nat. struct. mol. biol. 19, 62–71 (2012) an ankyrin-repeat ... · pdf filein this file...

nature structural amp molecular biology

Nat Struct Mol Biol 19 62ndash71 (2012)

An ankyrin-repeat ubiquitin-binding domain determines TRABIDrsquos specificity for atypical ubiquitin chainsJulien D F Licchesi Juliusz Mieszczanek Tycho E T Mevissen Trevor J Rutherford Masato Akutsu Satpal Virdee Farid El Oualid Jason W Chin Huib Ovaa Mariann Bienz amp David Komander

In the version of Supplementary Figure 5b originally posted online three images were incorrect those for DMSO K27only Ub MG132 K27only Ub and MG132 K29only Ub The correct figure is now shown The conclusions from this figure remain unchanged These errors have been corrected in this file as of 29 February 2012

C O R R E C T I O N N OT I C E

13 1

An Ankyrin-repeat ubiquitin binding domain determines TRABIDrsquos specificity for atypical ubiquitin

chains

Supplementary Material

Julien DF Licchesi1 Juliusz Mieszczanek1 Tycho ET 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 dkmrc-lmbcamacuk

Nature Structural amp Molecular Biology doi101038nsmb2169

HsTRABID

HsTRABIDMmTRABIDDmTRABIDHsA20

HsA20

HsTRABID 320 330 340 350 360 370

HsTRABID IR I L F T VGYTLVHLAIRFQRQDMLAILLTEVSQQAAKC PAMVCPELTEQ REIAAS HQRKGDFACY L DMmTRABID IR I L F T VGYTLVHLAIRFQRQDMLAILLTEVSQQAAKC PAMVCPELTEQ REIAAS HQRKGDFACY L DDmTRABID IR V L Y Q VGHTLIHLAIRFHREEMLPMLLDQISGSGPGIKR PSYVAPDLAAD RHFANT RLRKSGLPCH V KHsA20 IR A I F T MAEQVLPQ LYLSNMRKAVK ERTPED FKPTNGIIHH K MH

HsA20

HsTRABID380 390 400 410 420 430 440

HsTRABID T E DR Q LE NW E L G CL L L VL V I L RL A N L LV FT PADIEDLPPTVQ K FDE D KE EESPI SL ATRLDS Y W RTA D DSMmTRABID T E DR Q LE NW E L G CL L L VL V I L RL A N L LV FT PADIEDLPPTVQ K FDE D KE EESPI SL ATRLDS Y W RTA D DSDmTRABID T E DR Q LE NW E L G CL L L LL A L I RM V N L HA FA PAEIEELPIPIQ Q YDE D KQ TPPPA SL TARLSS F W RSA D DSHsA20 T E DR Q LE NW E L G CL M I LI I L V KL A T M RY LE FRTCQFCPQFR I HKA N AT SQKK CR R V K NGD N HA

HsA20

HsTRABID450 460 470 480 490 500 510

HsTRABID Q WG D D LR AL L F RW W I V K S H H QW ED IL LVL AT Y K S HD DCS W YT KDWESWYSQSFGLHFSLREE Q AF S MmTRABID Q WG D D LR AL L F RW W I V K S H H QW ED IL LVL AT Y K S HD DCS W YT KDWESWYSQSFGLHFSLREE Q AF S DmTRABID Q WG D D LR AL L F RW W V I R T H H QF ED LL LAM AT F R N AD QCG V FT KEYEMLQASMLHFTLEDS E ST S HsA20 Q WG D D LR AL L F RW W V V K T K R NW DE LI MTS YM Q T L FS ETDT N KF QLESLKSQEFVETGLCYDTR N DN K

HsA20

HsTRABID 520 530 540 550 560 570

HsTRABID A SLE HIF L ILRRPIIV K S G G YLPL W C G V K R V S SQP A QT AH YGV YY FR ETLGYT FQ L EQ F WMmTRABID A SLE HIF L ILRRPIIV K S G G YLPL W C G V K R V S SQP A QT AH YGV YY FR ETLGYT FQ L EQ F WDmTRABID A SLE HIF L ILRRPIIV K S G G YLPL W C G A K R V N GQP S QL AH YGV YV FR EDIGYA FE F DQ F THsA20 A SLE HIF L ILRRPIIV K S G G YLPL W C P V R K I Q STDT MARSGLQYN EI CN ISD ML LES SNFAPL VG H PA E Y

HsA20

HsTRABID 580 590 600 610 620 630 640

HsTRABID PI LGY HF LV PL L HFL E K A M I L V E V S S TRG SA A ENDGYGNRGAGANLNTDDDVT TF DS RKL H AQ LGNEMmTRABID PI LGY HF LV PL L HFL E K A M I L V E V S S TRG SA A ENDGYGNRGAGANLNTDDDVT TF DS RKL H AQ LGNEDmTRABID PI LGY HF LV PL L HFL E K A M V L M E I T S TRG SA P EPFTRIDGRRDDVED TY DC LKL P QS VGNHsA20 PI LGY HF LV PL L HFL E R V L I V V D V T Y DSH VP T KDSGPE RA NR RGRFED K DP NE

HsA20

HsTRABID 650 660 670 680 690

HsTRABID E L A LLR W V V V MVE W S EQQ K E DCCVTEGG L MQKSSRRRNHPL TQ K LDRYRQIRPCT LMmTRABID E L A LLR W V V V MVE W S EQQ K E DCCVTEGG L MQKSSRRRNHPL TQ K LDRYRQIRPCT LDmTRABID E L A MMR W L V V MLE W T E S Q DVCVTDGG L QQKLSKRPLL AQ E LNHYRRIAQVI APFIRRHsA20 E L A LLK Y I A I LVD Y Q MK K E MVIEIPVQGWDHGTTHL N KLDEANLPKE N D FELVQHEYKKW E

HsA20

HsTRABID 700

HsTRABID S D DGEEDE DEDE MmTRABID S D DGEEDE DEDE DmTRABID T D PQI HYSSDG SDEE HsA20 N E SEQGRR

HsA20

αA0 αA1 αA2 αB0

αB1 αB2 α0 α1 β1 β2

α2 α3 α3 β3 α4

α5 α6 α7

α8 β4 β5 η1

β6 β7 β8 α9

α10 β11 β12 α11

α1 β1 β2

α2 α3 β3

α4 α5 α6 α7

α8 β4 β5 η1

β6 β7 β8 β9 β10 α9

α10 β11 β12 η2 α11

250 260 270 280 290 300 310

DW LNAC GVVE A EAY S GG AR LT E LNR SAFD V K K KN K F V I Q V L SKEELE DF KL QI RM KT L GDLA K S DI AD R P DW LNAC GVVE A EAY S GG AR LT E LNR SAFD V K K KN K F V I Q V L TKEELE DF KL QI RM KT L GDLA K S DI AD R P DW LNAC GVVE A EAY S GG AR LT E LNR SAFD L R R RQ R W L V S I A INNCDT QE QE RQ IR QV Q NNYS L C NP ST A N

VG TL HLAIRF R ML LL S K VG TL HLAIRF R ML LL S K VG TL HLAIRF R ML LL S K

α4

α1rsquo

Supplementary Figure 1

a

b

c

CCHFV vOTU

Ub in S1 site of vOTU

superposition with Trabid


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

(httptcoffeevital-itchcgi-binTcoffeetcoffee_cgiindexcgi) and coloured

with ESPript (httpespriptibcpfrESPriptESPript) (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



ENSP00000352676_Hsap_245-340 250 260 270 280 290 300 310 320 330 340

ENSP00000352676_Hsap_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSTGUP00000011812_Tgut_248-343 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V VE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQHAAENSGALP00000037851_Ggal_248-343 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V VE Q S V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQHAAENSMODP00000012802_Mdom_246-341 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQHAAENSMEUP00000003869_Meug_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQHAAENSETEP00000014727_Etel_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V VE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS G Y A RFQ QD A TEVSQQAAENSOCUP00000006113_Ocun_113-208 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSSTOP00000012708_Stri_246-341 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSLAFP00000012747_Lafr_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSMUSP00000101763_Mmus_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSPPYP00000003205_Ppyg_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSTTRP00000011999_Ttru_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSSSCP00000011448_Sscr_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSECAP00000015318_Ecab_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSCJAP00000023013_Cjac_271-366 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSMMUP00000029009_Mmul_264-359 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSCAFP00000018785_Cfam_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSMLUP00000000645_Mluc_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSBTAP00000004401_Btau_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSRNOP00000023257_Rnor_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSGGOP00000016444_Ggor_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M V LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSPVAP00000016914_Pvam_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKV QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSVPAP00000009918_Vpac_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L S GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSTSYP00000000113_Tsyr_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L N GVVEGDLAS AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSCPOP00000007544_Cpor_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M I LE DFKKL QI RM KTD L S GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSPTRP00000005350_Ptro_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q T V M V LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VRL N PS V Y A RFQ QD A TEVSQQAAENSOPRP00000014251_Opri_244-336 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V VE Q T V M L LE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI AD MRL PS D Y A LQ QD A TEVSQQAAENSPCAP00000005868_Pcap_245-340 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F M IE Q T V M V LE DFKKL QI RM KTD L S GVVEGDLAA AYKSSG DI R AD VRL N PS A Y A RFQ QD A TEVSQQAAENSGACP00000012744_Gacu_274-369 W L AC G A L E L R AFD G TL HL I R L LL V K KN R F V VE Q T V M V LE DFKKL QI RM RTD L N GVVEGDLAA AYKSSG DI R SD VRL N PS D F A RFQ QD A TEVSQQAAENSTNIP00000000075_Tnig_257-352 W L AC G A L E L R AFD G TL HL I R L LL V K KN R F V VE Q T V M V LE DFKKL QI RM RTD L N AVVEGDLAA AYKSSG DI R AD VRL N PS D F A RFQ QD A TEVSQQAAENSORLP00000007119_Olat_263-358 W L AC G A L E L R AFD G TL HL I R L LL V K KN R F V VE Q T V M V LE DFKKL QI RM RTD L N GVVEGDLGA AYKSSG DI R SD VRL N PS D F A RFQ QD A TEVSQQAAENSTRUP00000003439_Trub_292-388 W L AC G A L E L R AFD G TL HL I R L LL V K KN R F V VE Q S V M V LE DFKKL QI RM RTD L N EGVVEGDLAA AYKSSG DI R SD VRL N PS D F A RFQ QD A TEVSQQAAENSEEUP00000003089_Eeur_202-297 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F V IE Q S V M V LE DFKKL QI RM KPD L N GVVEGDLAA AYKASG DI R AD AHL N PS V Y A RFQ QD A TEVSQQAAENSTRUP00000024457_Trub_252-347 W L AC G A L E L R AFD G TL HL I R L LL V K RN K F A VE Q T V M I QE DFKKL QI RM KTD L N GVVEGDLAA AYKSSG DI R AD VQL N SS A Y S RFQ QD A TEVSQQAAENSXETP00000050986_Xtro_238-333 W L AC G A L E L R AFD G TL HL I R L LL L K KN R F V VE Q S V M I FE DLKKL QI RM KTD L N GVVEGDLSA AYKTSG DI R AD VRL N PS V Y S RFQ QD A TEVSQHAAENSTNIP00000022624_Tnig_251-346 W L AC G A L E L R AFD G TL HL I R L LL V K RN K F A VE Q T V M I QE DFKKL QI RM KID L N GVVEGDLAA AYKSSG DI R AD VQL N SS A Y S RFQ QD A TEVSQQAAENSORLP00000012164_Olat_239-331 W L AC G A L E L R AFD G TL HL I R L LL V K RN R F A VE Q T V M I QE DFKKL QI RM KTD L S GVVEGDLAA AYKSSD DI R AD VQL N SS V F A RFQ QD A TEVSLENSDARP00000079149_Drer_226-321 W L AC G A L E L R AFD G TL HL I R L LL M K KN R F A VE Q N V M I EE DFKKI QI RM KTD L N GVVEGDLSA AYKSSG DI R AD VRL N PS S F A RFQ QD A TEVSQRAVENSGACP00000003400_Gacu_251-346 W L AC G A L E L R AFD G TL HL I R L LL V K RN R F A IE Q T V M I QD DFKKL QI RM KTD L N GVVEGDLAA AYKASG DI R AD VQL S SS V F A RFQ QD A TEVNQQAAENSDARP00000086546_Drer_253-348 W L AC G A L E L R AFD G TL HL I R L LL A K RN R F A VE Q T V M V QE DFKKL QI RM RSD L N GVVEGDLAA AYKSSG DI R AD VRI N PS A F A RFQ QD A TEVSQQTAENSACAP00000014249_Acar_245-338 W L AC G A L E L R AFD G TL HL I R L LL V K KN K F I IE Q T V M V LE DFKKL QI RM KTD L N GKFFGDLAA YKIIRE DI R AD ST N P V Y A RFQ QD A TEVSQHAAENSCINP00000024265_Cint_214-308 W L AC G A L E L R AFD G TL HL I R L LL L R KT R F I VD T T V I L SR EILDN SM RS NQN L K GVVEENAEA AYLANG NI R LD VNL N PS V H A RFQ HG A NPEATHFBpp0081569_Dmel_318-413 W L AC G A L E L R AFD G TL HL I R L LL L R RQ R W L VE S T I M M DT QERQE RQ IR QVD Q N GVVENNYSA AYLSCG NP R ST IAA N NS V H A RFH EE P DQISGSGP

αA0 αA1 αA2 αB0 αB1 αB2

b AnkUBD conservation

a AnkUBD NMR 13C15N labelled AnkUBD + unlabeled Ub

10

10

8

8

6

6

ω2 - 1H (ppm)

130 130

120 120

110 110

ω1

- 15

N (

ppm

)

F266

N268

A269

C270

V274

D277

I281

E282

K285

S286

D290

I291

A292

R293

Q294L295

A308

F309

D310

V311

G312

Y313

T314

L315

V316

H317L318

A319

I320

R321

F322

R324

Q325

D326

M327L328

A329

I330

L331

L332

T333

E334

V335

S336

Q337

250 μM Ub

1 mM Ub

no ubiquitin


Supplementary Figure 2 AnkUBD NMR spectra and AnkUBD conservatio

(a) 1H15N-HSQC spectra for 13C15N-labeled AnkUBD in absence (blue) or

presence of 250 microM (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

(httpwwwensemblorgindexhtml) Secondary structure elements are

indicated and labeled Invariant residues (blue background) in this alignment

are colored red in Fig 3f


Supplementary Figure 3 (1)

AnkUBD H317A

AnkUBD wild type

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100ω

1 -

15N

(pp

m)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48

Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

100 μM

250 μM

400 μM

535 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

75 μM

150 μM

250 μM

370 μM

a

b



AnkUBD L332E

AnkUBD I320D

no AnkUBD250 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD250 μM422 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

c

d


Supplementary Figure 3 Titration experiments of labeled Ub with unlabeled

AnkUBD variants 1H15N-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



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM

Supplementary Figure 4 Additional in vitro DUB assays (a) The TRABID OTU domainis less efficient compared to AnkOTU at similar concentration and is hardly active atan enzyme concentration of [02 microg] in contrast to AnkOTU at the same concentration(see Fig 1b 5a) (b) Catalytic activity of bacterially produced Trabid variants againstLys29- (top) Lys33- (middle) and Lys63-linked (bottom) diUb Lys63-cleavage was assayed at higher enzyme concentration of 1 microM Enzyme concentrations are indicated


GFP-Cezanne C194S


DAPI Mergea

b

MergeGFP FL C443SFLAG-Ub wt (α-Flag)

c

+MG132

d GFP FL ∆Ank C443SHA FL C443S DAPI Merge

+MG132+DMSO +MG132+DMSO

wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub

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 Cy3reg goat anti-rat IgG (Invitrogen)

13


AnkOTU (low)

AnkOTU (high)

OTU

MergeFL C443S (GFP) FLAG-Ub (α-FLAG) K6only

K27only

K48only

a

b


FL C443S (GFP) DAPI Merge

Supplementary Figure 6 Colocalization of GFP-C443S with ubiquitin mutants andin vivo DUB assay (a) Puncta-forming GFP-C443S (left image) was co-expressedwith FLAG-Ub single-Lys (Konly) mutants K6 K27 or K48 (middle images) The merged image is shown to the right (b) Dissolving TRABID assemblies requiresthe AnkUBD Puncta-forming GFP-tagged full-length TRABID C443S (left) wasco-transfected with either Flag-AnkOTU or Flag-OTU in COS-7 cells and thepresence of GFP puncta was assessed Note that two cells are shown forFlag-AnkOTU corresponding to either low or high level of Flag-AnkOTU expressionNuclei are stained using DAPI and the merged image is shown to the right


13 2

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 20 Technology (Clontech)

Point mutants were generated by site directed mutagenesis using the

QuikChangereg technology (Stratagene) For mammalian expression full-

length 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 EcoR1Xho1 sites

For immunofluorescence full-length TRABID (2-708) TRABID AnkOTU (245-

708) TRABID OTU (339-708) were subcloned into pEGFP-C1 using

Xho1EcoR1 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 QuikChangereg 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 4degC TRABID AnkOTU (245-697)

and OTU (339-697) constructs were expressed in Arctic Xpress cells

(Stratagene) Cells were induced at an OD600 of 06-08 with 150 microM IPTG

and grown overnight at 16 degC Cells from 6 L culture were lysed by sonication

in 50 ml lysis buffer (270 mM sucrose 50 mM Tris [pH 80] 1 mM EDTA 1

mM EGTA 10 mM sodium β-glycerophosphate 50 mM sodium fluoride 10

mM β-mercaptoethanol 1 mM benzamidine 01 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 85] 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 microg GST-

tagged PreScission protease overnight Cleaved protein was diluted to ~50


13 3

mM NaCl with 25 mM Tris (pH 85) and subjected to anion exchange

chromatography (MonoQ 550 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 82] 1 mM EDTA 5

mM DTT) The protein was concentrated to 35 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 23degC

from 150 mM NaCl 100 mM NaOAc 5 mM MgCl2 50 mM MES [pH 59] For

synchrotron data collection crystals were soaked in mother liquor containing

275 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

225 Aring resolution and a peak wavelength SAD dataset at 3 Aring 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 SHELXhkl2map 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


13 4

NMR titration analysis

For titration experiments HSQC spectra were recorded for 50 microM TRABID

alone and in presence of 250 microM and 1 mM unlabeled Ub For the reverse

experiment spectra were recorded for 50 microM 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 fast-

HSQC experiments 8 and defined as D1H + (D15N5) [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

(httpstructbiovanderbilteduchazinwisdomkdcalchtm) 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


13 5

Supplementary References

1 Bricogne G Vonrhein C Flensburg C Schiltz M amp Paciorek W

Generation representation and flow of phase information in structure

determination recent developments in and around SHARP 20 Acta

Crystallogr D Biol Crystallogr 59 2023-30 (2003)

2 Akutsu M Ye Y Virdee S Chin JW amp Komander D Molecular

basis for ubiquitin and ISG15 cross-reactivity in viral ovarian tumor

domains Proc Natl Acad Sci U S A 108 2228-33 (2011)

3 Tran H Hamada F Schwarz-Romond T amp Bienz M Trabid a new

positive regulator of Wnt-induced transcription with preference for

binding and cleaving K63-linked ubiquitin chains Genes Dev 22 528-

42 (2008)

4 Evans PC et al Isolation and characterization of two novel A20-like

proteins Biochem J 357 617-23 (2001)

5 Pape T amp Schneider TR Hkl2map a graphical user interface for

macromolecular phasing with shelx programs J Appl Cryst 37 843-

844 (2004)

6 Emsley P amp Cowtan K Coot model-building tools for molecular

graphics Acta Crystallogr D Biol Crystallogr 60 2126-32 (2004)

7 Adams PD et al PHENIX building new software for automated

crystallographic structure determination Acta Crystallogr D Biol

Crystallogr 58 1948-54 (2002)

8 Mori S Abeygunawardana C Johnson MO amp van Zijl PC

Improved sensitivity of HSQC spectra of exchanging protons at short

interscan delays using a new fast HSQC (FHSQC) detection scheme

that avoids water saturation J Magn Reson B 108 94-8 (1995)


13 6

9 Hajduk PJ et al NMR-based discovery of lead inhibitors that block

DNA binding of the human papillomavirus E2 protein J Med Chem 40

3144-50 (1997)

10 Schwarz-Romond T Merrifield C Nichols BJ amp Bienz M The Wnt

signalling effector Dishevelled forms dynamic protein assemblies rather

than stable associations with cytoplasmic vesicles J Cell Sci 118

5269-77 (2005)


13 1

An Ankyrin-repeat ubiquitin binding domain determines TRABIDrsquos specificity for atypical ubiquitin

chains

Supplementary Material

Julien DF Licchesi1 Juliusz Mieszczanek1 Tycho ET 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 dkmrc-lmbcamacuk


HsTRABID


HsA20

HsTRABID 320 330 340 350 360 370


HsA20

HsTRABID380 390 400 410 420 430 440


HsA20

HsTRABID450 460 470 480 490 500 510


HsA20

HsTRABID 520 530 540 550 560 570


HsA20

HsTRABID 580 590 600 610 620 630 640


HsA20

HsTRABID 650 660 670 680 690


HsA20

HsTRABID 700


HsA20

αA0 αA1 αA2 αB0


α2 α3 α3 β3 α4

α5 α6 α7

α8 β4 β5 η1

β6 β7 β8 α9

α10 β11 β12 α11

α1 β1 β2

α2 α3 β3

α4 α5 α6 α7

α8 β4 β5 η1

β6 β7 β8 β9 β10 α9

α10 β11 β12 η2 α11

250 260 270 280 290 300 310



α4

α1rsquo


a

b

c

CCHFV vOTU


























ENSP00000352676_Hsap_245-340 250 260 270 280 290 300 310 320 330 340





10

10

8

8

6

6

ω2 - 1H (ppm)

130 130

120 120

110 110

ω1

- 15

N (

ppm

)

F266

N268

A269

C270

V274

D277

I281

E282

K285

S286

D290

I291

A292

R293

Q294L295

A308

F309

D310

V311

G312

Y313

T314

L315

V316

H317L318

A319

I320

R321

F322

R324

Q325

D326

M327L328

A329

I330

L331

L332

T333

E334

V335

S336

Q337

250 μM Ub

1 mM Ub

no ubiquitin















AnkUBD H317A

AnkUBD wild type

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100ω

1 -

15N

(pp

m)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48

Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

100 μM

250 μM

400 μM

535 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

75 μM

150 μM

250 μM

370 μM

a

b



AnkUBD L332E

AnkUBD I320D

no AnkUBD250 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76


9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

c

d







shown in Fig 4



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


HsTRABID


HsA20

HsTRABID 320 330 340 350 360 370


HsA20

HsTRABID380 390 400 410 420 430 440


HsA20

HsTRABID450 460 470 480 490 500 510


HsA20

HsTRABID 520 530 540 550 560 570


HsA20

HsTRABID 580 590 600 610 620 630 640


HsA20

HsTRABID 650 660 670 680 690


HsA20

HsTRABID 700


HsA20

αA0 αA1 αA2 αB0


α2 α3 α3 β3 α4

α5 α6 α7

α8 β4 β5 η1

β6 β7 β8 α9

α10 β11 β12 α11

α1 β1 β2

α2 α3 β3

α4 α5 α6 α7

α8 β4 β5 η1

β6 β7 β8 β9 β10 α9

α10 β11 β12 η2 α11

250 260 270 280 290 300 310



α4

α1rsquo


a

b

c

CCHFV vOTU


























ENSP00000352676_Hsap_245-340 250 260 270 280 290 300 310 320 330 340





10

10

8

8

6

6

ω2 - 1H (ppm)

130 130

120 120

110 110

ω1

- 15

N (

ppm

)

F266

N268

A269

C270

V274

D277

I281

E282

K285

S286

D290

I291

A292

R293

Q294L295

A308

F309

D310

V311

G312

Y313

T314

L315

V316

H317L318

A319

I320

R321

F322

R324

Q325

D326

M327L328

A329

I330

L331

L332

T333

E334

V335

S336

Q337

250 μM Ub

1 mM Ub

no ubiquitin















AnkUBD H317A

AnkUBD wild type

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100ω

1 -

15N

(pp

m)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48

Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

100 μM

250 μM

400 μM

535 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

75 μM

150 μM

250 μM

370 μM

a

b



AnkUBD L332E

AnkUBD I320D

no AnkUBD250 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76


9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

c

d







shown in Fig 4



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)
























ENSP00000352676_Hsap_245-340 250 260 270 280 290 300 310 320 330 340





10

10

8

8

6

6

ω2 - 1H (ppm)

130 130

120 120

110 110

ω1

- 15

N (

ppm

)

F266

N268

A269

C270

V274

D277

I281

E282

K285

S286

D290

I291

A292

R293

Q294L295

A308

F309

D310

V311

G312

Y313

T314

L315

V316

H317L318

A319

I320

R321

F322

R324

Q325

D326

M327L328

A329

I330

L331

L332

T333

E334

V335

S336

Q337

250 μM Ub

1 mM Ub

no ubiquitin















AnkUBD H317A

AnkUBD wild type

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100ω

1 -

15N

(pp

m)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48

Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

100 μM

250 μM

400 μM

535 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

75 μM

150 μM

250 μM

370 μM

a

b



AnkUBD L332E

AnkUBD I320D

no AnkUBD250 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76


9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

c

d







shown in Fig 4



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)















AnkUBD H317A

AnkUBD wild type

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100ω

1 -

15N

(pp

m)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48

Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

100 μM

250 μM

400 μM

535 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33

E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

no AnkUBD

75 μM

150 μM

250 μM

370 μM

a

b



AnkUBD L332E

AnkUBD I320D

no AnkUBD250 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76


9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

c

d







shown in Fig 4



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)



AnkUBD L332E

AnkUBD I320D

no AnkUBD250 μM

9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76


9

9

8

8

7

7

6

6

ω2 - 1H (ppm)

130 130

125 125

120 120

115 115

110 110

105 105

100 100

ω1

- 15

N (

ppm

)

Q2

I3

F4

V5

K6

T7

L8

T9

G10

K11

T12

I13

T14

L15

E16

V17

E18

S20

D21

T22

I23 N25V26

K27

A28

K29I30

Q31

D32

K33E34

G35

I36

D39

Q40Q41

R42

L43

I44

F45

A46

G47

K48Q49

L50

E51

D52 R54

T55

L56

S57

D58

Y59N60

I61

Q62

K63

E64S65

T66

L67

H68

L69

V70

L71

R72 L73

R74

G75

G76

c

d







shown in Fig 4



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)







shown in Fig 4



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)



0 5 30Lys11

0 5 30Lys27

0 5 30Lys29

0 5 30Lys33

0 5 30Lys48

0 5 30Lys63

0 5 30Linear

M

OTU (339-697)

DiUb

MonoUb

0 5 30Lys6

min

a OTU 697 339

b

0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Lys63 Ub2

Ub

min

Lys33 Ub2

Ub

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

Ub

Lys29 Ub2

min0 5 30AnkOTU

0 5 30OTU

0 5 30H317A

0 5 30I320A

0 5 30I320D

0 5 30L332A

0 5 30L332E

INPM

AnkOTU

OTUAnkOTU

[E] 01 μM

[E] 01 μM

[E] 1 μM

[E] 025 μM



GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


GFP-Cezanne C194S


DAPI Mergea

b


c

+MG132



wt Ub

K6only Ub

K11only Ub

K27only Ub

K29only Ub

K33only Ub

K48only Ub

K63only Ub




















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)





















13


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


AnkOTU (low)

AnkOTU (high)

OTU


K27only

K48only

a

b





13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


13 2
































13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


13 3




























13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


13 4
































13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


13 5












42 (2008)





844 (2004)





Crystallogr 58 1948-54 (2002)






13 6



3144-50 (1997)




5269-77 (2005)


13 6



3144-50 (1997)




5269-77 (2005)


nat. struct. mol. biol. 19, 62–71 (2012) an ankyrin-repeat ... · pdf filein this file...

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