Article
Extended Data Fig. 1 | Variant BTB domains of KEAP1 are recognized by
SCFFBXL17. a, Mutations in KEAP1’s BTB domain result in efficient recognition by
SCFFBXL17. The same mutations as in Fig. 1a were introduced into the KEAP1S172A
variant that had previously been used for crystallization.^35 S-labelled double
mutants, but not the KEAP1S172A single mutant, were retained by immobilized
MBPFBXL17, as detected by gel electrophoresis and autoradiography. This
experiment was performed once. b, SCFFBXL17 strongly prefers mutant over
wild-type BTB domains. Increasing concentrations of MBPFBXL17 were
immobilized on amylose beads and incubated with 100 nM wild-type or mutant
KEAP1. Depletion of KEAP1 from the supernatant was measured by quantitative
LiCor imaging of Coomassie-stained SDS–PAGE gels. The affinity of FBXL17 to
wild-type KEAP1 was too low to be determined reliable by this method. Three
independent experiments were performed with similar results. c, Mutant BTB
domains form homodimers in vitro. Recombinant BTB domains of KEAP1,
KEAP1F64A, and KEAPV98A (MW about 15 kDa) were analysed by size exclusion
chromatography detecting A 280. Expected position of BTB dimer versus
monomer, as well as of control proteins with known MW are shown on top.
Three independent experiments were performed with similar results. d, BTB
domains of wild-type KEAP1 and mutant KEAP1F64A form homodimers in
solution, as determined by SEC-MALS. This experiment was performed twice.
e, Mutant BTB domains unfold via an intermediate species. Wild-type or
mutant BTB domains of KEAP1 (0.04 mg/ml) were incubated with various
concentrations of urea, equilibrated overnight, and their resulting secondary
structure was monitored by ellipticity at 222 nm using circular dichroism. The
experiment was performed once for the mutants and twice for the wild-type
KEAP1. f, The intermediate seen in the unfolding of KEAP1V98A probably ref lects
local conformational changes, rather than monomerization. Urea-dependent
unfolding curves for the BTB domain of KEAP1V98A were repeated at tenfold
higher BTB concentration (low: 0.04 mg/ml; high: 0.4 mg/ml); only the
second transition shifted to higher urea concentrations, identifying it as a
dimer-unfolded transition. This experiment was performed once. g. Mutation
of Phe64 or Val98 to Ala in the BTB domain of KEAP1 reduces contacts between
helices of two interacting subunits of the KEAP1 dimer.