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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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Screening of HECT E3 ligases to identify enzymes that assemble Lys33-linked polyUb(A) Ubiquitylation assays of HECT E3 ligases in the presence of UBE1, Ub and different E2 enzymes: UBE2D1, UBE2D2, UBE2D3 and UBE2L3. (B) Ubiquitylated products generated by HECT E3 ligases with the optimal E2 were analysed by pRM LC–MS/MS for the abundance of Ub linkages as described in ‘Materials and Methods’ (Supplementary Figure S1B). Each Ub linkage assembled by the HECT E3 ligases was plotted as a bar graph where the y-axes are summed ion current values for the relevant daughter ions of each precursor mass analysed (Supplementary Table S2). No signal was observed for Met1 and Lys27 linkages for any of the ligases tested.
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Figure 1: Screening of HECT E3 ligases to identify enzymes that assemble Lys33-linked polyUb(A) Ubiquitylation assays of HECT E3 ligases in the presence of UBE1, Ub and different E2 enzymes: UBE2D1, UBE2D2, UBE2D3 and UBE2L3. (B) Ubiquitylated products generated by HECT E3 ligases with the optimal E2 were analysed by pRM LC–MS/MS for the abundance of Ub linkages as described in ‘Materials and Methods’ (Supplementary Figure S1B). Each Ub linkage assembled by the HECT E3 ligases was plotted as a bar graph where the y-axes are summed ion current values for the relevant daughter ions of each precursor mass analysed (Supplementary Table S2). No signal was observed for Met1 and Lys27 linkages for any of the ligases tested.

Mentions: PolyUb chains were digested with trypsin and analysed on an LTQ-Velos mass spectrometer (Thermo) fitted with an Easy-Spray Source (Thermo) and utilizing a Dionex RSLC HPLC system. Standard diUb chains were purchased from Boston Biochemicals and a synthetic peptide AK(GG)IQDK representing the tryptic Ub K29 linkage was purchased from Pepceuticals. Digests (prepared in 0.1% TFA (trifluoroacetic acid)/water) were concentrated on a 20×0.1 mm nanotrap column (Thermo) equilibrated in 0.1% TFA/water (10 μl/min) and washed with 10 μl of the same buffer. The samples were loaded and washed in TFA buffers, as the trap column in the presence of formic acid did not retain the tryptic peptide containing the Lys29 linkage. Peptides were then separated on a 150×0.075 mm PepMap C18, 3 μm Easy-Spray column (Thermo) equilibrated with 2% acetonitrile/0.1% formic acid/water at 300 nl/min, employing a stepped gradient of buffer B (80% acetonitrile/0.1% formic acid/water) as follows: 0–14 min=1%–30% B, 14–15 min=30%–80% B, 15–20 min=80% B. LC–MS data was acquired in data-independent mode with one full scan (m/z=350–1800) followed by eight product ion scans as described below. Parameters used: Easy-Spray column voltage was 1.9 kV; isolation width was set to 1 Da; normalized collision energy was 35, and the activation time was 10 ms. The ion current for the daughter ions was summed using Xcalibur software (Thermo) for each precursor mass analysed (Supplementary Table S2). The resultant summed intensities provide the y-axis values for Figure 1(B) and Supplementary Figure S2. This method was more specific than solely using the extracted ion current for the precursor mass for each Ub chain peptide.


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)

Screening of HECT E3 ligases to identify enzymes that assemble Lys33-linked polyUb(A) Ubiquitylation assays of HECT E3 ligases in the presence of UBE1, Ub and different E2 enzymes: UBE2D1, UBE2D2, UBE2D3 and UBE2L3. (B) Ubiquitylated products generated by HECT E3 ligases with the optimal E2 were analysed by pRM LC–MS/MS for the abundance of Ub linkages as described in ‘Materials and Methods’ (Supplementary Figure S1B). Each Ub linkage assembled by the HECT E3 ligases was plotted as a bar graph where the y-axes are summed ion current values for the relevant daughter ions of each precursor mass analysed (Supplementary Table S2). No signal was observed for Met1 and Lys27 linkages for any of the ligases tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Screening of HECT E3 ligases to identify enzymes that assemble Lys33-linked polyUb(A) Ubiquitylation assays of HECT E3 ligases in the presence of UBE1, Ub and different E2 enzymes: UBE2D1, UBE2D2, UBE2D3 and UBE2L3. (B) Ubiquitylated products generated by HECT E3 ligases with the optimal E2 were analysed by pRM LC–MS/MS for the abundance of Ub linkages as described in ‘Materials and Methods’ (Supplementary Figure S1B). Each Ub linkage assembled by the HECT E3 ligases was plotted as a bar graph where the y-axes are summed ion current values for the relevant daughter ions of each precursor mass analysed (Supplementary Table S2). No signal was observed for Met1 and Lys27 linkages for any of the ligases tested.
Mentions: PolyUb chains were digested with trypsin and analysed on an LTQ-Velos mass spectrometer (Thermo) fitted with an Easy-Spray Source (Thermo) and utilizing a Dionex RSLC HPLC system. Standard diUb chains were purchased from Boston Biochemicals and a synthetic peptide AK(GG)IQDK representing the tryptic Ub K29 linkage was purchased from Pepceuticals. Digests (prepared in 0.1% TFA (trifluoroacetic acid)/water) were concentrated on a 20×0.1 mm nanotrap column (Thermo) equilibrated in 0.1% TFA/water (10 μl/min) and washed with 10 μl of the same buffer. The samples were loaded and washed in TFA buffers, as the trap column in the presence of formic acid did not retain the tryptic peptide containing the Lys29 linkage. Peptides were then separated on a 150×0.075 mm PepMap C18, 3 μm Easy-Spray column (Thermo) equilibrated with 2% acetonitrile/0.1% formic acid/water at 300 nl/min, employing a stepped gradient of buffer B (80% acetonitrile/0.1% formic acid/water) as follows: 0–14 min=1%–30% B, 14–15 min=30%–80% B, 15–20 min=80% B. LC–MS data was acquired in data-independent mode with one full scan (m/z=350–1800) followed by eight product ion scans as described below. Parameters used: Easy-Spray column voltage was 1.9 kV; isolation width was set to 1 Da; normalized collision energy was 35, and the activation time was 10 ms. The ion current for the daughter ions was summed using Xcalibur software (Thermo) for each precursor mass analysed (Supplementary Table S2). The resultant summed intensities provide the y-axis values for Figure 1(B) and Supplementary Figure S2. This method was more specific than solely using the extracted ion current for the precursor mass for each Ub chain peptide.

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