Fig 1: Schematic showing the domain architecture of KEAP1 and CUL3 and predicted model for their interaction. KEAP1 is shown as a homodimer in red and blue. Substrate degrons (purple) are shown bound to the Kelch domains. The CUL3 (light brown) C-terminal domain (CTD) is bound to RBX1 (claret). The CUL3 N-terminal domain (NTD) and 22 residue N-terminal extension (N22) are predicted to bind to the KEAP1 BTB and 3-box domains (model based on the previously determined structure of a KLHL11-CUL3 complex [29]). The BTB domain was first identified as a conserved motif in the Drosophila proteins bric-à-brac, tramtrack and broad complex (reviewed in Ref. [34]). Likewise, the Kelch repeat domain was first identified in the Drosophila Kelch protein (reviewed in Ref. [35]). The BACK domain (for BTB and C-terminal Kelch) is also known as the intervening region (IVR) in KEAP1 and includes the 3-box motif at it N-terminus [36].
Fig 2: Structure of the crystallised KEAP1-CUL3 complex. The structure shown in two orientations reveals a stoichiometry of 2:2 for KEAP1BTB-3-box binding to the Cul3NTD.
Fig 3: Comparison of the 3-box hydrophobic groove in the respective KEAP1 and KLHL11 bound CUL3 complexes. (A) Superposition of KEAP1 (dark blue) and KLHL11 (orange) showing conservation of a hydrophobic groove between the BTB domain (α5) and 3 box (α7). KLHL11 Leu211 is replaced by Phe190 in KEAP1. (B) Superposition of CUL3 complexes of KEAP1 and KLHL11 with the KEAP1-CDDO complex. A surface representation of the 3-box hydrophobic groove in the KLHL11-CUL3 complex is shown (green with blue and red areas denoting hydrogen bond donor and acceptor positions, respectively) with interacting CUL3 residues shown as yellow sticks. Superposition predicts clashes between KEAP1 Phe190 (protruding through this surface) and CUL3 Ala20 as well as between KEAP1-bound CDDO and CUL3 Phe21.
Fig 4: A generalizable TR-FRET platform for profiling BTB domain-containing proteins. (A) Dose-response titration of KLHL11BTB-BACK and small molecule KEAP1-Kelch domain inhibitor KI-696 (positive control) in KEAP1FL dimerization assay [44,59], using a mixture of CoraFluor-1- and FITC-labeled Nrf2-derived peptides (LDEETGEFL-CONH2), showing that KLHL11BTB-BACK does not heterodimerize with KEAP1FL at concentrations up to 25 μM. Also see Fig. S3 (B) Dose-response titration of KEAP1BTB-3-box and KEAP1BTB-BACK-Kelch in KLHL11BTB-BACK-CUL3NTD TR-FRET protein displacement assay provides evidence that the complete BTB-BACK domain does not meaningfully contribute to the binding affinity of the KEAP1-CUL3NTD interaction.
Fig 5: Examining the role of the CUL3NTD N-terminal extension on binding affinity toward KLHL11 and KEAP1. (A–B) Dose-response titration of CUL3NTD and CUL3NTDΔ22 in TR-FRET protein displacement assay between AF488-labeled CUL3NTD and (A) CoraFluor-1-labeled KLHL11BTB-BACK, or (B) CoraFluor-1-labeled KEAP1FL. Deletion of the N-terminal 22 amino acid residues results in a ∼100-fold loss of binding affinity of CUL3NTD toward both proteins.
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