Article
Extended Data Fig. 3 | SCFFBXL17 binds the BTB domain, but its active site is
next to the Kelch repeats of KEAP1. a, Elution profile of the SCFFBXL17-KEAP1V99A
complex by size exclusion chromatography detecting A 280. Control proteins
with known MW are shown on top. This experiment was performed three
times. b, Cryo-EM density map of the SCFFBXL17-KEAP1V98A complex. Dark grey:
CUL11–450; light grey: SKP1; orange: FBXL17; blue: KEAP1. c, Despite an
overlap in binding sites, CUL3 does not compete with SCFFBXL17 for substrate
ubiquitylation.^35 S-labelled wild-type or mutant KEAP1 were ubiquitylated by
SCFFBXL17 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, SCFFBXL17 ubiquitylates full-length BTB
proteins with Kelch repeats better than isolated BTB domains.^35 S-labelled
full-length KEAP1F64A, the BTB domain of KEAP1F64A, full-length KLHL12V50A, or
the BTB domain of KLHL12V50A 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.^35 S-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.