Abstract
Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.
Highlights
Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria
In combination with biochemical experiments, our analysis reveals the mechanism of ATP hydrolysis that is required to generate the catalytic phosphate in the active site, and provides insights into the allosteric regulatory role of the Dop-loop on Pup-binding and ATP hydrolysis
This is important, since the binding of Pup to Dop stimulates the generation of the inorganic phosphate that must be present in the active site for catalysis[19]
Summary
In order to deduce the catalytic mechanism of the depupylase enzyme, a structure of Dop in complex with Pup is crucial. This is important, since the binding of Pup to Dop stimulates the generation of the inorganic phosphate that must be present in the active site for catalysis[19]. Pup in complex with Dop exhibits a significantly more extensive network of interactions than Pup bound to the ligase PafA, with differences especially pronounced at the C-terminal residue of Pup (Fig. 1c, d). The C-terminal glutamine residue of Pup in the Dop-Pup complex is well defined in the electron density map (Supplementary Fig. 1)
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