Prokaryotic Ubiquitin-Like Protein Pup Is Intrinsically Disordered

doi:10.1016/j.jmb.2009.07.018

Journal of Molecular Biology

Volume 392, Issue 1, 11 September 2009, Pages 208-217

https://doi.org/10.1016/j.jmb.2009.07.018 Get rights and content

Abstract

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.

Introduction

Proteasomes are ATP-dependent, multi-subunit proteases found in all domains of life. Like their eukaryotic counterparts, prokaryotic proteasomes are self-compartmentalized proteases.¹ To date, only bacteria found in the class Actinomycetes are known to have proteasomes.2, 3, 4, 5 Despite the presence of bacterial proteases that are structurally and biochemically similar to eukaryotic proteasomes, it was not understood how proteins were targeted for degradation, as ubiquitin, the posttranslational modifier that tags proteins for degradation, is found only in eukaryotes.⁶ Proteins with a ubiquitin-like fold are present in bacteria; however, none has demonstrated covalent attachment to other proteins. Recently, a small protein modifier, prokaryotic ubiquitin-like protein (Pup), was found to target proteins for proteolysis by the Mycobacterium tuberculosis proteasome.7, 8

Pup has a C-terminal glutamine, which is deamidated to glutamate by Dop.⁹ PafA⁹ ligates Pup to substrates to form an isopeptide bond between Pup's C-terminus and the ɛ-amino group of substrate lysines.7, 8 It also binds non-covalently to the Mycobacterium proteasomal ATPase Mpa,⁷ which forms a hexameric ring that presumably unfolds and translocates substrates into the bacterial 20S core particle for degradation. The Mpa:Pup interaction likely recruits pupylated substrates for degradation, and Mpa itself is also covalently modified by Pup to become a degradation substrate.⁷

Despite their functional similarity, secondary-structure prediction programs suggest that Pup does not have a canonical ubiquitin fold (Fig. 1a). We characterized Pup's structural and dynamic characteristics by NMR and circular dichroism (CD) spectroscopy to find that it is an intrinsically disordered protein. We have also found that it binds to Mpa through interactions that span S21–K61 with its strongest contacts towards the C-terminal end. We propose that Pup's stronger-binding C-terminal region serves as a targeting signal to dock degradation substrates to the prokaryotic proteasome complex while its unstructured N-terminal sequence contains the properties characteristic of a degradation initiation sequence.

Section snippets

Pup's migratory behavior suggests that it is intrinsically disordered

We found that Pup runs at 14 kDa on an SDS gel as demonstrated previously,⁷ rather than its calculated 6.9 kDa molecular mass. The delayed migration is most likely due to low SDS binding,¹⁴ as Pup's primary sequence has a low hydrophobic amino acid content and is 30% glutamic or aspartic acid (Fig. 1a). Pup also elutes earlier than expected during size-exclusion chromatography however. It directly follows the 16.7-kDa ubiquitin receptor Rpn13 and elutes significantly earlier than 8.6 kDa

Discussion

We have found that unlike ubiquitin, Pup is intrinsically disordered. Degradation by eukaryotic proteasome typically requires substrates to be covalently attached to ubiquitin and to either harbor or be complexed with a protein containing an unstructured region.25, 26, 27 It is possible that Pup fulfills these two requirements in prokaryotes by serving as an adaptor that tethers substrates to Mpa and by harboring intrinsically disordered segments (Fig. 5c). Disordered segments are significantly

Sample preparation

Mpa and Pup were expressed and purified from E. coli as fusion proteins with a histidine tag (for Mpa) and a chitin-binding domain and intein that undergoes self-cleavage in the presence of thiols (for Pup; New England Biolabs IMPACT™ Kit). Following cell lysis by sonication, the proteins were purified by affinity chromatography with a chitin column (New England Biolabs) for Pup and Ni-NTA agarose resin (Qiagen) for Mpa. On-column cleavage of Pup from the intein tag was achieved by incubating

Acknowledgements

We are grateful to Dr. Hiroshi Matsuo for useful discussions and his critical reading of the manuscript. NMR data were acquired in the NMR facilities of the University of Minnesota and the University of Georgia. We are grateful to Dr. John Glushka of the University of Georgia for setting up experiments for us. The EXSY pulse sequence was generously provided to us by Dr. Marco Tonelli of the National Magnetic Resonance Facility at Madison. Data processing and visualization occurred in the

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The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.

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Journal of Molecular Biology

Prokaryotic Ubiquitin-Like Protein Pup Is Intrinsically Disordered

Abstract

Introduction

Section snippets

Pup's migratory behavior suggests that it is intrinsically disordered

Discussion

Sample preparation

Acknowledgements

Curr. Opin. Struct. Biol.

J. Biol. Chem.

Methods Enzymol.

Trends Biochem. Sci.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

Curr. Opin. Struct. Biol.

J. Biol. Chem.

Methods Enzymol.

The 20S proteasome of Streptomyces coelicolor

J. Bacteriol.

Characterization of the 20S proteasome from the actinomycete Frankia

Mol. Microbiol.

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide

Science

Inactivation of the 20S proteasome in Mycobacterium smegmatis

Mol. Microbiol.

Modification of proteins by ubiquitin and ubiquitin-like proteins

Annu. Rev. Cell Dev. Biol.

Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis

Science

Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes

Nat. Struct. Mol. Biol.