Abstract
Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery.
Highlights
The interaction of PafA, the prokaryotic ubiquitin-like ligase, with its protein substrates is poorly understood
We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by prokaryotic ubiquitin-like protein (Pup) binding
By establishing a quantitative pupylation assay, we were able to measure the kinetics of M. smegmatis PafA pupylation in this study
Summary
The interaction of PafA, the prokaryotic ubiquitin-like ligase, with its protein substrates is poorly understood. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer These findings suggest that protein pupylation can be regulated at the level of PafA allostery. The Pup ligase, an enzyme termed PafA (proteasomal accessory factor A), both activates and conjugates Pup to protein substrates in a two-step reaction that is typical of glutamine synthetases and ␥-glutamyl cysteine synthetases [2, 14, 19]. Analysis of engineered chimeric substrates that differ in the number of pupylation sites suggested that PafA is active as a dimer. In view of these findings, we propose that protein pupylation can be regulated at the enzyme level via an allosteric mechanism
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