Prokaryotic Ubiquitin-Like Protein Pup Is Intrinsically Disordered

Abstract

The prokaryotic ubiquitin-like protein Pup targets substrates for degradation by the Mycobacterium tuberculosis proteasome through its interaction with Mpa, an ATPase that is thought to abut the 20S catalytic subunit. Ubiquitin, which is assembled into a polymer to similarly signal for proteasomal degradation in eukaryotes, adopts a stable and compact structural fold that is adapted into other proteins for diverse biological functions. We used NMR spectroscopy to demonstrate that unlike ubiquitin, the 64 amino acid protein Pup is intrinsically disordered with small helical propensity in the C-terminal region. We found that the Pup:Mpa interaction involves an extensive contact surface that spans S21–K61 and that the binding is in the “slow” exchange regime on the NMR time scale, thus demonstrating higher affinity than most ubiquitin:ubiquitin receptor pairs. Interestingly, during the titration experiment, intermediate Pup species were observable, suggesting the formation of one or more transient state(s) upon binding. Moreover, Mpa selected one configuration for a region undergoing chemical exchange in the free protein. These findings provide mechanistic insights into Pup’s functional role as a degradation signal.

Posttranslational Modification of Cellular Proteins by a Ubiquitin-like Protein in Bacteria

“…TtuB (homology modeling (supplemental Fig. S1B) (29)) and Ub/Ubls in eukaryotes have an ordered ␤-grasp fold in contrast to the intrinsically disordered Pup protein (21,22). The mechanism of conjugation of TtuB to target proteins, which utilizes the E1-like TtuC protein, also differs from that of pupylation.…”

“…Pup is an intrinsically disordered protein (21,22), in contrast to the ordered ␤-grasp fold of Ub/Ubls. This study focused on investigating whether a bacterial Ubl can function as a protein modifier in the bacterium Thermus thermophilus.…”

RhodococcusProkaryotic Ubiquitin-Like Protein (Pup) Is Degraded by Deaminase of Pup (Dop)

“…11,28) Pup has a disordered structure and is not highly conserved among actinomycetes (Fig. 2B), 30,31) as indicated by the various Pup cleavage patterns generated by Dop. Although Dop Re and Dop Mt showed similar endopeptidase activity (Table 3), only Pup Re failed to accumulate after depupylation, while Pup Mt could accumulate (Fig.…”

Prokaryotic Ubiquitin-Like Protein Provides a Two-Part Degron toMycobacteriumProteasome Substrates

“…NMR analysis determined that Mpa interacts with the Cterminal half of Pup but not with the disordered N terminus (4,14,19). In addition to providing an unstructured degron for proteasome substrates, we hypothesized that the N terminus of Pup interacts with enzymes of the pupylation pathway.…”