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An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains.

ABSTRACT: Eight different types of ubiquitin linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are mainly unknown. We here reveal how the human ovarian tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin. A crystal structure of the extended catalytic domain reveals an unpredicted ankyrin repeat domain that precedes an A20-like catalytic core. NMR analysis identifies the ankyrin domain as a new ubiquitin-binding fold, which we have termed AnkUBD, and DUB assays in vitro and in vivo show that this domain is crucial for TRABID efficiency and linkage specificity. Our data are consistent with AnkUBD functioning as an enzymatic S1' ubiquitin-binding site, which orients a ubiquitin chain so that Lys29 and Lys33 linkages are cleaved preferentially.

SUBMITTER: Licchesi JD

PROVIDER: S-EPMC5260945 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains.

Licchesi Julien D F JD Mieszczanek Juliusz J Mevissen Tycho E T TE Rutherford Trevor J TJ Akutsu Masato M Virdee Satpal S El Oualid Farid F Chin Jason W JW Ovaa Huib H Bienz Mariann M Komander David D

Nature structural & molecular biology 20111211 1

Eight different types of ubiquitin linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are mainly unknown. We here reveal how the human ovarian tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin. A crystal structure of the extended catalytic domain reveals an unpredicted ankyrin repeat domain that p ...[more]

PMID: 22157957

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Project description:The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins.Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.

Project description:Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of I?B?, the temporally regulated inhibitor of canonical NF?B signaling. Under native conditions, most of the I?B? molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NF?B(RelA/p50)-I?B? complex. Similar high FRET efficiencies were found when the I?B? molecules were either free or in complex with NF?B(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound I?B? molecules (0.67) than for the free I?B? molecules (0.74), apparently indicating that binding of NF?B(RelA/p50) stretches the ARD of I?B?. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NF?B(RelA/p50) and I?B? encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on I?B? as well as I?B? bound to dimerization-domain-only constructs or full-length NF?B(RelA/p50). Although we expected the stretched I?B? to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NF?B NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the I?B? ARD induced by interactions of the I?B? proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Dataset Information

An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains.

Publications

An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains.

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