Ligase PafA Research Articles

Electrostatic interactions guide substrate recognition of the prokaryotic ubiquitin-like protein ligase PafA

Pupylation, a post-translational modification found in Mycobacterium tuberculosis and other Actinobacteria, involves the covalent attachment of prokaryotic ubiquitin-like protein (Pup) to lysines on target proteins by the ligase PafA (proteasome accessory factor A). Pupylated proteins, like ubiquitinated proteins in eukaryotes, are recruited for proteasomal degradation. Proteomic studies suggest that hundreds of potential pupylation targets are modified by the sole existing ligase PafA. This raises intriguing questions regarding the selectivity of this enzyme towards a diverse range of substrates. Here, we show that the availability of surface lysines alone is not sufficient for interaction between PafA and target proteins. By identifying the interacting residues at the pupylation site, we demonstrate that PafA recognizes authentic substrates via a structural recognition motif centered around exposed lysines. Through a combination of computational analysis, examination of available structures and pupylated proteomes, and biochemical experiments, we elucidate the mechanism by which PafA achieves recognition of a wide array of substrates while retaining selective protein turnover.

A conserved loop sequence of the proteasome system depupylase Dop regulates substrate selectivity in Mycobacterium tuberculosis

Mycobacteria use a proteasome system that is similar to a eukaryotic proteasome but do not use ubiquitin to target proteins for degradation. Instead, mycobacteria encode a prokaryotic ubiquitin-like protein (Pup) that posttranslationally modifies proteins to mark them for proteolysis. Pupylation occurs on lysines of targeted proteins and is catalyzed by the ligase PafA. Like ubiquitylation, pupylation can be reversed by the depupylase Dop, which shares high structural similarity with PafA. Unique to Dop near its active site is a disordered loop of approximately 40 amino acids that is highly conserved among diverse dop-containing bacterial genera. To understand the function of this domain, we deleted discrete sequences from the Dop loop and assessed pupylation in mutant strains of Mycobacterium tuberculosis. We determined that various Dop loop mutations resulted in altered pupylome profiles, in particular when mutant dop alleles were overexpressed. Taken together, our data suggest these conserved amino acids play a role in substrate selectivity for Dop.

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.

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis

Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA.

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.

Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins

BackgroundThe post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate.ResultsUsing a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate.ConclusionWhen linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.

Crystal Structure of the Complex between Prokaryotic Ubiquitin-like Protein and Its Ligase PafA

Prokaryotic ubiquitin-like protein (Pup) is covalently attached to target proteins by the ligase PafA, tagging substrates for proteasomal degradation. The crystal structure of Pup in complex with PafA, reported here, reveals that a long groove wrapping around the enzyme serves as a docking site for Pup. Upon binding, the C-terminal region of the intrinsically disordered Pup becomes ordered to form two helices connected by a linker, positioning the C-terminal glutamate in the active site of PafA.

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.

Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway

Pupylation is a posttranslational protein modification occurring in mycobacteria and other actinobacteria that is functionally analogous to ubiquitination. Here we report the crystal structures of the modification enzymes involved in this pathway, the prokaryotic ubiquitin-like protein (Pup) ligase PafA and the depupylase/deamidase Dop. Both feature a larger amino-terminal domain consisting of a central β-sheet packed against a cluster of helices, a fold characteristic for carboxylate-amine ligases, and a smaller C-terminal domain unique to PafA/Dop members. The active site is located on the concave surface of the β-sheet with the nucleotide bound in a deep pocket. A conserved groove leading into the active site could have a role in Pup-binding. Nuclear magnetic resonance and biochemical experiments determine the region of Pup that interacts with PafA and Dop. Structural data and mutational studies identify crucial residues for the catalysis of both enzymes.

Mycobacterial Ubiquitin-like Protein Ligase PafA Follows a Two-step Reaction Pathway with a Phosphorylated Pup Intermediate

In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasome-targeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a γ-glutamyl phosphate-mixed anhydride intermediate that is formed on the C-terminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate.

Dop functions as a depupylase in the prokaryotic ubiquitin‐like modification pathway

Post-translational modification of proteins with prokaryotic ubiquitin-like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two-step process in which the carboxy-terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ-carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.