An Ankyrin-repeat ubiquitin binding domain
determines TRABID’s specificity for atypical ubiquitin
chains
Supplementary Material
Julien D.F. Licchesi1, Juliusz Mieszczanek1, Tycho E.T. Mevissen1, Trevor J.
Rutherford1, Masato Akutsu1, Satpal Virdee1, Farid El Oualid2, Jason W.
Chin1, Huib Ovaa2, Mariann Bienz1 and David Komander1*
1
Medical Research Council Laboratory of Molecular Biology, Hills Road,
Cambridge, CB2 0QH, UK.
2
Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands.
* Corresponding author: David Komander, dk@mrc-lmb.cam.ac.uk
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
1
Supplementary Figure 1
a
c
superposition
with Trabid
CCHFV vOTU
Ub in S1 site
of vOTU
b
αA0
αA1
αA2
αB0
HsTRABID
250
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
260
270
280
290
300
310
SKEELEVDFKKLKQIKNRMKKTDWLFLNACVGVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFD
TKEELEVDFKKLKQIKNRMKKTDWLFLNACVGVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFD
INNCDTLQERQERRQRQIRRQVDWQWLNACLGVVENNYSAVEAYLSCGGNPARSLTSTEIAALNRNSAFD
......................................................................
αB1
αB2
α0
α1
β1
β2
HsTRABID
320
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
330
340
350
360
370
VGYTLVHLAIRFQRQDMLAILLTEVSQQAA..KCIPAMVCPELTEQIRREIAASLHQRKGDFACYFLTD.
VGYTLVHLAIRFQRQDMLAILLTEVSQQAA..KCIPAMVCPELTEQIRREIAASLHQRKGDFACYFLTD.
VGHTLIHLAIRFHREEMLPMLLDQISGSGPGIKRVPSYVAPDLAADIRRHFANTLRLRKSGLPCHYVQK.
..........................MAEQVLPQALYLSNMRKAVKIRERTPEDIFKPTNGIIHHFKTMH
α1
α2
α1’
α3
β1
α3'
β2
β3
α4
HsTRABID
380
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
390
400
410
420
430
440
LVTFTLPADIEDLPPTVQEKLFDEVLDRDVQKELEEESPIINWSLELATRLDSRLYALWNRTAGDCLLDS
LVTFTLPADIEDLPPTVQEKLFDEVLDRDVQKELEEESPIINWSLELATRLDSRLYALWNRTAGDCLLDS
HATFALPAEIEELPIPIQEQLYDELLDRDAQKQLETPPPALNWSLEITARLSSRMFVLWNRSAGDCLLDS
RYTLEMFR.TCQFCPQFREIIHKALIDRNIQATLES.QKKLNWCREV.....RKLVALKTNGDGNCLMHA
α2
α3
β3
α5
α4
α6
α7
HsTRABID
450
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
460
470
480
490
500
510
VLQATWGIYDKDSVLRKALHDSLHD.CSHWFYTRWKDWESWYSQSFGLHFS...LREEQWQEDWAFILSL
VLQATWGIYDKDSVLRKALHDSLHD.CSHWFYTRWKDWESWYSQSFGLHFS...LREEQWQEDWAFILSL
AMQATWGVFDRDNILRRALADTLHQ.CGHVFFTRWKEYEMLQASMLHFT.....LEDSQFEEDWSTLLSL
TSQYMWGVQDTDLVLRKALFSTLKETDTRNFKFRWQLESLKSQEFVETGLCYDTR...NWNDEWDNLIKM
α4
α5
α6
α8
α7
β4
β5
η1
HsTRABID
520
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
530
540
550
560
570
AS.QPG........ASLEQTHIFVLAHILRRPIIVYGVKYYKSFR.GETLGYTRFQGVYLPLLWEQSFCW
AS.QPG........ASLEQTHIFVLAHILRRPIIVYGVKYYKSFR.GETLGYTRFQGVYLPLLWEQSFCW
AG.QPG........SSLEQLHIFALAHILRRPIIVYGVKYVKSFR.GEDIGYARFEGVYLPLFWDQNFCT
ASTDTPMARSGLQYNSLEEIHIFVLCNILRRPIIVISDKMLRSLESGSNFAPLKVGGIYLPLHWPAQECY
α8
β6
β4
β5
β7
β8
η1
α9
HsTRABID
580
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
590
600
610
620
630
640
KSPIALGYTRGHFSALVAMENDGYGNRGAGANLNTDDDVTITFLPLVDSE...RKLLHVHFLSAQELGNE
KSPIALGYTRGHFSALVAMENDGYGNRGAGANLNTDDDVTITFLPLVDSE...RKLLHVHFLSAQELGNE
KSPIALGYTRGHFSALVPMEPFTRIDG......RRDDVEDVTYLPLMDCE...LKLLPIHFLTQSEVGN.
RYPIVLGYDSHHFVPLVTLKDS...............GPEIRAVPLVNRDRGRFEDLKVHFLTDPENE..
β6
β7
α10
β8
β11
β12
β9
β10
α9
α11
HsTRABID
650
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
660
α10
β11
HsTRABID
700
HsTRABID
MmTRABID
DmTRABID
HsA20
HsA20
670
680
690
EQQEKLLREWLDCCVTEG.........GVLVAMQKSSRRRNHPLVTQMVEKWLDRYRQIRPCTSL.....
EQQEKLLREWLDCCVTEG.........GVLVAMQKSSRRRNHPLVTQMVEKWLDRYRQIRPCTSL.....
..EESMMRQWLDVCVTDG.........GLLVAQQKLS..KRPLLVAQMLEEWLNHYRRIAQVITAPFIRR
.MKEKLLKEYLMVIEIPVQGWDHGTTHLINAAKLDEA..NLPKEIN.LVDDYFELVQHEYKKWQE.....
...SDGEEDEDDEDE
...SDGEEDEDDEDE
PQITHYSSDGDSDEE
...NSEQGRRE....
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
β12
η2
α11
Supplementary Figure 1 Electron density, sequence and structure analysis.
(a) Stereo representation of experimental 2|Fo|-|Fc| electron density maps
(blue) after solvent flattening in SHARP 1, contoured at 1σ. Electron density
for the four gold atoms that were used to phase the structure in SIRAS
experiments is shown as yellow anomalous difference map contoured at 10σ.
The refined model of TRABID is shown as a ribbon, coloured in orange for the
AnkUBD and blue for the OTU domain. (b) Sequence alignment for the
crystallized constructs of human (Hs), mouse (Mm) and Drosophila
melanogaster (Dm) Trabid, and human A20 OTU domain. Boxed residues are
similar, and residues on a solid background are identical, and colours are blue
for the AnkUBD and red for the OTU domain. Secondary structure elements
are indicated and labeled according to Fig. 1c, above for TRABID and below
for A20. The sequence alignments were prepared with T-coffee
(http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi) and coloured
with ESPript (http://espript.ibcp.fr/ESPript/ESPript/). (c) Structure and
superposition of the Ub complex of Crimean Congo Hemorrhagic Fever Virus
(CCHFV) OTU domain (vOTU) (pdb-id 1phw, 2) and TRABID. The left image
shows the vOTU~Ub complex, with vOTU in green and Ub drawn as a yellow
surface. The right image shows a superposition, in the same orientation and
coloring as in Fig. 2d.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
Supplementary Figure 2
a AnkUBD NMR: 13C,15N labelled AnkUBD + unlabeled Ub
10
8
6
110
110
T314
F309
G312
F322
ω 1 - 15N (ppm)
T333
N268
S286
I330
L332
V316
D310
C270
D326
120
I320
R324
H317
A329
L318
S336 L331
Y313
L328
M327
F266
E282
D290
D277
V335
Q337
A319
R321
A308
Q325
A269
Q294
L295
E334
A292
L315
V311
130
V274
I281
K285
120
R293
I291
130
no ubiquitin
250 μM Ub
1 mM Ub
10
8
6
ω 2 - H (ppm)
1
b AnkUBD conservation
αA0
αA1
ENSP00000352676_Hsap_/245-340
ENSP00000352676_Hsap_/245-340
ENSTGUP00000011812_Tgut_/248-343
ENSGALP00000037851_Ggal_/248-343
ENSMODP00000012802_Mdom_/246-341
ENSMEUP00000003869_Meug_/245-340
ENSETEP00000014727_Etel_/245-340
ENSOCUP00000006113_Ocun_/113-208
ENSSTOP00000012708_Stri_/246-341
ENSLAFP00000012747_Lafr_/245-340
ENSMUSP00000101763_Mmus_/245-340
ENSPPYP00000003205_Ppyg_/245-340
ENSTTRP00000011999_Ttru_/245-340
ENSSSCP00000011448_Sscr_/245-340
ENSECAP00000015318_Ecab_/245-340
ENSCJAP00000023013_Cjac_/271-366
ENSMMUP00000029009_Mmul_/264-359
ENSCAFP00000018785_Cfam_/245-340
ENSMLUP00000000645_Mluc_/245-340
ENSBTAP00000004401_Btau_/245-340
ENSRNOP00000023257_Rnor_/245-340
ENSGGOP00000016444_Ggor_/245-340
ENSPVAP00000016914_Pvam_/245-340
ENSVPAP00000009918_Vpac_/245-340
ENSTSYP00000000113_Tsyr_/245-340
ENSCPOP00000007544_Cpor_/245-340
ENSPTRP00000005350_Ptro_/245-340
ENSOPRP00000014251_Opri_/244-336
ENSPCAP00000005868_Pcap_/245-340
ENSGACP00000012744_Gacu_/274-369
ENSTNIP00000000075_Tnig_/257-352
ENSORLP00000007119_Olat_/263-358
ENSTRUP00000003439_Trub_/292-388
ENSEEUP00000003089_Eeur_/202-297
ENSTRUP00000024457_Trub_/252-347
ENSXETP00000050986_Xtro_/238-333
ENSTNIP00000022624_Tnig_/251-346
ENSORLP00000012164_Olat_/239-331
ENSDARP00000079149_Drer_/226-321
ENSGACP00000003400_Gacu_/251-346
ENSDARP00000086546_Drer_/253-348
ENSACAP00000014249_Acar_/245-338
ENSCINP00000024265_Cint_/214-308
FBpp0081569_Dmel_/318-413
αA2
αB0
αB1
αB2
.
250
260
270
280
290
300
310
320
330
340
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAVEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQHAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAVEAYKSSGGDIARQLSADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQHAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQHAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQHAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAVEAYKSSGGDIARQLTADEVRLLNRPSAFDGGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKVKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLSACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLASIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLSACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAILLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDVGYTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLNACV.GVVEGDLAAVEAYKSSGGDIA.QLTADEMRLL.RPSAFDDGYTLVHLAI.LQRQDMLALLLTEVSQQAA
LEVDFKKLKQIKNRMKKTDWLFLSACM.GVVEGDLAAIEAYKSSGGDIARQLTADEVRLLNRPSAFDAGYTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKLKQIKNRMRRTDWLFLNACV.GVVEGDLAAVEAYKSSGGDIARQLTSDEVRLLNRPSAFDDGFTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKLKQIKNRMRRTDWLFLNACV.AVVEGDLAAVEAYKSSGGDIARQLTADEVRLLNRPSAFDDGFTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKLKQIKNRMRRTDWLFLNACV.GVVEGDLGAVEAYKSSGGDIARQLTSDEVRLLNRPSAFDDGFTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKLKQIKNRMRRTDWLFLNACVEGVVEGDLAAVEAYKSSGGDIARQLSSDEVRLLNRPSAFDDGFTLVHLAIRFQRQDMLAVLLTEVSQQAA
LEVDFKKLKQIKNRMKKPDWLFLNACV.GVVEGDLAAIEAYKASGGDIARQLSADEAHLLNRPSAFDVGYTLVHLAIRFQRQDMLAVLLTEVSQQAA
QEVDFKKLKQIRNRMKKTDWLFLNACA.GVVEGDLAAVEAYKSSGGDIARQLTADEVQLLNRSSAFDAGYTLVHLSIRFQRQDMLAILLTEVSQQAA
FELDLKKLKQIKNRMRKTDWLFLNACV.GVVEGDLSAVEAYKTSGGDIARQLSADEVRLLNRPSAFDVGYTLVHLSIRFQRQDMLAILLTEVSQHAA
QEVDFKKLKQIRNRMKKIDWLFLNACA.GVVEGDLAAVEAYKSSGGDIARQLTADEVQLLNRSSAFDAGYTLVHLSIRFQRQDMLAILLTEVSQQAA
QEVDFKKLKQIRNRMRKTDWLFLSACA.GVVEGDLAAVEAYKSSDGDIARQLTADEVQLLNRSSAFDVGFTLVHLAIRFQRQDMLAILLTEVS...L
EEMDFKKIKQIKNRMRKTDWLFLNACA.GVVEGDLSAVEAYKSSGGDIARQLNADEVRLLNRPSAFDSGFTLVHLAIRFQRQDMLAILLTEVSQRAV
QDVDFKKLKQIRNRMRKTDWLFLNACA.GVVEGDLAAIEAYKASGGDIARQLTADEVQLLSRSSAFDVGFTLVHLAIRFQRQDMLAILLTEVNQQAA
QEADFKKLKQIRNRMRRSDWLFLNACA.GVVEGDLAAVEAYKSSGGDIARQLTADEVRILNRPSAFDAGFTLVHLAIRFQRQDMLAVLLTEVSQQTA
LEVDFKKLKQIKNRMKKTDWLFLNACI.GKFFGDLAAIEYKIIREGDIARQLTADES.TLNRP.AFDVGYTLVHLAIRFQRQDMLAVLLTEVSQHAA
SRLEILDNRSMKTRSRNQNWLFLKACI.GVVEENAEAVDAYLANGGNIARTLTLDEVNLLNRPSAFDVGHTLVHLAIRFQRHGILALLLNPEATH..
DTLQERQERRQRQIRRQVDWQWLNACL.GVVENNYSAVEAYLSCGGNPARSLTSTEIAALNRNSAFDVGHTLIHLAIRFHREEMLPMLLDQISGSGP
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
Supplementary Figure 2 AnkUBD NMR spectra and AnkUBD conservatio.
(a) 1H,15N-HSQC spectra for 13C,15N-labeled AnkUBD in absence (blue) or
presence of 250 µM (yellow) or 1 mM (red) unlabeled Ub, as in Fig. 3b. A full
assignment of all peaks from 3D experiments is shown in Fig. 3a. Here, only
significantly perturbed residues are labeled, and arrows indicate perturbation
path upon increasing Ub concentration. A weighted chemical shift map is
shown in Fig. 3c. (b) Species alignment of the AnkUBD from TRABID
containing species annotated in the Ensembl database
(http://www.ensembl.org/index.html). Secondary structure elements are
indicated and labeled. Invariant residues (blue background) in this alignment
are colored red in Fig. 3f.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
a
Supplementary Figure 3 (1)
AnkUBD wild type
9
100
8
7
6
100
A46
G47
S20
105
105
T9
T55
G35
T22
G10
110
110
ω 1 - 15N (ppm)
G75
D39 S57
E64
E34
115
G76
T7
V17
V5
S65
Q40
L56
Q41
D32
125
V70
I61
R54
K63 D52
K29
T12
I23
I30
N25
T14
R74
V26
R42
K48
Q2
E51 E16 L71
Q49
D21
L73 A28
L43 R72
Q31
L69
F45
D58
L15
L50
L8
I44
115
K33 Y59
N60
T66
F4
E18 K27
H68
120
I3
120
I36
K11
535 μM
Q62
125
400 μM
250 μM
I13
K6
L67
100 μM
no AnkUBD
130
9
AnkUBD H317A
9
100
7
6
8
7
6
100
A46
G47
S20
105
105
T9
T55
G35
T22
G10
110
110
G75
ω 1 - 15N (ppm)
b
8
ω 2 - 1H (ppm)
130
D39 S57
E64
E34
115
G76
T7
V17
I3
S65
T66
Q40
L56
F4
E18 K27
H68
D32
K63 D52
K29
T12
V5
I23
I30
L8
N25
R74
T14
V26
K48
I44 Q2
Q49 R42 E51 E16 L71
D21
L43 R72 Q31
L73 A28
L69
F45
D58
L15
L50
V70
120
125
115
K33 Y59
N60
Q41
I61
R54
120
I36
K11
370 μM
Q62
125
250 μM
150 μM
I13
K6
L67
75 μM
no AnkUBD
130
9
8
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
ω 2 - 1H (ppm)
7
6
130
c
AnkUBD I320D
Supplementary Figure 3 (2)
9
100
8
7
6
100
A46
G47
S20
105
105
T9
T55
G35
T22
G10
110
110
ω 1 - 15N
(ppm)
G75
D39 S57
E64
E34
115
G76
T7
I3
S65
T66
V17
Q40
L56
F4
E18 K27
H68
D32
K63 D52
K29
T12
V5
I23
I30
L8
N25
R74
T14
V26
K48
I44 Q2
Q49 R42 E51 E16 L71
D21
L43 R72 Q31
L73 A28
L69
D58
F45
L15
L50
V70
120
125
115
K33 Y59
N60
Q41
I61
R54
120
I36
K11
125
Q62
I13
K6
L67
250 μM
130
no AnkUBD
9
d
AnkUBD L332E
8
ω 2 - 1H
9
100
7
6
7
6
130
(ppm)
8
100
A46
G47
S20
105
105
T9
T55
G35
T22
G10
110
110
ω 1 - 15N
(ppm)
G75
D39 S57
E64
E34
115
G76
T7
V17
I3
S65
T66
Q40
L56
F4
E18 K27
H68
D32
K63 D52
K29
T12
V5
I23
I30
L8
N25
R74
T14
V26
K48
I44 Q2
Q49 R42 E51 E16 L71
D21
L43 R72 Q31
L73 A28
L69
D58
F45
L15
L50
V70
120
125
115
K33 Y59
N60
Q41
I61
R54
120
I36
K11
125
Q62
I13
K6
L67
422 μM
250 μM
130
no AnkUBD
9
7
8
ω2 -
1H
(ppm)
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
6
130
Supplementary Figure 3 Titration experiments of labeled Ub with unlabeled
AnkUBD variants.. 1H,15N-HSQC spectra of 15N-labeled ubiquitin in absence
(yellow) or presence of increasing concentrations of (a) unlabeled AnkUBD
and (b-d) AnkUBD mutants, colored according to the key in the right corner of
the image. The resulting chemical shift perturbations were quantified and are
shown in Fig. 4.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
Supplementary Figure 4
a
339
OTU
Lys6
697
Lys11
Lys27
Lys29
Lys33
Lys48
Lys63
Linear
M 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 min
OTU
(339-697)
DiUb
MonoUb
[E] 0.25 μM
b
AnkOTU
AnkOTU
OTU
H317A
I320A
I320D
L332A
L332E
M INP 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 min
Lys29 Ub2
Ub
[E] 0.1 μM
AnkOTU
AnkOTU
OTU
H317A
I320A
I320D
L332A
L332E
M INP 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 min
Lys33 Ub2
Ub
[E] 0.1 μM
AnkOTU
AnkOTU
OTU
H317A
I320A
I320D
L332A
L332E
M INP 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 min
AnkOTU
OTU
Lys63 Ub2
Ub
[E] 1 μM
Supplementary Figure 4 Additional in vitro DUB assays. (a) The TRABID OTU domain
is less efficient compared to AnkOTU at similar concentration, and is hardly active at
an enzyme concentration of [0.2 µg] in contrast to AnkOTU at the same concentration
(see Fig. 1b, 5a). (b) Catalytic activity of bacterially produced Trabid variants against
Lys29- (top), Lys33- (middle), and Lys63-linked (bottom) diUb. Lys63-cleavage was
assayed at higher enzyme concentration of 1 µM. Enzyme concentrations are indicated.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
Supplementary Figure 5
a
GFP-Cezanne C194S DAPI
Merge
b
+DMSO
+DMSO
+MG132
wt Ub
K29only Ub
K6only Ub
K33only Ub
K11only Ub
K48only Ub
K27only Ub
K63only Ub
c
GFP FL C443S
FLAG-Ub wt
(α-Flag)
+MG132
Merge
+MG132
d
HA FL C443S
GFP FL ∆Ank C443S
DAPI
Merge
Supplementary Figure 5 Localization of Cezanne and co-localization of
TRABID variants. (a) GFP-tagged full-length Cezanne C194S was expressed
in COS-7 cells, and visualized (left image) alongside with the cell nucleus
(DAPI stain, middle image). The right image shows a merged picture.
Inactivated Cezanne does not form a punctate pattern like TRABID. (b)
Selected cells from co-transfection experiments with FLAG-Ub variants and
GFP-C443S, which have only been transfected with FLAG-Ub but not with
GFP-FL C443S are shown, in absence (as in Fig. 7e) or presence of
proteasome inhibitor MG132. Red color indicated the Cy3-labeled anti-HA
staining, while blue indicates a DAPI staining showing the cell nucleus. All Ub
variants express and are diffusely distributed across the cytoplasm, and no
significant changes are seen upon proteasome inhibition. Different intensities
result from different overexpression levels. (c) Co-localization of GFP-TRABID
FL C443S with FLAG-Ub wt, performed as in Fig. 7e but in presence of
MG132. (d) Co-localization of HA-TRABID FL C443S (left) with GFP-FL ΔAnk
C443S (second from left). A DAPI stain reveals nuclei (second from right),
and the merged image is shown to the right. The localization of the HA-tag
was performed as for the FLAG-tag, using a rat anti-HA antibody (Roche) and
a Cy3® goat anti-rat IgG (Invitrogen).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
Supplementary Figure 6
a
FL C443S (GFP)
FLAG-Ub (α-FLAG)
K6only
Merge
K27only
K48only
b
FL C443S (GFP)
DAPI
Merge
AnkOTU (low)
AnkOTU (high)
OTU
Supplementary Figure 6 Colocalization of GFP-C443S with ubiquitin mutants and
in vivo DUB assay. (a) Puncta-forming GFP-C443S (left image) was co-expressed
with FLAG-Ub single-Lys (Konly) mutants K6, K27 or K48 (middle images). The
merged image is shown to the right. (b) Dissolving TRABID assemblies requires
the AnkUBD. Puncta-forming GFP-tagged full-length TRABID C443S (left) was
co-transfected with either Flag-AnkOTU or Flag-OTU in COS-7 cells and the
presence of GFP puncta was assessed. Note that two cells are shown for
Flag-AnkOTU corresponding to either low or high level of Flag-AnkOTU expression.
Nuclei are stained using DAPI and the merged image is shown to the right.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
Supplementary Methods
Cloning and mutagenesis
For bacterial expression, TRABID AnkOTU (245-697) and Ank (245-339)
constructs were cloned into the pOPIN-K vector, that comprises an N-terminal
PreScission-cleavable GST tag, using the Infusion 2.0 Technology (Clontech).
Point mutants were generated by site directed mutagenesis using the
QuikChange® technology (Stratagene). For mammalian expression, fulllength TRABID (2-708), full-length TRABID NZF mutant 3, TRABID AnkOTU
(245-708), TRABID OTU (339-708), full-length TRABID ΔAnk (Δ249-338),
were subcloned into pCMV-3xFLAG (Stratagene) using EcoR1/Xho1 sites.
For immunofluorescence, full-length TRABID (2-708), TRABID AnkOTU (245708), TRABID OTU (339-708) were subcloned into pEGFP-C1 using
Xho1/EcoR1 sites. GFP-tagged TRABID constructs 2-200, 2-345, FL-TRABID
ΔAnk (Δ249-338), TRABID Ank (245-345) as well as all other Ank mutants
used in this study were obtained by using the QuikChange® technology.
pEGFP-Cezanne wt 4 was used to derive pEGFP-Cezanne C194S.
Protein purification for biochemical, biophysical and structural studies
All protein purifications were performed at 4°C. TRABID AnkOTU (245-697)
and OTU (339-697) constructs were expressed in Arctic Xpress cells
(Stratagene). Cells were induced at an OD600 of 0.6-0.8 with 150 µM IPTG
and grown overnight at 16 °C. Cells from 6 L culture were lysed by sonication
in 50 ml lysis buffer (270 mM sucrose, 50 mM Tris [pH 8.0], 1 mM EDTA, 1
mM EGTA, 10 mM sodium β-glycerophosphate, 50 mM sodium fluoride, 10
mM β-mercaptoethanol, 1 mM benzamidine, 0.1 mg mL-1 DNAse 1, 1 mg mL-1
lysozyme), and cleared by centrifugation. The cleared lysate was incubated
with 4 ml equilibrated glutathione-S-sepharose 4B resin (GE Healthcare) for 1
h, and subsequently washed with 50 ml lysis buffer, 500 ml buffer A (25 mM
Tris [pH 8.5], 1 mM EDTA, 5 mM DTT) plus 500 mM NaCl, and 500 ml buffer
A plus 150 mM NaCl. The GST-tag was cleaved on the resin with 50 µg GSTtagged PreScission protease overnight. Cleaved protein was diluted to ~50
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
2
mM NaCl with 25 mM Tris (pH 8.5), and subjected to anion exchange
chromatography (MonoQ 5/50, GE Healthcare) where it eluted as a single
peak in a NaCl gradient from 50 to 500 mM.
For crystallography, peak fractions were pooled and subjected to gel filtration
(Superdex75) in buffer B (200 mM NaCl, 25 mM Tris [pH 8.2], 1 mM EDTA, 5
mM DTT). The protein was concentrated to 3.5 mg ml-1 using a VivaSpin 10
kDa MW cut-off concentrator and used in crystallization screening.
TRABID Ank domain constructs were expressed in Rosetta2 pLysS cells and
were lysed and incubated with glutathione sepharose as described above.
Crystallisation, data collection, phasing and refinement
The TRABID AnkOTU structure was determined from crystals grown at 23°C
from 150 mM NaCl, 100 mM NaOAc, 5 mM MgCl2, 50 mM MES [pH 5.9]. For
synchrotron data collection, crystals were soaked in mother liquor containing
27.5 % ethylene glycol, and frozen in liquid nitrogen. To obtain phase
information, crystals were soaked in 1 mM KAu(CN)2 for 1 h prior to freezing.
Diffraction data on the AnkOTU crystals were collected at the ESRF
(Grenoble), beamline ID23-2. Crystals displayed space group P212121 with
one molecule of AnkOTU in the asymmetric unit. A native dataset, collected to
2.25 Å resolution, and a peak wavelength SAD dataset at 3 Å resolution from
AuCN soaked crystals were used for phasing, using single isomorphous
replacement with anomalous scattering (SIRAS) An initial set of sites was
obtained with the SHELX/hkl2map suite 5, and site refinement was performed
in SHARP 1. Density modification within SHARP resulted in a high-quality
map, which was interpreted by WarpNTrace and manually rebuilt in Coot 6.
Refinement was performed using PHENIX 7, including simulated annealing
and TLS B-factor refinement. Final statistics can be found in Table 1.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
3
NMR titration analysis
For titration experiments, HSQC spectra were recorded for 50 µM TRABID
alone, and in presence of 250 µM and 1 mM unlabeled Ub. For the reverse
experiment, spectra were recorded for 50 µM of 15N-labelled Ub alone and in
complex with varying concentrations of unlabeled TRABID Ank domain either
wild-type, or bearing point mutations (H317A, I320D, L332E). Separate
samples were prepared for each measurement, such that no adjustment was
required to account for dilution with increasing volume. For chemical shift
mapping, weighted chemical shift perturbations were measured in 15N fastHSQC experiments 8 and defined as D1H + (D15N/5) [ppm] 9. To obtain
approximate KD values by NMR, peak frequencies (either 1H or 15N,
whichever varied greatest in the titration) for nine representative correlations
were plotted as a function of the concentration of unlabeled protein at four
different concentrations, and fitted to a quadratic expression for binding
equilibria, as described
(http://structbio.vanderbilt.edu/chazin/wisdom/kdcalc.htm). KD values were
obtained by least squares fitting of the experimental data to trial curves
varying in the simulated KD. Quoted KD values are the median of values
obtained for the nine peaks.
Photobleaching experiments
FRAP (fluorescence recovery after photobleaching) experiments in COS-7
were carried out as described previously 10. Briefly, cells were seeded onto
Lab-Tek II chambered cover-slides (Nalgene Nunc International, Rochester,
NY, USA) 24 h before transfection. Next, 100 ng of GFP-TRABID constructs
were transfected using lipofectamine, and photobleaching was conducted 18
h post transfection. GFP puncta were bleached with five maximum-intensity
scans with the 488 and 514 nm lines of a 40 mW argon laser (Zeiss
AXIOVERT 200M inverted confocal microscope). Fluorescence recovery was
monitored in images taken during the following 60 seconds. The fluorescence
intensity was normalized to the mean fluorescence intensity of the whole cell.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2169
4
Supplementary References
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