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Prokaryotic ubiquitin-like protein pup is intrinsically disordered.

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.

SUBMITTER: Chen X

PROVIDER: S-EPMC2734869 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Prokaryotic ubiquitin-like protein pup is intrinsically disordered.

Chen Xiang X Solomon William C WC Kang Yang Y Cerda-Maira Francisca F Darwin K Heran KH Walters Kylie J KJ

Journal of molecular biology 20090714 1

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 ubiquit ...[more]

PMID: 19607839

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Project description:The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1.

Dataset Information

Prokaryotic ubiquitin-like protein pup is intrinsically disordered.

Publications

Prokaryotic ubiquitin-like protein pup is intrinsically disordered.

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