Structural Biology Research On K27-Iinked Diubiquitin And Its Recognition Protein UCHL3

DNA damage repair is an important way for organisms to maintain genetic stability of the genome,which is essential for plant growth and development.Studying the molecular mechanism of DNA damage repair provides a theoretical basis for cultivating anti-UV plants and ensuring food production in harsh environments.In recent years,reports have shown that,as a kind of ubiquitin code,K27 linked ubiquitin chains are involved in the regulation of DNA damage repair processes.Therefore,the study of recognition and regulation mechanism of K27 linked ubiquitin chains is significant for cultivating new plants to ensure grain production under harsh environment.As one of the most important post-translational modifications,ubiquitination is widespread in eukaryotes,which plays an important regulatory role in the regulation of plant growth and development through modification of substrate proteins.Ubiquitin can form various ubiquitin chains with different structural features through isopeptide bonds,which is also called ubiquitin code.The function of different kinds of ubiquitin chains is mainly determined by their structures.Among all kinds of ubiquitination,K27 linked ubiquitin chains play an important role in DNA damage repair.UCHL3 is the only deubiquitinating enzyme that shows specific binding ability towards K27 linked diubiquitin（K27diub）.UCHL3 can participate in DNA damage repair by showing deubiquitinase activity towards RAD51.Notably,studies show that UCHL3 can only cut Ub-AMC in vitro,but show no activity towards any kinds of polyubiquitin chains so far.Phosphorylation at serine 75（Ser75）on UCHL3significantly enhances the activity of cleavage of polyubiquitin chains in cells,but this conclusion has not been supported by any evidence in vitro.This paper focuses on the recognition mechanism of K27linked ubiquitin chains by UCHL3,which participates in DNA damage repair.The findings are listed as followed:1.The specific recognition of K27 diub by UCHL3 was verified in vitro by Isothermal Titration Calorimetry.2.By analyzing the high-resolution crystal structure of UCHL3 and K27 diub complex,it was firstly discovered that UCHL3 actually had two ubiquitin binding sites,S1 and S2,respectively.The S1 site recognized the C-terminus of proximal ubiquitin and the S2 site formed classical I44 hydrophobic interaction with distal ubiquitin,and was complemented by His¹⁵³ and Glu¹⁵⁸ to form hydrogen bond networks.The interactions between K27 diub and different UCHL3 mutations were significantly reduced after key amino acids were mutated in UCHL3.3.It was found that the conformation of K27 diub was more stretched and combined with UCHL3,showing a relatively open state.4.Sequence alignment and phylogenetic tree showed that the phosphorylation site(Ser⁷⁵)was only present in UCHL3 and was absent in other UCH family members,suggesting that phosphorylation regulation of UCHL3 was specific in the UCH family.5.It was found that the phosphorylation site(Ser⁷⁵)was widely present in different species of UCHL3,suggesting that the phosphorylation regulation mechanism of UCHL3 was evolutionarily conserved.It was noted that the phosphorylation site Ser was mutated to Glu in the model organism Arabidopsis,suggesting UCHL3 may be constitutively activated form,which meant that it can show high activity towards polyubiquitin chains without phosphorylation by kinases.6.Simulated phosphorylation of UCHL3 displayed enhancing cleavage activity towards Ub-AMC and did not show any activity to di-ubiquitin chains.The work above firstly demonstrated the recognition mechanism of UCHL3 towards K27 linked ubiquitin chains,revealing the uniqueness of UCHL3 phosphorylation regulation,and laid a solid foundation for molecular regulation of corresponding agricultural production.