An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets

Regev, Ofir; Korman, Maayan; Hecht, Nir; Roth, Ziv; Forer, Nadav; Zarivach, Raz; Gur, Eyal

doi:10.1016/j.jmb.2016.07.021

Cited by 12 publications

(14 citation statements)

References 30 publications

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“…These predictions were verified using BLI, which showed that Pup-binding of PafA S119 variants or of PafA co-incubated with AEBSF was significantly reduced compared to that of WT PafA. These results are also consistent with the clearly defective pupylation of lysine, whose binding does not involve substantial contact with PafA ( Regev et al, 2016 ), by PafA S119 variants or PafA co-incubated with AEBSF, as compared to that of WT PafA. Thus, although we cannot completely rule out that the reduced pupylation may result from a catalytic defect, our data strongly support the proposal that the inhibitory effect of the interference of the PafA S119-pocket, a non-active site location, is due to a reduced binding of Pup.…”

Section: Discussionsupporting

confidence: 60%

“…To obtain a better understanding of defective Pup binding in Mtb PafA S119 mutants and to exclude possible effects on protein-substrate binding, we examined the ability of Mtb PafA to pupylate free lysine under different conditions. Clearly, the docking of free lysine in the PafA active site does not require most of the residues involved in the interaction of PafA with protein targets ( Regev et al, 2016 ). We found that PafA S119A shows a weakened pupylase activity on free lysine compared to that of the WT enzyme, while lysine pupylation by PafA S119F, S119Y and S119 W was undetectable ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionsupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…tuberculosis (Mtb) [3–6]. Careful analysis of the structures and activities of the pupylation enzymes, namely the ligase PafA and the deamidase/depupylase Dop, has led to a good understanding of the reaction mechanisms by which pupylation and depupylation occur [9, 12, 13, 15–17, 40]. Furthermore, several studies have been conducted to identify pupylated substrates of different actinobacterial organisms, generating a catalog of pupylation targets [8, 27–34].…”

Section: Discussionmentioning

confidence: 99%

“…The modification of cellular proteins with Pup involves two structurally homologous and evolutionarily related enzymes, the Pup ligase PafA (proteasome accessory factor A) and the Pup deamidase/depupylase Dop (deamidase of Pup) [9–11]. The Pup ligase attaches the side-chain carboxylate of the C-terminal glutamate residue of Pup to a lysine side chain in the target protein by forming an isopeptide bond [12–15]. In mycobacteria, Pup is encoded with a C-terminal glutamine that first must be deamidated to glutamate by the enzyme Dop to produce the side-chain carboxylate for ligation [10].…”

Section: Introductionmentioning

confidence: 99%

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

Juerg¹,

Cui²,

Weber‐Ban³

2019

PLoS ONE

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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.

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“…Furthermore, the specificity constant (kcat/Km) for a bona fide substrate is three orders of magnitude higher than the one observed for free lysine as a model substrate [73]. A recent study suggests that the flexible loop N-terminal of strand 7 at one edge of the -sheet cradle is involved in making contacts to pupylation substrate proteins, since mutations in this region (196-216) lead to diminished activity in protein pupylation but not in Pup amidation using ammonia as a model substrate [75]. In the crystal structure, the loop runs roughly parallel to -strand 7, partially projecting above the active site cradle [33].…”

Section: Reaction Mechanism Of Pupylation and Depupylationmentioning

confidence: 90%

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

Delley

Müller

Ziemski

et al. 2017

Journal of Molecular Biology

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Prokaryotic ubiquitin-like protein (Pup) and the modification enzymes involved in attaching Pup to or removing it from target proteins present a fascinating example of convergent evolution with respect to eukaryotic ubiquitination. Like ubiquitin (Ub), Pup is a small protein that can be covalently attached to lysine side chains of cellular proteins, and like Ub it can serve to recruit tagged proteins for proteasomal degradation. However, unlike Ub, Pup is conformationally highly dynamic, exhibits a different linkage connectivity to its target lysines and its ligase belongs to a different class of enzymes than the E1/E2/E3 cascade of ubiquitination. A specific feature of actinobacteria (aside from sporadic cases in a few other lineages), pupylation appears to have evolved to provide an advantage to the bacteria under certain environmental stresses rather than act as a constitutive modification. For Mycobacterium tuberculosis, pupylation and the recruitment of pupylated substrates to the proteasome supports persistence inside host macrophages during pathogenesis, rendering the Pup-proteasome system an attractive drug target.In this review, we consider the dynamic nature of Pup in relation to its function, discuss the reaction mechanism of ligation to substrates as well as cleavage from pupylated 2 substrates and put it in perspective of the evolutionary history of this post-translational modification.

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An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets

Cited by 12 publications

References 30 publications

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

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

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

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

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