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 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/528651v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@3db02dorg.highwire.dtl.DTLVardef@1f97940org.highwire.dtl.DTLVardef@1879542org.highwire.dtl.DTLVardef@5847d2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA new crystal structure defines KEAP1 BTB and 3-box domain interactions with CUL3 C_LIO_LIKEAP1 and CUL3 form a heteromeric 2:2 complex with a KD value of 0.2 {micro}M C_LIO_LIA generalizable TR-FRET platform enables multimodal profiling of BTB proteins C_LIO_LIThe investigational drug CDDO is a partial antagonist of the KEAP1-CUL3 interaction C_LI