Studies Into The Molecular Mechanism Of The Non-uncanonical Ubiquitination Catalyzed By The Legionella Effector MavC

Protein ubiquitination is one of the most common forms of post-translational modifications in eukaryotic cells,which virtually involves various processes of eukaryotic cellular activities.It plays an important regulatory role and is crucial for protein degradation,localization,function,and metabolism.Recently,the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination,that is,MavC links ubiquitin(Ub)to UBE2N through its glutamine residue at position 40(Gln40),thereby modifying UBE2N with ubiquitination.Here,we solved the structures of the MavC/UBE2N/Ub ternary complex and revealed this unique ubiquitination mechanism at the molecular level.To gain insights into the catalytic process of this non-canonical ubiquitination,we also solved the structures of the MavC/UBE2N-Ub(product)binary complex.Moreover,we also found that MavC itself also exhibits weak activity to catalyze the reverse reaction,that is,the deubiquitination of this non-canonical ubiquitination product UBE2N-Ub,cleaving the product UBE2N-Ub into UBE2N and Ub.Through the structural analysis of the above two complexes,we designed multiple mutants of MavC,UBE2N and Ub in the complex,purified the proteins,and carried out subsequent biochemical experiments such as in vitro ubiquitination,deubiquitination,and deamination experiments to verify their functions.The results above,combined with the structures reported in this study,revealed the mechanism of this unique ubiquitination at the molecular level and provided insights into the catalytic process of this non-canonical ubiquitination.In summary,this study provides important insights into the mechanism of this transglutaminase-induced ubiquitination,and lay the foundation for further functional studies into this unique ubiquitination.