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Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly
KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 in...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier Science
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10564622/ https://www.ncbi.nlm.nih.gov/pubmed/37156295 http://dx.doi.org/10.1016/j.freeradbiomed.2023.04.021 |
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author | Adamson, Roslin J. Payne, N Connor Bartual, Sergio G. Mazitschek, Ralph Bullock, Alex N. |
author_facet | Adamson, Roslin J. Payne, N Connor Bartual, Sergio G. Mazitschek, Ralph Bullock, Alex N. |
author_sort | Adamson, Roslin J. |
collection | PubMed |
description | KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function. |
format | Online Article Text |
id | pubmed-10564622 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Elsevier Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-105646222023-10-12 Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly Adamson, Roslin J. Payne, N Connor Bartual, Sergio G. Mazitschek, Ralph Bullock, Alex N. Free Radic Biol Med Article KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function. Elsevier Science 2023-08-01 /pmc/articles/PMC10564622/ /pubmed/37156295 http://dx.doi.org/10.1016/j.freeradbiomed.2023.04.021 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Adamson, Roslin J. Payne, N Connor Bartual, Sergio G. Mazitschek, Ralph Bullock, Alex N. Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly |
title | Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly |
title_full | Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly |
title_fullStr | Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly |
title_full_unstemmed | Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly |
title_short | Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly |
title_sort | structural and biochemical characterization establishes a detailed understanding of keap1-cul3 complex assembly |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10564622/ https://www.ncbi.nlm.nih.gov/pubmed/37156295 http://dx.doi.org/10.1016/j.freeradbiomed.2023.04.021 |
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