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

Barandun, Jonas; Delley, Cyrille L.; Ban, Nenad; Weber‐Ban, Eilika

doi:10.1021/ja4024012

Cited by 28 publications

(62 citation statements)

References 22 publications

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“…The second factor is tight binding of Pup E , the product of both deamidation and depupylation, by Dop. This finding is not surprising, given the extended Pup region involved in Dop binding (21,22 binding to the enzyme. The outcome of Pup E binding therefore is a reduction in the concentration of free enzyme available for substrate binding.…”

Section: Discussion Competition For Substrate Binding and Exclusion Omentioning

confidence: 84%

“…1), with pupylated proteins being rescued from degradation following depupylation by Dop (deamidase of Pup) (18,19). PafA (proteasome accessory factor A) and Dop are homologous enzymes, both binding Pup through interaction with its extended C-terminal region (20)(21)(22)(23). In mycobacteria and many related PPS-encoding species, Dop also functions as a Pup modifier that catalyzes the first step in the pupylation pathway (20).…”

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confidence: 99%

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Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Elharar

Roth

Hecht

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for highmolecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions. Dop | mycobacteria | PafA | proteasome | Pup

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Section: Discussion Competition For Substrate Binding and Exclusion Omentioning

confidence: 84%

mentioning

confidence: 99%

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Elharar

Roth

Hecht

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…In accordance with the energy requirement of isopeptide bond formation the ligation process requires the turnover of ATP. The structure of PafA shows that it has a similar active site arrangement as glutamine synthetase (GS), consisting of a curved anti-parallel ␤-sheet with ATP bound at one end of the ␤-sheet cradle and the triphosphate chain running along the strands toward the opposite side of the sheet, where the glutamate residue of Pup is bound (9,10). The ␥-carboxylate of the C-terminal glutamate of Pup binds in close proximity to the ␥-phosphate, allowing an attack of the glutamyl ␥-carboxylate oxygen on the ␥-phosphate of ATP to cleave off ADP and generate the ␥-glutamyl phosphate-mixed anhydride intermediate of Pup (11).…”

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confidence: 99%

“…This reaction is chemically similar to the activation of the glutamate side chain for the attack of ammonia in GS. However, whereas bacterial GS is an oligomeric assembly (a double ring of hexamers) with the active sites buried in deep pockets at the intra-ring subunit interfaces (12), the Pup ligase PafA is active as a monomer and features a broad, easily accessible active site (9,10).…”

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confidence: 99%

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Bolten

Vahlensieck

Lipp

et al. 2017

Journal of Biological Chemistry

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Edited by George N. DeMartinoAnalogous 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 ATPbinding 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.

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“…As will be further discussed below, Pup undergoes disorder-to-order transitions upon interaction with its dedicated binding partners, the Pup ligase PafA [32], the depupylase Dop [33] and the proteasomal ATPase Mpa [34]. It is not uncommon for an IDP to already in the unbound state sample conformational elements of the structure it will adopt during binding [31,35].…”

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confidence: 99%

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

Cited by 28 publications

References 22 publications

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

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