Extended Data Fig. 3: SCF^FBXL17 binds the BTB domain, but its active site is next to the Kelch repeats of KEAP1.

a, Elution profile of the SCF^FBXL17-KEAP1^V99A complex by size exclusion chromatography detecting A₂₈₀. Control proteins with known MW are shown on top. This experiment was performed three times. b, Cryo-EM density map of the SCF^FBXL17-KEAP1^V98A complex. Dark grey: CUL1^1–450; light grey: SKP1; orange: FBXL17; blue: KEAP1. c, Despite an overlap in binding sites, CUL3 does not compete with SCF^FBXL17 for substrate ubiquitylation. ³⁵S-labelled wild-type or mutant KEAP1 were ubiquitylated by SCF^FBXL17 in reticulocyte lysate either in the presence or absence of a CUL3 variant shown to bind BTB proteins. Ubiquitylated KEAP1 was detected by gel electrophoresis and autoradiography. Three independent experiments were performed with similar results. d, SCF^FBXL17 ubiquitylates full-length BTB proteins with Kelch repeats better than isolated BTB domains. ³⁵S-labelled full-length KEAP1^F64A, the BTB domain of KEAP1^F64A, full-length KLHL12^V50A, or the BTB domain of KLHL12^V50A were incubated in reticulocyte lysate with recombinant FBXL17, and ubiquitylation was detected by gel electrophoresis and autoradiography. Two independent experiments were performed with similar results. e, Full-length BTB proteins or isolated BTB domains bind similarly well to FBXL17. ³⁵S-labelled full-length BTB proteins or isolated BTB domains, as indicated on the right, were incubated with immobilized ^MBPFBXL17 and bound proteins were detected by gel electrophoresis and autoradiography. Two independent experiments were performed with similar results.