Fluorescent probes reveal a minimal ligase recognition motif in the prokaryotic ubiquitin-like protein from Mycobac-terium tuberculosis

摘要

The prokaryotic ubiquitin-like protein (Pup) based proteasomal system in the pathogen Mycobacterium tuberculosis (Mtb) is essential for its survival in a mammalian host. The Pup ligase enzyme, PafA, conjugates Pup to a suite of proteins targeted for proteasomal degradation, and is necessary for persistent infection by Mtb. We report the design and application of fluorescent probes toward elucidating the mechanisms of Pup and substrate recognition by PafA. Our studies reveal that the C-terminal 26-amino acid sequence of Pup is the minimal ligase recognition motif in Mtb. Specific hydrophobic residues within this sequence that are known to be important for Pup interaction with proteasomes are also critical for the activation of Pup by PafA.