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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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Overall structure of the OTUB2-Ub complex.(A) A ribbon diagram showing OTUB2-Ub complex with OTUB2 colored in blue and Ub in red. The active site residues are shown as sticks with carbon atoms colored in cyan; the last two residues of Ub and the covalent linker to Cys51 are drawn as orange sticks. (B) Representative /2Fo-Fc/ map contoured at 1σ showing well defined electron density for residues around the active site. (C) Comparison of ligand bound and apo (grey) structures of OTUB2, the large structural changes due to Ub binding are indicated by arrows. (D) Close-up view of the active site of ligand bound and apo OTUB2. (E) Electrostatic surfaces of OTUB2 (bottom panel) and Ub (top panel). Ub is moved upwards and rotated 90° to show the positively charged surface patch that has complementary interactions with OTUB2.
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pone.0115344.g001: Overall structure of the OTUB2-Ub complex.(A) A ribbon diagram showing OTUB2-Ub complex with OTUB2 colored in blue and Ub in red. The active site residues are shown as sticks with carbon atoms colored in cyan; the last two residues of Ub and the covalent linker to Cys51 are drawn as orange sticks. (B) Representative /2Fo-Fc/ map contoured at 1σ showing well defined electron density for residues around the active site. (C) Comparison of ligand bound and apo (grey) structures of OTUB2, the large structural changes due to Ub binding are indicated by arrows. (D) Close-up view of the active site of ligand bound and apo OTUB2. (E) Electrostatic surfaces of OTUB2 (bottom panel) and Ub (top panel). Ub is moved upwards and rotated 90° to show the positively charged surface patch that has complementary interactions with OTUB2.

Mentions: A protein complex of human OTUB2 and Ub, an active site directed probe, was obtained by the irreversible formation of the covalent thioether bond between the catalytic site cysteine 51 of OTUB2 and the ethane bromoethyl moiety at the C-terminus of Ub. Protein complexes purified to homogeneity yielded crystals which diffracted to a resolution of 2.05 Å. The structure was determined by molecular replacement method and refined against all the x-ray data to an R-factor 0.212 (R-free 0.269) with rsmd’s of 0.010 Å for bond lengths and 1.2° for bond angles from ideal values (S1 Table). The final refined model contains residues 2–232 of OTUB2 and 1–75 of Ub in each OTUB2-Ub complex (Fig. 1). In addition, six residues from the N-terminal region of each Ub are ordered and interact with symmetry related molecules. OTUB2 is comprised of a 6-stranded β-sheet sandwiched by two helical domains. The overall structure of the OTUB2-Ub complex shows an Ub binding mode similar to other DUBs in that the Ub C-terminal tail is extended and reaches to the catalytic centre (Fig. 1A). The covalent thioether bond formed between Ub-Gly75-bromo ethyl and OTUB2 Cys51 (Fig. 1B), and the catalytic residues Cys51, His224, Asn226 and Thr45 are all well defined in the electron density map and within sufficient proximity to engage in a hydrogen bonding network. A comparison of the OTUB2 apo [17] and Ub ligand bound structures illustrates only subtle movements of these residues within the catalytic centre (Fig. 1C and D). However, large conformational changes and local re-structuring occur at both sides of the Ub binding cleft of OTUB2 upon Ub binding. Residues 197–204 are disordered and 205–208 fold into the Ub binding cleft in the apo OTUB2 structure. In the complex, residues197–204 become ordered and 205–208 form a β-strand (β4) antiparallel to β3 with a movement of 12.6 Å for the Cα atom of residue 205. The newly ordered β3β4 loop packs against the inner face of α2 helix and introduces a subtle reorientation of the α1 and α2 helices and conformational changes of α1α2 loop and β2 strand. On the other side of the cleft there is a 5 Å sideways movement of α8α9 loop. 201 Cα atoms out of 232 liganded OTUB2 can be overlapped with the apo enzyme with an rmsd of 0.8 Å (Fig. 1C).


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)

Overall structure of the OTUB2-Ub complex.(A) A ribbon diagram showing OTUB2-Ub complex with OTUB2 colored in blue and Ub in red. The active site residues are shown as sticks with carbon atoms colored in cyan; the last two residues of Ub and the covalent linker to Cys51 are drawn as orange sticks. (B) Representative /2Fo-Fc/ map contoured at 1σ showing well defined electron density for residues around the active site. (C) Comparison of ligand bound and apo (grey) structures of OTUB2, the large structural changes due to Ub binding are indicated by arrows. (D) Close-up view of the active site of ligand bound and apo OTUB2. (E) Electrostatic surfaces of OTUB2 (bottom panel) and Ub (top panel). Ub is moved upwards and rotated 90° to show the positively charged surface patch that has complementary interactions with OTUB2.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0115344.g001: Overall structure of the OTUB2-Ub complex.(A) A ribbon diagram showing OTUB2-Ub complex with OTUB2 colored in blue and Ub in red. The active site residues are shown as sticks with carbon atoms colored in cyan; the last two residues of Ub and the covalent linker to Cys51 are drawn as orange sticks. (B) Representative /2Fo-Fc/ map contoured at 1σ showing well defined electron density for residues around the active site. (C) Comparison of ligand bound and apo (grey) structures of OTUB2, the large structural changes due to Ub binding are indicated by arrows. (D) Close-up view of the active site of ligand bound and apo OTUB2. (E) Electrostatic surfaces of OTUB2 (bottom panel) and Ub (top panel). Ub is moved upwards and rotated 90° to show the positively charged surface patch that has complementary interactions with OTUB2.
Mentions: A protein complex of human OTUB2 and Ub, an active site directed probe, was obtained by the irreversible formation of the covalent thioether bond between the catalytic site cysteine 51 of OTUB2 and the ethane bromoethyl moiety at the C-terminus of Ub. Protein complexes purified to homogeneity yielded crystals which diffracted to a resolution of 2.05 Å. The structure was determined by molecular replacement method and refined against all the x-ray data to an R-factor 0.212 (R-free 0.269) with rsmd’s of 0.010 Å for bond lengths and 1.2° for bond angles from ideal values (S1 Table). The final refined model contains residues 2–232 of OTUB2 and 1–75 of Ub in each OTUB2-Ub complex (Fig. 1). In addition, six residues from the N-terminal region of each Ub are ordered and interact with symmetry related molecules. OTUB2 is comprised of a 6-stranded β-sheet sandwiched by two helical domains. The overall structure of the OTUB2-Ub complex shows an Ub binding mode similar to other DUBs in that the Ub C-terminal tail is extended and reaches to the catalytic centre (Fig. 1A). The covalent thioether bond formed between Ub-Gly75-bromo ethyl and OTUB2 Cys51 (Fig. 1B), and the catalytic residues Cys51, His224, Asn226 and Thr45 are all well defined in the electron density map and within sufficient proximity to engage in a hydrogen bonding network. A comparison of the OTUB2 apo [17] and Ub ligand bound structures illustrates only subtle movements of these residues within the catalytic centre (Fig. 1C and D). However, large conformational changes and local re-structuring occur at both sides of the Ub binding cleft of OTUB2 upon Ub binding. Residues 197–204 are disordered and 205–208 fold into the Ub binding cleft in the apo OTUB2 structure. In the complex, residues197–204 become ordered and 205–208 form a β-strand (β4) antiparallel to β3 with a movement of 12.6 Å for the Cα atom of residue 205. The newly ordered β3β4 loop packs against the inner face of α2 helix and introduces a subtle reorientation of the α1 and α2 helices and conformational changes of α1α2 loop and β2 strand. On the other side of the cleft there is a 5 Å sideways movement of α8α9 loop. 201 Cα atoms out of 232 liganded OTUB2 can be overlapped with the apo enzyme with an rmsd of 0.8 Å (Fig. 1C).

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