Escherichia coli, one of the most well-studied gram-negative bacterial species, encodes two ubiquitin-like proteins (UBLs), ThiS and MoaD. The studies on prokaryotic UBLs such as Pup, and small archaeal modifier protein have revealed the function of UBLs. However, in gram-negative bacteria, the functions of UBLs in protein modification are still poorly understood to date. Here, we report that ThiS, which has a β-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, is able to form protein conjugates in vivo and in vitro. We also constructed in vitro ThiS conjugation (thisylation) system and identified the modified lysine sites by MS/MS, this provides an essential platform for studying the UBLs thisylation system in E. coli. The modification system is dependent on lysine 83 (ATPase activity site) and cysteine 169 (zinc binding site) in ThiF and three important substrates, GroEL, PriC, FtsA, were found to be covalently modified by this system in vitro. Taken together, this study provided evidence that the protein conjugation function of β-grasp fold UBLs is conserved in the three major evolutionary lineages of life.
Keywords: ThiF; ThiS; Thisylation.
Copyright © 2019. Published by Elsevier B.V.