A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

Abstract

Edited by Miguel De la RosaKeywords: Prokaryotic ubiquitin-like protein Mpa ARC Proteasome NMR Mycobacterium tuberculosis a b s t r a c tThe mycobacterial ubiquitin-like protein Pup is coupled to proteins, thereby rendering them as substrates for proteasome-mediated degradation. The Pup-tagged proteins are recruited by the proteasomal ATPase Mpa (also called ARC). Using a combination of biochemical and NMR methods, we characterize the structural determinants of Pup and its interaction with Mpa, demonstrating that Pup adopts a range of extended conformations with a short helical stretch in its C-terminal portion. We show that the N-terminal coiled-coil domain of Mpa makes extensive contacts along the central region of Pup leaving its N-terminus unconstrained and available for other functional interactions. Structured summary:MINT-7262427: pup (uniprotkb:B6DAC1) binds (MI:0407) to mpa (uniprotkb:Q0G9Y7) by pull down (MI:0096) MINT-7262440: mpa (uniprotkb:Q0G9Y7) and pup (uniprotkb:B6DAC1) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

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

“…Both linear fusions in mycobacterium mutants and overproduction of Pup lacking its N-terminal region strongly support this hypothesis. We found that the C-terminal half of Pup, in addition to the proteasomal ATPase Mpa, is required to interact with pupylation enzymes (4,14,17,19). Taken together, we propose a model where (i) the C-terminal half of Pup interacts with enzymes to facilitate pupylation, (ii) the same region interacts with Mpa to target the protein to the proteasome, and (iii) the N-terminal half of Pup initiates degradation by the proteasome.…”

“…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.…”

Allosteric Transitions Direct Protein Tagging by PafA, the Prokaryotic Ubiquitin-like Protein (Pup) Ligase

“…his 6 6 i27-tev-cys-pup E ). Labeling with 5-iodoacetamidofluorescein was carried out following binding of the protein to the Ni 2ϩ -NTA column.…”

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

“…7) Proteasome accessory factor A then ligates Pup to target proteins by catalyzing the formation of an isopeptide bond between the C-terminal glutamate of Pup and the "-amino group of lysines in the substrates. In degrading pupylated proteins, the proteasomal ATPase complex (ARC) recruits pupylated proteins by binding Pup to its N-terminal coiled-coil domains, 8,9) and the substrates are then translocated through the ATPase pore into the proteasomal catalytic chamber. 10) In this Pup-dependent proteolytic system, Dop functions not only as a deaminase of Pup but also as a depupylase, removing Pup from pupylated substrates by cleaving the isopeptide bond between Pup and the target proteins.…”