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The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

Altun M, Walter TS, Kramer HB, Herr P, Iphöfer A, Boström J, David Y, Komsany A, Ternette N, Navon A, Stuart DI, Ren J, Kessler BM - PLoS ONE (2015)

Bottom Line: Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2.The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs.In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains.

View Article: PubMed Central - PubMed

Affiliation: Target Discovery Institute, Nuffield Department of Medicine, Roosevelt Drive, University of Oxford, Oxford, OX3 7FZ, United Kingdom; Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 21 Stockholm, Sweden.

ABSTRACT
Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

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Comparison of OTUB2-Ub with other OTU-Ub complexes.Superposition of OTUB2-Ub (blue and red) with yeast OTU1-Ub (grey) [20] (A), vOTU-Ub (grey) [23] (B) and OTUB1-Ubal-UBC13-Ub (grey / yellow) [13] (C) complexes. The free donor Ub is shown in yellow and the UBC13 is omitted in (C) for clarity.
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pone.0115344.g003: Comparison of OTUB2-Ub with other OTU-Ub complexes.Superposition of OTUB2-Ub (blue and red) with yeast OTU1-Ub (grey) [20] (A), vOTU-Ub (grey) [23] (B) and OTUB1-Ubal-UBC13-Ub (grey / yellow) [13] (C) complexes. The free donor Ub is shown in yellow and the UBC13 is omitted in (C) for clarity.

Mentions: The yeast OTU1 (yOTU1)—Ub complex derived from forming a covalent bond with UbBr3 [20] shares many structural features with the human OTUB2—Ub enzyme—ligand molecule conformation (Fig. 3A). OTUB2 and yOTU1 can be imposed with 114 (out of 231) equivalent Cαs and an rmsd of 1.4Å. In particular, the Ub ligands in both complexes have a very similar overall conformation with a modest (18°) difference in orientation to the enzyme. This is in contrast to the CCHFV derived vOTU-Ub complex [23], in which the Ub molecule is rotated by ~90° as compared to Ub in complex with OTUB2 (Fig. 3B). Interestingly, this is achieved by small differences only between the core structures of vOTU and OTUB2, represented by an rmsd of 1.7Å and 120 equivalent Cαs (out of 156). A major hallmark of the vOTU complex is the two extra β-strands of vOTU which are involved in direct contacts with the Ub β-sheet, which in the case of OTUB2 is contacting the α8 helix. This feature appears to be unique to vOTU and may be partly responsible, in addition to the orthogonal orientation of the Ub substrate, for allowing the accommodation of both deubiquitylating and deISGylating activity [33]. Consistent with this notion, OTUB2 does not process ISG15, but Lys (K) 48/63-linked poly-Ubs and neural precursor cell expressed, developmentally downregulated 8 (NEDD8) as substrates ([5, 19], S1A Fig.). This is in contrast to OTUB1 which has a slower cleavage kinetics (S1B Fig.) and preferential specificity for Lys48-linked poly-Ub [19] [18, 19], despite a considerable structural overlap with OTUB2 (Fig. 3A and C).


The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

Altun M, Walter TS, Kramer HB, Herr P, Iphöfer A, Boström J, David Y, Komsany A, Ternette N, Navon A, Stuart DI, Ren J, Kessler BM - PLoS ONE (2015)

Comparison of OTUB2-Ub with other OTU-Ub complexes.Superposition of OTUB2-Ub (blue and red) with yeast OTU1-Ub (grey) [20] (A), vOTU-Ub (grey) [23] (B) and OTUB1-Ubal-UBC13-Ub (grey / yellow) [13] (C) complexes. The free donor Ub is shown in yellow and the UBC13 is omitted in (C) for clarity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295869&req=5

pone.0115344.g003: Comparison of OTUB2-Ub with other OTU-Ub complexes.Superposition of OTUB2-Ub (blue and red) with yeast OTU1-Ub (grey) [20] (A), vOTU-Ub (grey) [23] (B) and OTUB1-Ubal-UBC13-Ub (grey / yellow) [13] (C) complexes. The free donor Ub is shown in yellow and the UBC13 is omitted in (C) for clarity.
Mentions: The yeast OTU1 (yOTU1)—Ub complex derived from forming a covalent bond with UbBr3 [20] shares many structural features with the human OTUB2—Ub enzyme—ligand molecule conformation (Fig. 3A). OTUB2 and yOTU1 can be imposed with 114 (out of 231) equivalent Cαs and an rmsd of 1.4Å. In particular, the Ub ligands in both complexes have a very similar overall conformation with a modest (18°) difference in orientation to the enzyme. This is in contrast to the CCHFV derived vOTU-Ub complex [23], in which the Ub molecule is rotated by ~90° as compared to Ub in complex with OTUB2 (Fig. 3B). Interestingly, this is achieved by small differences only between the core structures of vOTU and OTUB2, represented by an rmsd of 1.7Å and 120 equivalent Cαs (out of 156). A major hallmark of the vOTU complex is the two extra β-strands of vOTU which are involved in direct contacts with the Ub β-sheet, which in the case of OTUB2 is contacting the α8 helix. This feature appears to be unique to vOTU and may be partly responsible, in addition to the orthogonal orientation of the Ub substrate, for allowing the accommodation of both deubiquitylating and deISGylating activity [33]. Consistent with this notion, OTUB2 does not process ISG15, but Lys (K) 48/63-linked poly-Ubs and neural precursor cell expressed, developmentally downregulated 8 (NEDD8) as substrates ([5, 19], S1A Fig.). This is in contrast to OTUB1 which has a slower cleavage kinetics (S1B Fig.) and preferential specificity for Lys48-linked poly-Ub [19] [18, 19], despite a considerable structural overlap with OTUB2 (Fig. 3A and C).

Bottom Line: Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2.The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs.In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains.

View Article: PubMed Central - PubMed

Affiliation: Target Discovery Institute, Nuffield Department of Medicine, Roosevelt Drive, University of Oxford, Oxford, OX3 7FZ, United Kingdom; Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 21 Stockholm, Sweden.

ABSTRACT
Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

Show MeSH
Related in: MedlinePlus