Structural Insights Into The Mechanism And Inhibition Of The Non-canonical Ubiquitination By The Lehionella Effector MavC

Protein ubiquitination is one of the most prevalent post-translational modifications,controlling virtually every process in eukaryotic cells.Recently,the Legionella effector MavC was found to mediate a unique ubiquitination through trans-glutamination,linking ubiquitin(Ub)to UBE2N through UbGln40 in a process that can be inhibited by another Legionella effector Lpg2149.UBE2N is a ubiquitin-conjugating enzyme(E2),which plays an important role in the ubiquitin-proteasome pathway in the host cell.The ubiquitination effect catalyzed by MavC interferes with the role of UBE2N as a ubiquitin-conjugating enzyme.The pathogen Legionella pneumophila invades the host cell through this interference.Here we report the structures of MavC/UBE2N/Ub ternary complex,MavC/UBE2N-Ub(product)binary complex,and MavC/Lpg2149 binary complex through protein crystallography.We studied the interaction between MavC/Ub and between MavC/UBE2N through the MavC/UBE2N/Ub structure.By comparing the MavC/UBE2N-Ub and the MavC/UBE2N/Ub complex structure,we found that the loop containing the modification site K92 of UBE2N undergoes marked conformational change during the reaction.For the inhibition mechanism of the inhibitor Lpg2149,we found that its inhibits this ubiquitination by competing with Ub for binding to MavC,but does not interfere with the binding of substrate UBE2N.Moreover,we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination.This deubiquitination activity is specific to UBE2N-Ub,since MavC cannot catalyze the deubiquitination of K48/K63 diubiquitin.Together,our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC,but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination.