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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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Crystal structure of Lys33-linked diUb(A) The crystal structure of Lys33 diUb in two orientations. (B) Lys33 diUb is shown in ribbon and the residues at the interface are shown in stick representation. (C) Leu8 residue of Lys33-linked diUb contributes to Ile44 patch. Proximal Ub of Lys33 diUb and distal Ub of Lys33 diUb, Lys11 diUb (PDB 3NOB [10]) and Lys6 diUb (PDB 2XK5 [8]) were superposed with monoUb (PDB 1UBQ [41]) and coloured light cyan. The position of Leu8 (red), Ile36 (green) and Ile44 (blue) are indicated. As reference, the Leu8 of monoUb is not coloured differently. (D) A semi-transparent surface, coloured blue for Ile44 patch (Ile44, Leu8, His68 and Val70) and green for Ile36 patch (Ile36, Leu71 and Leu73) of diUb linked via Lys33, Lys6, Lys48 and Lys11 (PDB 2XK5, 3NOB, 1AAR [8,10,12]).
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Figure 3: Crystal structure of Lys33-linked diUb(A) The crystal structure of Lys33 diUb in two orientations. (B) Lys33 diUb is shown in ribbon and the residues at the interface are shown in stick representation. (C) Leu8 residue of Lys33-linked diUb contributes to Ile44 patch. Proximal Ub of Lys33 diUb and distal Ub of Lys33 diUb, Lys11 diUb (PDB 3NOB [10]) and Lys6 diUb (PDB 2XK5 [8]) were superposed with monoUb (PDB 1UBQ [41]) and coloured light cyan. The position of Leu8 (red), Ile36 (green) and Ile44 (blue) are indicated. As reference, the Leu8 of monoUb is not coloured differently. (D) A semi-transparent surface, coloured blue for Ile44 patch (Ile44, Leu8, His68 and Val70) and green for Ile36 patch (Ile36, Leu71 and Leu73) of diUb linked via Lys33, Lys6, Lys48 and Lys11 (PDB 2XK5, 3NOB, 1AAR [8,10,12]).

Mentions: The topology of polyUb of different linkage types and potentially the length of the polyUb chains determine specificity and outcome of polyUb recognition. We therefore wanted to structurally characterize Lys33-linked polyUb chains. We obtained crystals of Lys33-linked diUb at 9 mg/ml and the crystals diffracted to 1.65 Å (1 Å=0.1 nm) resolution. The structure was solved by molecular replacement and refined to the statistics shown in Table 1. The asymmetric unit (ASU) contains one Lys33-linked diUb (Figure 3A). The flexible isopeptide linkage formed between the C-terminus of the distal Ub and Lys33 of the proximal Ub is not fully resolved in the electron density maps and no clear electron density is present for Gly76.


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)

Crystal structure of Lys33-linked diUb(A) The crystal structure of Lys33 diUb in two orientations. (B) Lys33 diUb is shown in ribbon and the residues at the interface are shown in stick representation. (C) Leu8 residue of Lys33-linked diUb contributes to Ile44 patch. Proximal Ub of Lys33 diUb and distal Ub of Lys33 diUb, Lys11 diUb (PDB 3NOB [10]) and Lys6 diUb (PDB 2XK5 [8]) were superposed with monoUb (PDB 1UBQ [41]) and coloured light cyan. The position of Leu8 (red), Ile36 (green) and Ile44 (blue) are indicated. As reference, the Leu8 of monoUb is not coloured differently. (D) A semi-transparent surface, coloured blue for Ile44 patch (Ile44, Leu8, His68 and Val70) and green for Ile36 patch (Ile36, Leu71 and Leu73) of diUb linked via Lys33, Lys6, Lys48 and Lys11 (PDB 2XK5, 3NOB, 1AAR [8,10,12]).
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Related In: Results  -  Collection

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Figure 3: Crystal structure of Lys33-linked diUb(A) The crystal structure of Lys33 diUb in two orientations. (B) Lys33 diUb is shown in ribbon and the residues at the interface are shown in stick representation. (C) Leu8 residue of Lys33-linked diUb contributes to Ile44 patch. Proximal Ub of Lys33 diUb and distal Ub of Lys33 diUb, Lys11 diUb (PDB 3NOB [10]) and Lys6 diUb (PDB 2XK5 [8]) were superposed with monoUb (PDB 1UBQ [41]) and coloured light cyan. The position of Leu8 (red), Ile36 (green) and Ile44 (blue) are indicated. As reference, the Leu8 of monoUb is not coloured differently. (D) A semi-transparent surface, coloured blue for Ile44 patch (Ile44, Leu8, His68 and Val70) and green for Ile36 patch (Ile36, Leu71 and Leu73) of diUb linked via Lys33, Lys6, Lys48 and Lys11 (PDB 2XK5, 3NOB, 1AAR [8,10,12]).
Mentions: The topology of polyUb of different linkage types and potentially the length of the polyUb chains determine specificity and outcome of polyUb recognition. We therefore wanted to structurally characterize Lys33-linked polyUb chains. We obtained crystals of Lys33-linked diUb at 9 mg/ml and the crystals diffracted to 1.65 Å (1 Å=0.1 nm) resolution. The structure was solved by molecular replacement and refined to the statistics shown in Table 1. The asymmetric unit (ASU) contains one Lys33-linked diUb (Figure 3A). The flexible isopeptide linkage formed between the C-terminus of the distal Ub and Lys33 of the proximal Ub is not fully resolved in the electron density maps and no clear electron density is present for Gly76.

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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