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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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Ion-mobilitymass spectrometry data for the ASB9–EloBC–Cul5NTD quaternary complex. (A) Native mass spectrum and ion-mobilitydrift-time plot of the ASB9–EloBC–Cul5NTD sample. The quaternary complex (cyan) partially dissociates. ASB9–EloBC(purple) and Cul5NTD (monomeric (red) and dimeric (yellow))are also present but are separated by their varying drift times. (B)Experimentally measured collision cross section (CCS) areas of fourcharge states (17+ to 20+) of ASB9–EloBC–Cul5NTD. (C) Average CCS values for the ASB9–EloBC–Cul5NTD quaternary complex were determined to be 6000 ± 200Å2, which is in good agreement with that calculated(5895 Å2) from the model shown in panel D.
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fig5: Ion-mobilitymass spectrometry data for the ASB9–EloBC–Cul5NTD quaternary complex. (A) Native mass spectrum and ion-mobilitydrift-time plot of the ASB9–EloBC–Cul5NTD sample. The quaternary complex (cyan) partially dissociates. ASB9–EloBC(purple) and Cul5NTD (monomeric (red) and dimeric (yellow))are also present but are separated by their varying drift times. (B)Experimentally measured collision cross section (CCS) areas of fourcharge states (17+ to 20+) of ASB9–EloBC–Cul5NTD. (C) Average CCS values for the ASB9–EloBC–Cul5NTD quaternary complex were determined to be 6000 ± 200Å2, which is in good agreement with that calculated(5895 Å2) from the model shown in panel D.

Mentions: Attempts to obtain the atomic-levelstructure of the ASB9–EloBC–Cul5NTD quaternarycomplex in our laboratory have so far been unsuccessful, and previouslyreported models of quaternary ECS-type CRL complexes have not beenexperimentally validated. To provide structural validation of ourquaternary complex model, ion-mobility mass spectrometry (IM–MS)experiments were performed on the ASB9–EloBC–Cul5NTD complex purified by size-exclusion chromatography. Undernative conditions, drift times were measured for four charge states(17+ to 20+) of the intact monomeric heterocomplexASB9–EloBC–Cul5NTD (Figure 5), and the average collision cross section (CCS) was determinedto be 6000 ± 200 Å2 (Figure 5B,C), which is in very good agreement with the CCS calculatedfrom the model of ASB9–EloBC–Cul5NTD (5895Å2). This implies that our model reflects a highlyprobable conformation of the quaternary complex and therefore providesinsight into the physiological structure of this multimeric system.


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)

Ion-mobilitymass spectrometry data for the ASB9–EloBC–Cul5NTD quaternary complex. (A) Native mass spectrum and ion-mobilitydrift-time plot of the ASB9–EloBC–Cul5NTD sample. The quaternary complex (cyan) partially dissociates. ASB9–EloBC(purple) and Cul5NTD (monomeric (red) and dimeric (yellow))are also present but are separated by their varying drift times. (B)Experimentally measured collision cross section (CCS) areas of fourcharge states (17+ to 20+) of ASB9–EloBC–Cul5NTD. (C) Average CCS values for the ASB9–EloBC–Cul5NTD quaternary complex were determined to be 6000 ± 200Å2, which is in good agreement with that calculated(5895 Å2) from the model shown in panel D.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3756526&req=5

fig5: Ion-mobilitymass spectrometry data for the ASB9–EloBC–Cul5NTD quaternary complex. (A) Native mass spectrum and ion-mobilitydrift-time plot of the ASB9–EloBC–Cul5NTD sample. The quaternary complex (cyan) partially dissociates. ASB9–EloBC(purple) and Cul5NTD (monomeric (red) and dimeric (yellow))are also present but are separated by their varying drift times. (B)Experimentally measured collision cross section (CCS) areas of fourcharge states (17+ to 20+) of ASB9–EloBC–Cul5NTD. (C) Average CCS values for the ASB9–EloBC–Cul5NTD quaternary complex were determined to be 6000 ± 200Å2, which is in good agreement with that calculated(5895 Å2) from the model shown in panel D.
Mentions: Attempts to obtain the atomic-levelstructure of the ASB9–EloBC–Cul5NTD quaternarycomplex in our laboratory have so far been unsuccessful, and previouslyreported models of quaternary ECS-type CRL complexes have not beenexperimentally validated. To provide structural validation of ourquaternary complex model, ion-mobility mass spectrometry (IM–MS)experiments were performed on the ASB9–EloBC–Cul5NTD complex purified by size-exclusion chromatography. Undernative conditions, drift times were measured for four charge states(17+ to 20+) of the intact monomeric heterocomplexASB9–EloBC–Cul5NTD (Figure 5), and the average collision cross section (CCS) was determinedto be 6000 ± 200 Å2 (Figure 5B,C), which is in very good agreement with the CCS calculatedfrom the model of ASB9–EloBC–Cul5NTD (5895Å2). This implies that our model reflects a highlyprobable conformation of the quaternary complex and therefore providesinsight into the physiological structure of this multimeric system.

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
Related in: MedlinePlus