Extended Data Fig. 8: Binding and destabilization of BTB dimers by SCF^FBXL17.

a, A FRET-based assay to monitor BTB dimer formation. Blue curve: The BTB domain of KEAP1^F64A was labelled with Alexa 555, then denatured and refolded. Red curve: The BTB domain of KEAP1^F64A was labelled with Alexa 647, then denatured and refolded. Green curve: Two separate BTB domain pools of KEAP1^F64A were labelled with either Alexa 555 or Alexa 647, mixed in equimolar concentrations, denatured, and then refolded. About 50% of dimers are labelled with distinct fluorophores in each BTB subunit, giving rise to donor fluorescence quenching and acceptor emission as indication of FRET. Three independent experiments were performed with similar results. b, KEAP1^F64A dimers dissociate very slowly and inefficiently. BTB domains of KEAP1^F64A were labelled with either Alexa 555 or Alexa 647, respectively. The labelled BTB domains were then mixed, incubated overnight, and analysed for FRET that results from stochastic rebinding of BTB monomers, leading to formation of BTB dimers containing one subunit labelled with Alexa 555 and the other subunit labelled with Alexa 647. However, in comparison to complex reformation by refolding (see above), little FRET was detected. This experiment was performed twice. c, FBXL17 can modulate BTB complex composition in vitro. The KLHL12 locus was tagged with a 3xFLAG epitope by CRISPR/Cas9-mediated genome editing. Endogenous KLHL12^3xFLAG complexes were affinity-purified from 293T cells and incubated with recombinant MBP (control), FBXL17, or inactive FBXL17^ΔCTH. Proteins that remained bound to KLHL12^3xFLAG were determined by mass spectrometry. d, Overexpression of FBXL17^ΔFBOX, which can bind but not ubiquitylate BTB proteins, prevents BTB heterodimerization. The endogenous KLHL12^3xFLAG was affinity-purified either in the presence or absence of FBXL17^ΔFbox, and bound proteins were determined by mass spectrometry. e, FBXL17 can bind BTB dimers. ^FLAGKLHL12 was affinity-purified from 293T cells also expressing ^MYCKLHL12 and FBXL17^HA. ^FLAGKLHL12 complexes were eluted with FLAG-peptide and FBXL17^HA-containing complexes were then purified over αHA-agarose. Bound ^MYCKLHL12, indicative of FBXL17 associating with KLHL12 dimers, was then detected by western blotting. This experiment was performed once. f, Binding of FBXL17 to BTB dimers requires its CTH to be disengaged from its binding site at the BTB dimer interface. A structural model of a KEAP1 BTB dimer bound to FBXL17 was generated using the KEAP1^F64A dimer and the FBXL17-KEAP1^F64A complex structures. Clashes are predominantly at the CTH of FBXL17. g, Residues of FBXL17, which in the structural model of a FBXL17-BTB dimer complex are in proximity to the leaving BTB subunit, contribute to stable substrate recognition. Indicated FBXL17 residues at the interface with the leaving BTB subunit (above) were mutated in the sensitized background of the FBXL17^C680D variant and analysed for binding to endogenous BTB proteins by affinity purification and western blotting. This experiment was performed once.