Nature - USA (2020-10-15)

Nature | Vol 586 | 15 October 2020 | 455

KEAP1

a

F139

I145S144 A143

Y54 L56

F64

A17^0

KEAP1mutant

KEAP1

KEAP1

MBP–FBXL17

Coomassie

Autoradiography

Autoradiography

PD: MBP

Input

WTY54D/L56

D

F139DA143D/S144D/I145DS166DA170DF64AF64A

/Y54D

/L56D

F64A/F139

D

F64A

/A143D/S14

4D/I145D

F64A

/S166

D

F64A/A170D

d

N-terminal

β-strand N-terminal

β-strand

KEAP1

BTB1

KEAP1

BTB1

KEAP1

BTB2 FBXL17

Conservation

Most

Least

MBP–FBXL17

Coomassie

PD: MBP

Input

KLHL12–KEAP1

KLHL12–KEAP1I19AL20FI19A/L20FI49V

Mutant KLHL12–KEAP1 heterodimer

N-swapI19A/L20F +

N-swa

p

KEAP1

–KEAP

1

Autoradiography

KLHL12–KEAP1

Autoradiography

KEAP1–KEAP1

Autoradiography

Autoradiography

Coomassie

MBP–FBXL17

KEAP1–KEAP1

PD: MBP

Input

KEAP1–KEAP1KLH

L12–KEAP

1

A63I/F64LN-swa

p

A63I/F64L

+ N-swap

g

h

i

BTB BTB BTB BTB

Wild type β-strand mutant

BTB1 BTB2 BTB2

Heterodimer I19A/L20F + N-swap

BTB BTB

Homodimer

BTB BTB

A63I/F64L + N-swap

β-strand

BTB1

KLHL12

N-swap

I19A/L20F

BTB BTB

AF 55514 ÅAF 647

FRET

FBXL17

No FRET:

donor uoresence up

FBXL17

0

5

10

15

Incr

ease in donor

uor

escence (%)

20

25

FBXL17(

ΔCTH)

Overnight c

f

BTB

FBXL1

7

e

Autoradiography

Coomassie

MBP–FBXL17

SKP1

BTB–BTB

BTB

FLAG–BTB (cut)

BTB (cut)

++++++WT KLHL12

++++++KLHL12(V50A)

Unfused Fused Fusedand cut

Unfused Fused Fused

and cut

Input PD: MBP

BTB BTB

Unfused

BTB BTB

Fused

BTB BTB

Fused and cut

0

10

20

30

Change in donoruor

escence (AU)

5255075100 120

Time (min)

FBXL17

FBXL17(ΔCTH)

MBP

KEAP1(F64A)

KEAP1(V98A)GroEL

Sinks (BTB–GroEL)

Controls (MBP;

FBXL17–ΔCTH)

KEAP1(F64A)

b

Interacting

residues

Dimer interface

S166

–5

Fig. 4 | A domain-swapped β-sheet in BTB domains controls access to
F BX L 1 7. a, FRET assay. KEAP1(F64A) BTB dimers labelled with distinct
f luorophores dissociate, as shown by loss of donor f luorescence quenching,
upon incubation with FBXL17. Inactive FBXL17(ΔCTH), excess unlabelled BTB
domains (KEAP1(F64A) and KEAP1(V98A)), GroEL or maltose-binding protein
(MBP) did not have strong effects. Dissociation by FBXL17 was measured three
times. AF 555, Alexa Fluor 555; AF 647, Alexa Fluor 647; AU, arbitrary units.
b, Overnight incubation of FRET-labelled KEAP1(F64A) BTB dimers with
FBXL17, FBXL17(ΔCTH) or excess unlabelled KEAP1(F64A) BTB domain.
Dissociation was measured two or three times. c, The N-terminal β-strand of
the KEAP1 BTB domain forms an intermolecular sheet in dimers, but adopts an
intramolecular conformation in the BTB monomer bound to FBXL17. d, Binding
of^35 S-labelled KEAP1 variants with mutations in the domain-swapped β-sheet
to MBP–FBXL17. This experiment was performed once. e,^35 S-labelled unfused

BTB domains of KLHL12(V50A), fused BTB domains of KLHL12(V50A) or fused

BTB domains that were cut within the linker were bound to MBP–FBXL17. Two

independent experiments were performed with similar results. f, Structural

model of a KLHL12–KEAP1 heterodimer shows clashes at the dimerization

helix region and N-terminal β-strand. g,^35 S-labelled KLHL12–KEAP1 BTB

heterodimers, mutant heterodimers and KEAP1 BTB homodimers were bound

to MBP–FBXL17. Two independent experiments were performed with similar

results. h,^35 S-labelled homodimeric KEAP1 (green), mutants with helix

and β-strand residues of KLHL12 placed into the first subunit of a KEAP1

homodimer (blue), or KLHL12–KEAP1 heterodimers were incubated with

MBP–FBXL17 and analysed for binding by gel electrophoresis and

autoradiography. Two independent experiments were performed with similar

results. i, The N-terminal β-strand and its interaction residues in the partner

BTB domain evolve rapidly (blue, high conservation; red, no conservation).

BTBBTB

BTB1 BTB2

FBXL17

Ub

Homodimer

Stable heterodimer,
mutant dimer

Unstable

heterodimer

BTB1BTB2

Ub

FBXL17

BTB

FBXL17

BTB

Fig. 5 | Model of the DQC mechanism. BTB

homodimers have identical N-terminal β-strands

mostly in the domain-swapped position.

This prevents FBXL17 from engaging and

ubiquitylating the homodimer. BTB heterodimers

or mutant BTB dimers have poorly compatible

helices and β-strands. Their N-terminal β-strands

are mostly displaced, enabling capture of these

aberrant dimers by FBXL17. FBXL17 may further

destabilize these dimers or rely on spontaneous

dimer dissociation to associate with a monomeric

BTB subunit for ubiquitylation and degradation.

Ub, ubiquitin.