Prokaryotic Ubiquitin-like Protein Pup Research Articles

Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

Pupylated proteins are subject to broad proteasomal degradation specificity and differential depupylation.

In actinobacteria, post-translational modification of proteins with prokaryotic ubiquitin-like protein Pup targets them for degradation by a bacterial proteasome assembly consisting of the 20S core particle (CP) and the mycobacterial proteasomal ATPase (Mpa). Modification of hundreds of cellular proteins with Pup at specific surface lysines is carried out by a single Pup-ligase (PafA, proteasome accessory factor A). Pupylated substrates are recruited to the degradative pathway by binding of Pup to the N-terminal coiled-coil domains of Mpa. Alternatively, pupylation can be reversed by the enzyme Dop (deamidase of Pup). Although pupylated substrates outcompete free Pup in proteasomal degradation, potential discrimination of the degradation complex between the various pupylated substrates has not been investigated. Here we show that Mpa binds stably to an open-gate variant of the proteasome (oCP) and associates with bona fide substrates with highly similar affinities. The proteasomal degradation of substrates differing in size, structure and assembly state was recorded in real-time, showing that the pupylated substrates are processed by the Mpa-oCP complex with comparable kinetic parameters. Furthermore, the members of a complex, pupylated proteome (pupylome) purified from Mycobacterium smegmatis are degraded evenly as followed by western blotting. In contrast, analysis of the depupylation behavior of several pupylome members suggests substrate-specific differences in enzymatic turnover, leading to the conclusion that largely indiscriminate degradation competes with differentiated depupylation to control the ultimate fate of pupylated substrates.

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

Induced Folding Under Membrane Mimetic and Acidic Conditions Implies Undiscovered Biological Roles of Prokaryotic Ubiquitin-Like Protein Pup.

Ubiquitin-like proteins play important roles in diverse biological processes. In Mycobacterium tuberculosis, Pup (prokaryotic ubiquitin-like protein), a functional homologue of eukaryotic ubiquitin, interacts with the proteasome ATPase subunit Mpa to recognize and unfold substrates, and then translocate them into the proteasome core for degradation. Previous studies revealed that, Pup, an intrinsically disordered protein (IDP), adopts a helical structure upon binding to the N-terminal coiled-coil domain of Mpa, at its disordered C-terminal region. In the present study, using circular dichroism (CD), surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR), we show that membrane mimetic and acidic conditions also induce Pup to adopt helical conformations. Moreover, at low pH, Pup, via both of its N- and C-terminal regions, binds to Mpa on sites from the N-terminal region in addition to the C-terminal region of the coiled-coil domain. Our results imply Pup may play undiscovered roles in some biological processes e.g. those involve in membrane.

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

Pupylation-dependent and -independent proteasomal degradation in mycobacteria.

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway.

Pupylierung — ein bakterielles Pendant zur Ubiquitinylierung

Pupylation is a reversible process in which the prokaryotic ubiquitin-like protein Pup is attached to target proteins by an isopeptide bond. It occurs mainly in Actinobacteria and was discovered in Mycobacterium tuberculosis, where pupylated proteins are targeted for proteasomal degradation, thus resembling ubiquitination in eukaryotes. Interestingly, species without a proteasome like Corynebacterium glutamicum also perform pupylation. Key features of this protein modification are summarized here.

Bacterial proteasome activator bpa (rv3780) is a novel ring-shaped interactor of the mycobacterial proteasome.

The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins.

Activity of the Mycobacterial Proteasomal ATPase Mpa Is Reversibly Regulated by Pupylation

Pupylation is a bacterial post-translational modification of target proteins on lysine residues with prokaryotic ubiquitin-like protein Pup. Pup-tagged substrates are recognized by a proteasome-interacting ATPase termed Mpa in Mycobacterium tuberculosis. Mpa unfolds pupylated substrates and threads them into the proteasome core particle for degradation. Interestingly, Mpa itself is also a pupylation target. Here, we show that the Pup ligase PafA predominantly produces monopupylated Mpa modified homogeneously on a single lysine residue within its C-terminal region. We demonstrate that this modification renders Mpa functionally inactive. Pupylated Mpa can no longer support Pup-mediated proteasomal degradation due to its inability to associate with the proteasome core. Mpa is further inactivated by rapid Pup- and ATPase-driven deoligomerization of the hexameric Mpa ring. We show that pupylation of Mpa is chemically and functionally reversible. Mpa regains its enzymatic activity upon depupylation by the depupylase Dop, affording a rapid and reversible activity control over Mpa function.

Prokaryotic Ubiquitin-Like Protein Pup Is Intrinsically Disordered

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.