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Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

Thomas JC, Matak-Vinkovic D, Van Molle I, Ciulli A - Biochemistry (2013)

Bottom Line: This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex.The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family.The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, United Kingdom.

ABSTRACT
Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

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ASB9 is part of a Cullin-RINGE3 ubiquitin ligase complex. (A)Cartoon showing the assembly of Cullin-RING E3 ubiquitin ligases.ASB9 acts as a substrate recognition subunit (SRS) utilizing ElonginB and Elongin C as adaptor proteins to bind the Cullin N-terminaldomain. The Cullin C-terminal domain binds RING-box protein (Rbx)to bring the ubiquitin (Ub)-loaded E2 enzyme into close proximitywith substrate (S), allowing polyubiquitination to occur. (B) Crystalstructures of the ASB9–EloBC ternary complex (PDB 3ZKJ) and Cullin 5 N-terminaldomain (Cul5NTD, PDB 2WZK). ASB9 is shown in purple, EloC, in green,and EloB, in cyan. The ASB9 BC-box is shown in pink and the Cullin-boxin orange. Cul5NTD is shown in red. (C) Sequence schematicof ASB9 highlighting the C-terminal SOCS-box domain. The EloBC-bindingregion (BC-box) is underlined in pink, and the Cullin 5-binding boxunderlined in orange. The canonical Cullin 5-binding motif is shownunderneath and is also underlined in orange (φ is a hydrophobicresidue and X is any residue).
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fig1: ASB9 is part of a Cullin-RINGE3 ubiquitin ligase complex. (A)Cartoon showing the assembly of Cullin-RING E3 ubiquitin ligases.ASB9 acts as a substrate recognition subunit (SRS) utilizing ElonginB and Elongin C as adaptor proteins to bind the Cullin N-terminaldomain. The Cullin C-terminal domain binds RING-box protein (Rbx)to bring the ubiquitin (Ub)-loaded E2 enzyme into close proximitywith substrate (S), allowing polyubiquitination to occur. (B) Crystalstructures of the ASB9–EloBC ternary complex (PDB 3ZKJ) and Cullin 5 N-terminaldomain (Cul5NTD, PDB 2WZK). ASB9 is shown in purple, EloC, in green,and EloB, in cyan. The ASB9 BC-box is shown in pink and the Cullin-boxin orange. Cul5NTD is shown in red. (C) Sequence schematicof ASB9 highlighting the C-terminal SOCS-box domain. The EloBC-bindingregion (BC-box) is underlined in pink, and the Cullin 5-binding boxunderlined in orange. The canonical Cullin 5-binding motif is shownunderneath and is also underlined in orange (φ is a hydrophobicresidue and X is any residue).

Mentions: The E1 (activating),E2 (conjugating),and E3 (ligating) ubiquitination cascade plays a key role in controllingcellular protein levels by catalyzing the polyubiquitination of substrateproteins, leading to their subsequent proteasomal degradation.1−3 The E3 ubiquitin ligases impart specificity for this process andact by bringing a ubiquitin-loaded E2 enzyme and the substrate intoclose proximity to allow the ubiquitin transfer to occur.4 The Cullin-RING ligases (CRLs) function as multisubunitprotein complexes (Figure 1A). They are thelargest family of E3 ligases in eukaryotes, consisting of ∼400members5 that target ∼20% of theproteins degraded by the proteasome.6 CRLsare therefore involved in a myriad of cellular processes, includingcell-cycle control, gene transcription, and signal transduction,7 so it is of great interest to study how thesecomplex molecular machines assemble and function.


Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

Thomas JC, Matak-Vinkovic D, Van Molle I, Ciulli A - Biochemistry (2013)

ASB9 is part of a Cullin-RINGE3 ubiquitin ligase complex. (A)Cartoon showing the assembly of Cullin-RING E3 ubiquitin ligases.ASB9 acts as a substrate recognition subunit (SRS) utilizing ElonginB and Elongin C as adaptor proteins to bind the Cullin N-terminaldomain. The Cullin C-terminal domain binds RING-box protein (Rbx)to bring the ubiquitin (Ub)-loaded E2 enzyme into close proximitywith substrate (S), allowing polyubiquitination to occur. (B) Crystalstructures of the ASB9–EloBC ternary complex (PDB 3ZKJ) and Cullin 5 N-terminaldomain (Cul5NTD, PDB 2WZK). ASB9 is shown in purple, EloC, in green,and EloB, in cyan. The ASB9 BC-box is shown in pink and the Cullin-boxin orange. Cul5NTD is shown in red. (C) Sequence schematicof ASB9 highlighting the C-terminal SOCS-box domain. The EloBC-bindingregion (BC-box) is underlined in pink, and the Cullin 5-binding boxunderlined in orange. The canonical Cullin 5-binding motif is shownunderneath and is also underlined in orange (φ is a hydrophobicresidue and X is any residue).
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fig1: ASB9 is part of a Cullin-RINGE3 ubiquitin ligase complex. (A)Cartoon showing the assembly of Cullin-RING E3 ubiquitin ligases.ASB9 acts as a substrate recognition subunit (SRS) utilizing ElonginB and Elongin C as adaptor proteins to bind the Cullin N-terminaldomain. The Cullin C-terminal domain binds RING-box protein (Rbx)to bring the ubiquitin (Ub)-loaded E2 enzyme into close proximitywith substrate (S), allowing polyubiquitination to occur. (B) Crystalstructures of the ASB9–EloBC ternary complex (PDB 3ZKJ) and Cullin 5 N-terminaldomain (Cul5NTD, PDB 2WZK). ASB9 is shown in purple, EloC, in green,and EloB, in cyan. The ASB9 BC-box is shown in pink and the Cullin-boxin orange. Cul5NTD is shown in red. (C) Sequence schematicof ASB9 highlighting the C-terminal SOCS-box domain. The EloBC-bindingregion (BC-box) is underlined in pink, and the Cullin 5-binding boxunderlined in orange. The canonical Cullin 5-binding motif is shownunderneath and is also underlined in orange (φ is a hydrophobicresidue and X is any residue).
Mentions: The E1 (activating),E2 (conjugating),and E3 (ligating) ubiquitination cascade plays a key role in controllingcellular protein levels by catalyzing the polyubiquitination of substrateproteins, leading to their subsequent proteasomal degradation.1−3 The E3 ubiquitin ligases impart specificity for this process andact by bringing a ubiquitin-loaded E2 enzyme and the substrate intoclose proximity to allow the ubiquitin transfer to occur.4 The Cullin-RING ligases (CRLs) function as multisubunitprotein complexes (Figure 1A). They are thelargest family of E3 ligases in eukaryotes, consisting of ∼400members5 that target ∼20% of theproteins degraded by the proteasome.6 CRLsare therefore involved in a myriad of cellular processes, includingcell-cycle control, gene transcription, and signal transduction,7 so it is of great interest to study how thesecomplex molecular machines assemble and function.

Bottom Line: This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex.The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family.The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, United Kingdom.

ABSTRACT
Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Show MeSH