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Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations.

Kristariyanto YA, Choi SY, Rehman SA, Ritorto MS, Campbell DG, Morrice NA, Toth R, Kulathu Y - Biochem. J. (2015)

Bottom Line: In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation.These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system.Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.

View Article: PubMed Central - PubMed

Affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.

ABSTRACT
Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6-AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.

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Assembly of Lys33-linked polyUb(A) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or Ub mutants that have only one or no lysine residue. (B) Large-scale assembly of polyUb chains by AREL1 in the presence of UBE1, UBE2D1 and Ub. The addition of DUBs, Cezanne EK and OTUB1, releases free polyUb chains. (C) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or lysine-to-arginine Ub mutants. DUBs, Cezanne EK and OTUB1, were added after 3 h of reaction. (D) Auto-ubiquitylation assays of AREL1 as in (A) with wild-type Ub. DUBs, Cezanne EK, OTUB1 and TRABID, were added after 3 h reaction as indicated. (E) Purification of Lys33-linked chains of defined lengths by cation-exchange chromatography. (F) The Lys33-linked diUb and triUb purified in (D) were visualized in silver-stained SDS gel.
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Figure 2: Assembly of Lys33-linked polyUb(A) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or Ub mutants that have only one or no lysine residue. (B) Large-scale assembly of polyUb chains by AREL1 in the presence of UBE1, UBE2D1 and Ub. The addition of DUBs, Cezanne EK and OTUB1, releases free polyUb chains. (C) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or lysine-to-arginine Ub mutants. DUBs, Cezanne EK and OTUB1, were added after 3 h of reaction. (D) Auto-ubiquitylation assays of AREL1 as in (A) with wild-type Ub. DUBs, Cezanne EK, OTUB1 and TRABID, were added after 3 h reaction as indicated. (E) Purification of Lys33-linked chains of defined lengths by cation-exchange chromatography. (F) The Lys33-linked diUb and triUb purified in (D) were visualized in silver-stained SDS gel.

Mentions: Whereas AREL1 makes Lys33 chains, it also assembles Lys11 and Lys48 linkages (Figures 1B and 2A). To obtain pure Lys33-linked polyUb, the other linkages assembled by AREL1 have to be removed, for which linkage-selective DUBs are required. Cezanne mainly hydrolyses Lys11 linkages, whereas OTUB1 only cleaves Lys48 linkages [24]. We used a mutant version of Cezanne (Cezanne EK) that hydrolyses Lys6, Lys11, Lys48 and Lys63 linkages (Supplementary Figure S2). When Cezanne EK and OTUB1 were included in the assembly reaction, the end product was enriched in free polyUb chains and almost 90% of the input Ub was converted into unanchored or free polyUb chains (Figure 2B). In order to confirm the linkage type of the resulting polyUb chains, we performed a linkage type analysis using Ub mutants containing lysine-to-arginine substitutions. In the presence of Cezanne EK and OTUB1, free polyUb chain formation was not impaired with K6R, K11R, K27R, K29R, K48R or K63R mutants (Figure 2C). In contrast, formation of polyUb chains was significantly reduced with the K33R mutant, suggesting that this method generates polyUb chains that are Lys33 linked (Figure 2C). Moreover, when incubated with the DUB TRABID that specifically hydrolyses Lys29 and Lys33 linkages [23], the assembled polyUb chains were cleaved down to monoUb, confirming the presence of Lys33 linkages (Figure 2D). Taken together, these results demonstrate that an Ub chain editing complex made up of the enzymes AREL1, UBE2D1, Cezanne EK and OTUB1 can be used to assemble Lys33-linked polyUb chains.


Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations.

Kristariyanto YA, Choi SY, Rehman SA, Ritorto MS, Campbell DG, Morrice NA, Toth R, Kulathu Y - Biochem. J. (2015)

Assembly of Lys33-linked polyUb(A) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or Ub mutants that have only one or no lysine residue. (B) Large-scale assembly of polyUb chains by AREL1 in the presence of UBE1, UBE2D1 and Ub. The addition of DUBs, Cezanne EK and OTUB1, releases free polyUb chains. (C) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or lysine-to-arginine Ub mutants. DUBs, Cezanne EK and OTUB1, were added after 3 h of reaction. (D) Auto-ubiquitylation assays of AREL1 as in (A) with wild-type Ub. DUBs, Cezanne EK, OTUB1 and TRABID, were added after 3 h reaction as indicated. (E) Purification of Lys33-linked chains of defined lengths by cation-exchange chromatography. (F) The Lys33-linked diUb and triUb purified in (D) were visualized in silver-stained SDS gel.
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Figure 2: Assembly of Lys33-linked polyUb(A) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or Ub mutants that have only one or no lysine residue. (B) Large-scale assembly of polyUb chains by AREL1 in the presence of UBE1, UBE2D1 and Ub. The addition of DUBs, Cezanne EK and OTUB1, releases free polyUb chains. (C) Ubiquitylation assays of AREL1 in the presence of UBE1, UBE2D1 and wild-type Ub or lysine-to-arginine Ub mutants. DUBs, Cezanne EK and OTUB1, were added after 3 h of reaction. (D) Auto-ubiquitylation assays of AREL1 as in (A) with wild-type Ub. DUBs, Cezanne EK, OTUB1 and TRABID, were added after 3 h reaction as indicated. (E) Purification of Lys33-linked chains of defined lengths by cation-exchange chromatography. (F) The Lys33-linked diUb and triUb purified in (D) were visualized in silver-stained SDS gel.
Mentions: Whereas AREL1 makes Lys33 chains, it also assembles Lys11 and Lys48 linkages (Figures 1B and 2A). To obtain pure Lys33-linked polyUb, the other linkages assembled by AREL1 have to be removed, for which linkage-selective DUBs are required. Cezanne mainly hydrolyses Lys11 linkages, whereas OTUB1 only cleaves Lys48 linkages [24]. We used a mutant version of Cezanne (Cezanne EK) that hydrolyses Lys6, Lys11, Lys48 and Lys63 linkages (Supplementary Figure S2). When Cezanne EK and OTUB1 were included in the assembly reaction, the end product was enriched in free polyUb chains and almost 90% of the input Ub was converted into unanchored or free polyUb chains (Figure 2B). In order to confirm the linkage type of the resulting polyUb chains, we performed a linkage type analysis using Ub mutants containing lysine-to-arginine substitutions. In the presence of Cezanne EK and OTUB1, free polyUb chain formation was not impaired with K6R, K11R, K27R, K29R, K48R or K63R mutants (Figure 2C). In contrast, formation of polyUb chains was significantly reduced with the K33R mutant, suggesting that this method generates polyUb chains that are Lys33 linked (Figure 2C). Moreover, when incubated with the DUB TRABID that specifically hydrolyses Lys29 and Lys33 linkages [23], the assembled polyUb chains were cleaved down to monoUb, confirming the presence of Lys33 linkages (Figure 2D). Taken together, these results demonstrate that an Ub chain editing complex made up of the enzymes AREL1, UBE2D1, Cezanne EK and OTUB1 can be used to assemble Lys33-linked polyUb chains.

Bottom Line: In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation.These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system.Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.

View Article: PubMed Central - PubMed

Affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.

ABSTRACT
Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6-AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.

Show MeSH