Pup, a prokaryotic ubiquitin-like protein, is an intrinsically disordered protein

Abstract

Pup (prokaryotic ubiquitin-like protein) from Mycobacterium tuberculosis is the first ubiquitin-like protein identified in non-eukaryotic cells. Although different ubiquitin-like proteins from eukaryotes share low sequence similarity, their 3D (three-dimensional) structures exhibit highly conserved typical ubiquitin-like folds. Interestingly, our studies reveal that Pup not only shares low sequence similarity, but also presents a totally distinguished structure compared with other ubiquitin-like superfamily proteins. Diverse structure predictions combined with CD and NMR spectroscopic studies all demonstrate that Pup is an intrinsically disordered protein. Moreover, 1H-15N NOE (nuclear Overhauser effect) data and CSI (chemical shift index) analyses indicate that there is a residual secondary structure at the C-terminus of Pup. In M. tuberculosis, Mpa (mycobacterium proteasomal ATPase) is the regulatory cap ATPase of the proteasome that interacts with Pup and brings the substrates to the proteasome for degradation. In the present paper, SPR (surface plasmon resonance) and NMR perturbation studies imply that the C-terminus of Pup, ranging from residues 30 to 59, binds to Mpa probably through a hydrophobic interface. In addition, phylogenetic analysis clearly shows that the Pup family belongs to a unique and divergent evolutionary branch, suggesting that it is the most ancient and deeply branched family among ubiquitin-like proteins. This might explain the structural distinction between Pup and other ubiquitin-like superfamily proteins.

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.…”