Nature - USA (2020-10-15)

454 | Nature | Vol 586 | 15 October 2020

Article

caught by FBXL17, the amino-terminal β-strand folds back onto its own
C-terminal β-strand (Fig. 4c). As the intermolecular β-sheet must be
dismantled for FBXL17 to capture BTB monomers, differences in its
stability might allow substrate discrimination.
Indeed, mutations that disrupt the domain-swapped β-sheet strongly
promoted substrate recognition by FBXL17 (Fig. 4d), similar to deletion
of the amino-terminal β-strand^2. FBXL17 binding was also stimulated by
dimer-interface mutations, such as V50A in KLHL12 or F64A in KEAP1
(Fig. 1a, b, Extended Data Fig. 1a–d). Combining V50A in KLHL12 or
F64A in KEAP1 with mutations in the β-strand did not further enhance
substrate recognition by FBXL17 (Fig. 4d), suggesting that altering the
dimer interface displaces the domain-swapped β-strand. Conversely,
if we fused the amino terminus of KLHL12(V50A) to the C terminus of
another BTB subunit to lock the β-strand in its dimer position, substrate
recognition by FBXL17 was lost (Fig. 4e).
A structural model of KLHL12–KEAP1 heterodimers showed clashes
only at the domain-swapped β-sheet and at neighbouring residues of
the dimer interface (Fig. 4f). If we replaced clashing KLHL12 residues
with those of KEAP1 to anchor the domain-swapped β-strand, the het-
erodimer escaped capture by FBXL17 (Fig. 4g). Conversely, if we intro-
duced clashing KLHL12 residues into one subunit of KEAP1 homodimers
to release the β-strand, the resulting complexes were readily detected
by FBXL17 (Fig. 4h). When we transferred the same residues of KEAP1
into KLHL12, the chimeric KLHL12 formed heterodimers with KEAP1
in cells (Extended Data Fig. 9a), which were strongly impaired in their
association with targets of either KLHL12 or KEAP1 (Extended Data

Fig. 9b, c). Thus, the domain-swapped β-sheet of BTB domains guides

BTB dimerization and target selection by SCF–FBXL17. The β-sheet

residues are highly divergent across BTB domains, with not a single

β-strand being identical to another (Fig. 4i, Extended Data Fig. 10).

This raises the possibility that the N-terminal β-strand evolved rapidly

to constitute a molecular barcode for BTB dimerization that controls

access to SCF–FBXL17.

On the basis of these findings, we propose that BTB homodimers form

a robust domain-swapped β-sheet around their N-terminal β-strands

to escape capture by SCF–FBXL17 (Fig.  5 ). By contrast, heterodimers

or mutant dimers do not stabilize this β-sheet, which licenses them

for detection and further destabilization by SCF–FBXL17. Dimer dis-

sociation produces an unbound BTB subunit that can be immediately

captured by other SCF–FBXL17 molecules. We expect that SCF–FBXL17

also binds BTB monomers that emerge upon spontaneous dissociation

of heterodimers composed of distantly related BTB domains. SCF–

FBXL17 finally wraps around and ubiquitylates single BTB domains

that are similar in shape, but not necessarily in sequence. SCF–FBXL17

therefore selects its targets through a mechanism analogous to subunit

exchange^22 –^27.

By probing complementarity and shape of BTB domains, SCF–FBXL17

discriminates complexes independently of the nature of specific subu-

nits. Together with sequence variation accommodated by its large

substrate-binding surface, this enables SCF–FBXL17 to target hun-

dreds of heterodimers while ignoring the respective homodimers.

This approach could be extended to other interaction modules, such

a

b

e

d

C680

L684

T683

L677

W626

C574

C627

N572

A109

P105

K108

T112

V98

H96

M67 L70A63

0 100

KEAP1 binding (%)

WTW692AA687DL684DL684AN572A/C574A/L575AV676D/L677DT683DW626A/L677AD623A/G625D/W626

D

C574A/W626AΔCTHN572A/W626AN572A/C680DW626A/C680DC680D/T683D/L684D

FBXL17

mutant

c

W626A/L677AN572

A

W626

A

W692

A

C574A/W626

A

L684

D

C680DL677AD623A/G625D/W626DC680D/T683D/L684DΔCTHN572A/W626

A

N572A/C680DW626A/C680

D

FBXL17

mutant

+ FBXL17

+ FBXL17 +

dnCUL1

(^0100)
KEAP1 levels (%)
0
WTT112A/G114DP105D/K108DA63D/M67D/L70DS124KT112AL70D/K108DV98D/K108DH96D/V98DL70D/V98DA63D/M67D/L70D/H96D/V98DA63D/M67D/L70D/T112A/G114
D
G114 H96D/V98D/T112A/G114DH96D/V98D/P105K/K108DA63D/M67D/L70D/P105D/K108DL70D/V98D/K108D
D
100
KEAP1 binding (%)
KEAP1
mutant
KEAP1
mutant
P105DT112A/G114DM67DH96DK108DP105D/K108DV98DA63DH96D/V98DA63D/M67D/L70DL70DA63D/M67D/L70D/T112A/G114DH96D/V98D/P105K/K108DA63D/M67D/L70D/H96D/V98DA63D/M67D/L70D/P105D/K108DH96D/V98D/T112A/G114D
0 100
KEAP1 levels (%)

• FBXL17


• FBXL17 +
  dnCUL1
  Control
  f
  WT FBXL17
  ΔCTH
  C680D/T683D/L684D
  C574A/W626A
  N572A/W626A
  FBXL17CUL1SKP1CUL3KLHL9KLH
  L13
  KEAP1KLHL7KLHL
  12
  EN
  C2
  KLH
  L21
  ZBTB10KBTBD4KLHL2
  6
  KB
  TBD6
  KBTBD7NUDCD3KLHL20BA
  CH1
  KBTBD8KLHL
  17
  KLHL24ZBT
  B33
  ENC1KLHL2ZB
  TB
  5
  KLHL28ZBTB19ZB
  TB3
  KLHL1
  4
  ZBTB8KC
  TD5
  ABTB2KLHL2
  2
  ZB
  TB43
  BCL6ZBT
  B46
  BTBD7KCTD17
  Fig. 3 | Multiple surfaces of FBXL17 contribute to substrate binding.
  a, Detailed view of the interface between FBXL17 (orange) and the BTB domain
  of KEAP1(F64A) (blue). b, Combined mutation of FBXL17 residues in LRRs and
  CTH prevents recognition of haemagglutinin (HA)-tagged KEAP1 (HA–KEAP1),
  as shown by FBXL17–Flag affinity purification and quantitative western
  blotting with HA antibody. c, Combined mutations in FBXL17 interfere with
  proteasomal degradation of KEAP1, as monitored by quantitative western
  blotting. dnCUL1, dominant-negative CUL1. d, Mutations in FBXL17 prevent
  recognition of endogenous BTB proteins, as determined by affinity
  purification and mass spectrometry. The heat map shows total spectral counts
  normalized to FBXL17. e, Mutation of residues in HA–KEAP1 inhibits binding to
  FBXL17, as seen by FBXL17–Flag affinity purification and quantitative western
  blotting. f, Combined mutations in KEAP1 inhibit FBXL17-dependent
  degradation, as monitored by quantitative western blotting.