilv H ACT domain ?1,Lys55,Lys62,and Lys66 of ilv H ACT domain ?2 are the key of the interaction interface.And the study predicts the holoenzyme's interaction structural model between the catalytic subunit ilv I and the regulatory subunit ilv H.Ran GTPase-related proteins,such as Ran,Ran BP1 and RCC1,regulate the nucleocytoplasmic transport through the nuclear pore complex,and control the cell cycle process through microtubule polymerization and formation of the mitotic spindle.This study uses the GST pulldown to prove that Ran interacts directly with Ran BP1,Ran interacts directly with RCC1,Ran BP1 interacts with RCC1 indirectly through Ran.Ran,Ran BP1,RCC1 protein pulls down with 293 T cell lysate complex to conduct XL-MS,respectively.The results shows: The protein cross-linked with Ran include DNA helicase RAD54L2,the initial recognition complex subunit ORC6.The protein cross-linked with RCC1 includes Ras-related protein RALB.The protein cross-linked with Ran BP1 include upstream controlling element binding protein FUBP3 and Ral GTPase activate protein subunits RALGAPA2,which confirmed interact proteins with Ran,Ran BP1 and RCC1,relating to DNA transcription and cell cycle regulation.And the proteins expand its interaction network.