of KEAP1(F64A), KEAP1(S172A/F64A/R116C) was first exchanged into
filtered, degassed DPBS using SEC and subsequently concentrated to
7 mg ml−1. A 20 mM stock of Alexa Fluor 555 C2 maleimide (donor,
Thermo Fisher, A20346) and Alexa Fluor 647 C2 maleimide (accep-
tor, Thermo Fisher, A20347) were independently created by dissolv-
ing 1 mg of solid dye in DMSO. KEAP1 was labelled in an overnight
reaction in DPBS containing 100 μM protein, 20% degassed glycerol,
and 1 mM of labelling dye. The reactions were quenched by adding
β-mercaptoethanol to a final concentration of 10 mM, spun down at
21,000g for 10 min at 4 °C to eliminate any precipitation, and loaded
onto a Superdex 200 Increase 10/300 GL equilibrated with 150 mM
NaCl, 50 mM Tris-HCl 8.0, and 1 mM TCEP. Protein fractions contain-
ing dimeric KEAP1 were pooled and concentrated. Based on spectral
comparisons of KEAP1(F64A/S172A) and KEAP1(F64A/S172A/R116C)
labelling reactions, we estimate that >90% of the labelling was specific
to the introduced cysteine and that >80% of molecules were labelled.
The resulting homolabelled KEAP1(F64A) were then subjected to 8 M
urea denaturation and refolding via dialysis overnight at room tempera-
ture, protected from light. The protein was then concentrated, spun at
21,000g for 10 min at 4 °C to eliminate any precipitation, and ran on a
Superdex 200 Increase 10/300 GL column equilibrated with 150 mM
NaCl, 50 mM Tris-HCl 8.0 and 1 mM TCEP. The fractions containing
dimeric KEAP1 were collected and concentrated.
To obtain heterolabelled KEAP1(F64A), we mixed equimolar amounts
of acceptor- and donor-labelled KEAP1(F64A/S172A/R116C), denatured
by adding urea to 8 M, and refolded via dialysis overnight at room tem-
perature in the dark. The protein was similarly concentrated, spun, and
purified by SEC. The fractions containing dimeric KEAP1 were collected
and concentrated.
Fluorescence resonance energy transfer assay and analysis
All FRET assays were conducted at 4 °C using a QuantaMaster QM4CW
(PTI) fluorimeter and Hellma 105.251-QS cuvettes. Time courses
followed the donor channel using an excitation of 555 nm and emission
of 570 nm. Recordings were taken every 30 s for 2 h once 100 nM het-
erolabelled KEAP1 was mixed with 5 μM of chase protein. GroEL was
obtained from Sigma-Aldrich (C7688). Overnight incubations were
performed by taking spectra (555 nm excitation) of 100 nM heterola-
belled KEAP1 dimer, followed by an overnight 4 °C incubation with 5
μM of chase protein. Similar measurements were performed for KEAP1
dimers labelled with either FRET donor or acceptor and then mixed.
The bar graphs depicting percent increase in donor fluorescence upon
addition of FBXL17, FBXL17(ΔCTH) or KEAP1(F64A) were calculated
from emission at 570 nm. Calculations corrected for the dilution upon
adding the chase protein.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
The atomic coordinates of the CUL1–SKP1–FBXL17–KEAP1(V99A) com-
plex has been deposited to PDB with accession number 6WCQ. The
respective cryo-EM map has been deposited to the Electron Microscopy
Data Bank with accession number EMD-21617. The atomic coordinates
of the X-ray crystal structures have been deposited to the PDB with the
following accession numbers: 6W66 (KEAP1(S172A/F64A)–FBXL17–
SKP1 complex), 6W67 (KEAP1(S172A)), 6W68 (KEAP1(S172A/V98A))
and 6W69 (KEAP1(S172A/F64A)).
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Acknowledgements We thank C. Nixon, S. Costello and S. Marqusee for their generous help
with circular dichroism; members of the A. Martin laboratory for their help with FRET; L. Nocka
for help with SEC–MALS; F. Rodriguez Perez for help with R; A. Patel and D. Toso for help with
graphene oxide grid preparation and cryo-EM data collection, respectively; J. Holton and
G. Meigs at the Advanced Light Source Beamline 8.3.1 for assistance with data collection;
J. Schaletzky for her comments on the manuscript; and members of the Rape, Nogales and
Kuriyan laboratories for discussion and suggestions. Beamline 8.3.1 at the Advanced Light
Source is operated by the University of California Office of the President, Multicampus
Research Programs and Initiatives grant MR-15-328599, the National Institutes of Health (R01
GM124149 and P30 GM124169), Plexxikon and the Integrated Diffraction Analysis Technologies
program of the US Department of Energy Office of Biological and Environmental Research.
The Advanced Light Source (Berkeley) is a national user facility operated by Lawrence
Berkeley National Laboratory on behalf of the US Department of Energy under contract
number DE-AC02-05CH11231, Office of Basic Energy Sciences. E.L.M. was funded by an NSF
predoctoral scholarship (ID 2013157149). B.J.G. was supported by fellowships from the Swiss
National Science Foundation (projects P300PA_160983, P300PA_174355). P.J. was funded by a
Siebel Institute postdoctoral fellowship. D.A. was funded by an NIH F32 postdoctoral
fellowship. E.N., J.K. and M.R. are investigators with the Howard Hughes Medical Institute.
Author contributions E.L.M., B.G.L. and D.A. purified recombinant proteins and performed
SEC, SEC–MALS, CD and FRET experiments. E.L.M., B.G.L. and C.L.G. performed
crystallization experiments. B.J.G. and E.L.M. performed cryo-EM experiments. P.J.
performed immunoprecipitation, degradation and mass spectrometry analyses. E.L.M. and
P.J. performed in vitro binding assays. P.J. and E.L.M. performed in vitro ubiquitylation
assays. P.J. and D.A. performed in vitro titration assays. All authors interpreted the data and
wrote the manuscript.
Competing interests M.R. and J.K. are founders and consultants of Nurix, a biotechnology
company working in the ubiquitin field.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2636-7.
Correspondence and requests for materials should be addressed to M.R.
Reprints and permissions information is available at http://www.nature.com/reprints.