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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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Structural model of the quaternary complex ASB9–EloBC–Cul5NTD. The model was constructed using the crystal structureof FboxSkp2–Skp1–Cul1NTD as atemplate (PDB 1LDK).34 The Cul5NTD crystal structure(PDB 2WZK) wasaligned with Cul1NTD, and EloC of the ASB9–EloBCcrystal structure (PDB 3ZKJ) was aligned with Skp1 of the FboxSkp2–Skp1–Cul1NTD structure. ASB9 is shown in purple, EloC, in green, EloB,in cyan, Cul5NTD, in red, Cul1NTD, in orange,Skp1, in dark pink, and FboxSkp2, in light pink. (A) Surfacerepresentation of the ASB9–EloBC–Cul5NTD quaternarycomplex. (B) Structural alignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the variation in the Cullin NTD orientations. The kink atCul1NTD residue Gly212 is labeled. (C) Close-up structuralalignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the highstructural conservation at the interface between the adaptor protein(Skp1/EloC) and Cullin (Cul1/Cul5, respectively).
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fig4: Structural model of the quaternary complex ASB9–EloBC–Cul5NTD. The model was constructed using the crystal structureof FboxSkp2–Skp1–Cul1NTD as atemplate (PDB 1LDK).34 The Cul5NTD crystal structure(PDB 2WZK) wasaligned with Cul1NTD, and EloC of the ASB9–EloBCcrystal structure (PDB 3ZKJ) was aligned with Skp1 of the FboxSkp2–Skp1–Cul1NTD structure. ASB9 is shown in purple, EloC, in green, EloB,in cyan, Cul5NTD, in red, Cul1NTD, in orange,Skp1, in dark pink, and FboxSkp2, in light pink. (A) Surfacerepresentation of the ASB9–EloBC–Cul5NTD quaternarycomplex. (B) Structural alignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the variation in the Cullin NTD orientations. The kink atCul1NTD residue Gly212 is labeled. (C) Close-up structuralalignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the highstructural conservation at the interface between the adaptor protein(Skp1/EloC) and Cullin (Cul1/Cul5, respectively).

Mentions: We constructed a structural model of the ASB9–EloBC–Cul5NTD quaternary complex using the published crystal structureof the homologous FboxSkp2–Skp1–Cul1NTD complex as a template.34 Thefirst α-helical bundle of the crystal structure of Cul5NTD(PDB 2WZK) was aligned with the corresponding region of Cul1NTD (sequence identity 29%, rmsd of all atoms 2.31 Å), whereasthe EloC subunit of the ASB9–EloBC ternary-complex crystalstructure (PDB 3ZKJ) was aligned with the homologous Skp1 subunit (sequence identity38%, rmsd of all atoms 1.19 Å) to give the quaternary complexshown in Figure 4A. Similar models of otherECS-type CRL quaternary complexes have previously been reported, includingthose for SOCS2–EloBC–Cul5,47 RAR3–EloBC–Cul5,44 HIV-1Vif–EloBC–Cul5,48 and VHL–EloBC–Cul2.44 Our model shows that the Cul5NTD stalkis oriented in the opposite direction relative to the ankyrin repeatsof ASB9.


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)

Structural model of the quaternary complex ASB9–EloBC–Cul5NTD. The model was constructed using the crystal structureof FboxSkp2–Skp1–Cul1NTD as atemplate (PDB 1LDK).34 The Cul5NTD crystal structure(PDB 2WZK) wasaligned with Cul1NTD, and EloC of the ASB9–EloBCcrystal structure (PDB 3ZKJ) was aligned with Skp1 of the FboxSkp2–Skp1–Cul1NTD structure. ASB9 is shown in purple, EloC, in green, EloB,in cyan, Cul5NTD, in red, Cul1NTD, in orange,Skp1, in dark pink, and FboxSkp2, in light pink. (A) Surfacerepresentation of the ASB9–EloBC–Cul5NTD quaternarycomplex. (B) Structural alignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the variation in the Cullin NTD orientations. The kink atCul1NTD residue Gly212 is labeled. (C) Close-up structuralalignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the highstructural conservation at the interface between the adaptor protein(Skp1/EloC) and Cullin (Cul1/Cul5, respectively).
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fig4: Structural model of the quaternary complex ASB9–EloBC–Cul5NTD. The model was constructed using the crystal structureof FboxSkp2–Skp1–Cul1NTD as atemplate (PDB 1LDK).34 The Cul5NTD crystal structure(PDB 2WZK) wasaligned with Cul1NTD, and EloC of the ASB9–EloBCcrystal structure (PDB 3ZKJ) was aligned with Skp1 of the FboxSkp2–Skp1–Cul1NTD structure. ASB9 is shown in purple, EloC, in green, EloB,in cyan, Cul5NTD, in red, Cul1NTD, in orange,Skp1, in dark pink, and FboxSkp2, in light pink. (A) Surfacerepresentation of the ASB9–EloBC–Cul5NTD quaternarycomplex. (B) Structural alignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the variation in the Cullin NTD orientations. The kink atCul1NTD residue Gly212 is labeled. (C) Close-up structuralalignment of ASB9–EloBC–Cul5NTD with FboxSkp2–Skp1–Cul1NTD showing the highstructural conservation at the interface between the adaptor protein(Skp1/EloC) and Cullin (Cul1/Cul5, respectively).
Mentions: We constructed a structural model of the ASB9–EloBC–Cul5NTD quaternary complex using the published crystal structureof the homologous FboxSkp2–Skp1–Cul1NTD complex as a template.34 Thefirst α-helical bundle of the crystal structure of Cul5NTD(PDB 2WZK) was aligned with the corresponding region of Cul1NTD (sequence identity 29%, rmsd of all atoms 2.31 Å), whereasthe EloC subunit of the ASB9–EloBC ternary-complex crystalstructure (PDB 3ZKJ) was aligned with the homologous Skp1 subunit (sequence identity38%, rmsd of all atoms 1.19 Å) to give the quaternary complexshown in Figure 4A. Similar models of otherECS-type CRL quaternary complexes have previously been reported, includingthose for SOCS2–EloBC–Cul5,47 RAR3–EloBC–Cul5,44 HIV-1Vif–EloBC–Cul5,48 and VHL–EloBC–Cul2.44 Our model shows that the Cul5NTD stalkis oriented in the opposite direction relative to the ankyrin repeatsof ASB9.

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