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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 triUb(A) The crystal structure of Lys33-linked triUb. (B) Representative diagram of how a Ub moiety in an ASU is arranged into triUb (left) and how Ile44 and Ile36 patches are located in Lys33-linked triUb (right) (C) Superposition of the distal Ub moieties (white) of Lys33-linked diUb and Lys33-linked triUb, shown in cartoon and surface representations. Hydrophobic patches are coloured as in Figure 3D. Only two Ub moieties are shown for Lys33-linked triUb. In comparison with the proximal Ub of Lys33 diUb (teal), the proximal Ub of Lys33 triUb (orange) moves approximately 65° from to the more open conformation. (D–F) A semi-transparent surface of Lys33 triUb (D), Met1 diUb (E) and Lys63 diUb (F) coloured as in Figure 3D (PDB 2W9N, 2JF5 [13]).
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Figure 4: Crystal structure of Lys33-linked triUb(A) The crystal structure of Lys33-linked triUb. (B) Representative diagram of how a Ub moiety in an ASU is arranged into triUb (left) and how Ile44 and Ile36 patches are located in Lys33-linked triUb (right) (C) Superposition of the distal Ub moieties (white) of Lys33-linked diUb and Lys33-linked triUb, shown in cartoon and surface representations. Hydrophobic patches are coloured as in Figure 3D. Only two Ub moieties are shown for Lys33-linked triUb. In comparison with the proximal Ub of Lys33 diUb (teal), the proximal Ub of Lys33 triUb (orange) moves approximately 65° from to the more open conformation. (D–F) A semi-transparent surface of Lys33 triUb (D), Met1 diUb (E) and Lys63 diUb (F) coloured as in Figure 3D (PDB 2W9N, 2JF5 [13]).

Mentions: The presence of a symmetric interface raises the question of how chain extension can be achieved and what structure longer Lys33 polyUb chains adopt. To address this question we purified milligram quantities of Lys33-linked triUb for crystallization studies. Lys33 triUb crystallized in a different space group with unit cell dimensions different from that of Lys33 diUb crystals. Diffraction data were obtained at 1.4 Å resolutions and the structure solved by molecular replacement and refined to the final statistics shown in Table 1. Although we crystallized triUb, the ASU only contains one Ub molecule (Figures 4A and 4B, chain B). In the crystal lattice, neighbouring Ub molecules complete the trimer where the C-terminus of a symmetry-related molecule (chain C) is close to Lys33 of chain B; and the C-terminus of chain B is positioned close to Lys33 residue of chain A (Figure 4A). Clear electron density is visible for the isopeptide linkage connecting the Ub moieties via Lys33 (Supplementary Figure S3).


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 triUb(A) The crystal structure of Lys33-linked triUb. (B) Representative diagram of how a Ub moiety in an ASU is arranged into triUb (left) and how Ile44 and Ile36 patches are located in Lys33-linked triUb (right) (C) Superposition of the distal Ub moieties (white) of Lys33-linked diUb and Lys33-linked triUb, shown in cartoon and surface representations. Hydrophobic patches are coloured as in Figure 3D. Only two Ub moieties are shown for Lys33-linked triUb. In comparison with the proximal Ub of Lys33 diUb (teal), the proximal Ub of Lys33 triUb (orange) moves approximately 65° from to the more open conformation. (D–F) A semi-transparent surface of Lys33 triUb (D), Met1 diUb (E) and Lys63 diUb (F) coloured as in Figure 3D (PDB 2W9N, 2JF5 [13]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390085&req=5

Figure 4: Crystal structure of Lys33-linked triUb(A) The crystal structure of Lys33-linked triUb. (B) Representative diagram of how a Ub moiety in an ASU is arranged into triUb (left) and how Ile44 and Ile36 patches are located in Lys33-linked triUb (right) (C) Superposition of the distal Ub moieties (white) of Lys33-linked diUb and Lys33-linked triUb, shown in cartoon and surface representations. Hydrophobic patches are coloured as in Figure 3D. Only two Ub moieties are shown for Lys33-linked triUb. In comparison with the proximal Ub of Lys33 diUb (teal), the proximal Ub of Lys33 triUb (orange) moves approximately 65° from to the more open conformation. (D–F) A semi-transparent surface of Lys33 triUb (D), Met1 diUb (E) and Lys63 diUb (F) coloured as in Figure 3D (PDB 2W9N, 2JF5 [13]).
Mentions: The presence of a symmetric interface raises the question of how chain extension can be achieved and what structure longer Lys33 polyUb chains adopt. To address this question we purified milligram quantities of Lys33-linked triUb for crystallization studies. Lys33 triUb crystallized in a different space group with unit cell dimensions different from that of Lys33 diUb crystals. Diffraction data were obtained at 1.4 Å resolutions and the structure solved by molecular replacement and refined to the final statistics shown in Table 1. Although we crystallized triUb, the ASU only contains one Ub molecule (Figures 4A and 4B, chain B). In the crystal lattice, neighbouring Ub molecules complete the trimer where the C-terminus of a symmetry-related molecule (chain C) is close to Lys33 of chain B; and the C-terminus of chain B is positioned close to Lys33 residue of chain A (Figure 4A). Clear electron density is visible for the isopeptide linkage connecting the Ub moieties via Lys33 (Supplementary Figure S3).

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