07254 JLGGC

Supplementary Table S1 Kinetic Analyses of the AMSH-LP mutants AMSH-LP KM (μM) kcat x 10-3 (s-1)

WT 71.8 ± 6.3 860 ± 65.4

T353A 76.8 ± 11.7 46.3 ± 3.7

F355A 58.9 ± 10.4 5.33 ± 0.30

S358A 75.1 ± 8.2 82.8 ± 5.3

C402S 33.9 ± 2.4 2.14 ± 0.12

proximal binding

F407A 30.2 ± 4.6 24.9 ± 1.7

E329A 209 ± 49.6 440 ± 19.7 F332A 897 ± 25.8 528 ± 60.9

distal binding

M370A 1277 ± 110.4 487 ± 50.8

E292A N.D. N.D.

S357A 71.2 ± 6.7 24.6 ± 0.89 active site

D360A N.D. N.D.

N.D., not detectable; the mean values±s.e.m. in three independent experiments are shown.

SUPPLEMENTARY INFORMATION

doi: 10.1038/nature07254

www.nature.com/nature 1

Supplementary Table S2 Data collection, phasing, and refinement statistics

AMSH-LPE292A•K63-Ub2 AMSH-LP

Native Native Zn SAD

Data Collection

X-ray source PF-AR NW12A PF-AR NW12A

Wavelength (Å) 1.00000 1.00000 1.28214

Space group P21 P65 P65

Unit cell parameter a = 38.1 Å b = 97.4 Å c = 87.9 Å

β = 97.5˚

a = b = 81.9 Å

c = 64.7 Å

a = b = 82.1 Å

c = 64.9 Å

Resolution (Å) 50.0-1.60 (1.62-1.60) 50.0-1.20 (1.21-1.20) 50.0-1.99 (2.04-1.99)

Unique reflections 83,683 76,845 17,081

Total relections 3,804,643 1,738,510 1,312,874

Completeness (%) 97.3 (90.7) 99.4 (98.3) 99.0 (100.0)

I / σ(I) 16.9 (3.20) 34.3 (14.1) 162 (147)

Rsym 0.068 (0.267) 0.046 (0.158) 0.106 (0.153)

Phasing Statistics

Number of zinc sites 2

Phasing power 2.953

Rculis 0.438

Mean overall figure of merit (Centric/Acentric) 0.118/0.565

Refinement Statistics

Number of atoms: protein; ligand/ion 5072; 619 1432; 328

Rmsd bond length (Å) 0.010 0.008

Rmsd bond angle (˚) 1.265 1.261

Average B factors (Å2) :protein; liand/ion 13.9; 25.1 11.4; 23.3

Residues in core region (%) 94.3 91.7

Residues in additionally allowed region (%) 5.5 8.3

Residues in generously allowed region (%) 0.0 0.0

Residues in disallowed region (%) 0.2 0.0

Rwork, Rfree 0.185, 0.215 0.149, 0.165

The numbers in parentheses are for the highest resolution shell.

Rsym = Σ|Iavg - Ii|/ΣIi.

Rcullis = Σ||FPH - FP| - |FH(calc)|| /Σ|FPH|.

Rwork = Σ|Fo - Fc|/ΣFo for reflections of working set.

Rfree = Σ|Fo - Fc|/ΣFo for reflections of test set (5% of total unique reflections).

doi: 10.1038/nature07254 SUPPLEMENTARY INFORMATION


Supplementary Figure Legends

Supplementary Figure S1 Specific cleavage of Lys63-linked polyubiquitin chains by the

AMSH-LP DUB domain and by full-length AMSH-LP.

a, The AMSH-LP DUB domain can cleave Lys63-linked polyubiquitin chains but not Lys48-linked

chains. Reaction mixtures were analyzed by SDS-PAGE after 20-hour reactions.

b, Full-length AMSH-LP can cleave Lys63-linked polyubiquitin chains but not Lys48-linked

chains. In contrast, the USP family DUB, UBPY, can cleave both Lys48- and Lys63-linked

polyubiquitin chains. Reaction mixtures were analyzed by SDS-PAGE after 20-hour reactions.

Supplementary Figure S2 Comparison of AfJAMM and the AMSH-LP DUB domain.

a, Sequence alignment of AfJAMM and the AMSH-LP DUB domain. The secondary structure of

AMSH-LP is shown above the alignment. Identical residues are highlighted by red background.

Gray, orange, and green bars below the alignment correspond to the JAMM core, Ins-1, and

Ins-2, respectively.

b, Crystal structures of AfJAMM and the AMSH-LP DUB domain are shown as cartoon models in

a similar orientation. Zn2+ and the coordinating water molecule in the active site are shown as

grey and red spheres, respectively. Residues involved in Zn2+ coordination are shown as stick

models. Ins-1 and Ins-2 of AMSH-LP are colored orange and green, respectively.

Supplementary Figure S3 Comparison of the DUB domains of AMSH-LP and AMSH.

a, Sequence alignment of the DUB domains of AMSH-LP and AMSH from representative

organisms. The drawing schemes are the same as in Fig. 5a.

b, Conservation of the DUB domains of AMSH and AMSH-LP is mapped on the AMSH-LP

surface. The drawing schemes are the same as in Fig. 5b.



Supplementary Figure S4 The closed active site of AMSH-LP.

AMSH-LP and K63-Ub2 are shown as molecular surface and cartoon representations,

respectively. The side chains of Asp321 and Phe407, which cover the active site, are colored

orange and green, respectively. The proximal and distal ubiquitins are colored pink and cyan,

respectively.

Supplementary Figure S5 Cleavage of Lys48-linked polyubiquitin chains by the DUB domain

of AMSH-LP mutants

Supplementary Figure S6 Sequence alignment of 10 human JAMM proteins (6 DUBs and 4

non-DUB proteins). Common sequences for JAMM DUBs are surrounded by rectangles. The

secondary structure of AMSH-LP and putative signature sequences (φ; aliphatic residues, X; any

residues) are shown above the alignment. MYSM1 has a 23-residue insertion between α3 and

β7.

Supplementary Figure S7 Sequence alignment of the DUB domains of AMSH-LP, AMSH,

and POH1/Rpn11 from the representative organisms

The secondary structure of AMSH-LP is shown above the alignment. 100% and more than 60%

identical residues are highlighted by red and yellow backgrounds, respectively. Gray, orange,

and green bars below the alignment correspond to the JAMM core, Ins-1, and Ins-2, respectively.

Green squares indicate the residues involved in Zn2+ coordination. Pink and cyan triangles

indicate residues that form hydrophobic interactions with the proximal and distal ubiquitins,

respectively. Pink and cyan circles indicate residues that form hydrogen bonds with the proximal

and distal ubiquitins, respectively. Open circles indicate that only the main chain atoms are

involved in the hydrogen bonding, whereas filled circles indicate that only the side chain atoms or

both the side chain and main chain atoms are involved in the hydrogen bonding.


4

Supplementary Figure S8 Sample images of tricine SDS-PAGE gels stained by coomassie

brilliant blue for quantification of ubiquitin monomers produced by DUB reactions.



Lys63Lys48linkage linkage

AMSH-LP DUB

polyubiquitinchain

polyubiquitinchain

ubiquitin monomer

- + - +

66.4 -44.3 -

29.0 -

20.1 -

14.3 -

6.5 -

(kDa)

ubiquitin monomer

UBPY

AMSH

-LP

- - UBPY

AMSH

-LP

Lys48 Lys63

Fukai, S. Supplementary Figure S1

#2008-05-04441A

AMSH-LPDUB

AMSH-LPUBPY

a b



H.sapiens AMSH-LP A.fulgidus JAMM

disordered regionIns-2 Ins-1

264 270 280 290 300 310 320 330 340 350

360 370 380 390 400 410 420 430 436

a

b

H.sapiens_AMSH-LP L L A S E L G I G G H HP EGLRCVVLPED CHKF QL E NTVRGI TCGI C KLTHNEFTITHV VPKQSA PDYCDMENVEELFNVQDQHDLLTL WI T TA.fulgidus_JAMM L L A S E L G I G G H HP ..GSSMKISRG LKTI EA K AH..PD FIAL S SK....DVMDEL FLPFVS SVSAVIHLDMLPIG......MKVF TV S S

H.sapiens_AMSH-LP-ssβ1 α1 β2 β3 β4 β5 α2 β6

H.sapiens_AMSH-LP S DL I V DQTAFL SV HTHCSYQLMLPEAIA VCS..PKHKDTGIFRLTNAGMLEVSACKKKGFHPHTKEPRLFSICKHVL K. IKIIVLDLRA.fulgidus_JAMM S DL I V DPSCRP EE SLFTRFG.....KYH IVCYPYDENSWKCYNR........................KGEEVELEV EK .........

H.sapiens_AMSH-LP-ssα3 β7 β8 α4 β9 β10 β11

A.fulgidus_JAMM-ss

A.fulgidus_JAMM-ss


#2008-05-04441A



proximal ubiquitin binding sitedistal ubiquitin binding site

90

Ins-2

Ins-1

Ins-2

Ins-1active site

active site

a

b

proximalubiquitin

proximalubiquitin

distalubiquitin

distalubiquitin

H.sapiens_AMSH-LP LR FL NT ETCG L G L ITH P Q D C F QD L TLGWIHTHPTQT G VVLP DL LA RGI I C K NEFT V K SAGP Y EN EEL V QH LE C E CHK Q ES V TH IV DM V N DM.musculus_AMSH-LP LR FL NT ETCG L G L ITH P Q D C F QD L TLGWIHTHPTQT G VVL DL LA RGI I C K NEFT V K SAGP Y EN EEL V QH LE C SR CHK L DS V TH VV DV V N GX.tropicalis_AMSH-LP LR FL NT ETCG L G L ITH P Q D C F QD L TLGWIHTHPTQT G VVLP DL LA RGI I C K EFT V K SAGP Y EN EEL V QH LD P R SQR Q EA S THD IV DM V N NH.sapiens_AMSH LR FL NT ETCG L G L ITH P Q D C F QD L TLGWIHTHPTQT G VV P L LA RG I C K NEFT V K SAG Y EN EEL QD H V GR CPQ Q SA A V MR LI S NT E LI QG ID.melanogaster_AMSH LR FL NT ETCG L G L ITH P Q D C F QD L TLGWIHTHPTQT V P D LA I N P E V QGS L YV G TMEV K LA SKN V A H SQ QLY IIT Q QGT S NTMHE QI D MQ IS.pombe_AMSH LR FL NT ETCG L G L ITH P Q D C F QD L TLGWIHTHPTQT LP L I C K N F A L H LKP TIY KL KKV DVVKP KKNL RQ A F LVI L E TS T GTTDEAS EF K N

H.sapiens_AMSH-LP F SSVDLHTHCSYQ M PE AIV P G F L G C G FHPH H DLRA L L L AI CS K T I R T E S KK K P LF C V V D K V HKD .NA ML V A K . T. E R SI K L K. I II L ..M.musculus_AMSH-LP F SSVDLHTHCSYQ M PE AIV P G F L G C G FHPH H DLRA L L L AI CS K T I R T E S KK K P LF C V V D K V HKD .NA ML V T K . T. D K SI S L K. I TT L ..X.tropicalis_AMSH-LP F SSVDLHTHCSYQ M PE AIV P G F L G C G FHPH H DLRA L L L AI CS K T I R T E S KK K P F C V V D V HND .SA ML V A K . S. E RQ NT R T Q. AGIT L ..H.sapiens_AMSH F SSVDLHTHCSYQ M PE AIV P G F L G C G FHPH H DLRA L L CS K T T E S K K P LF C V V D M SV FQE F K .DH LE I S RQ . S. D P CS S T V. RAVTIT ..D.melanogaster_AMSH F SSVDLHTHCSYQ M PE AIV P G F L G C G FHPH H DLRA L A C K T T P LF K V I M L A YNT F I PHY LDYIAQ RQS . P.ND P MEAQ IRMDNQA IK I R.S.pombe_AMSH F SSVDLHTHCSYQ M PE AIV P G F L G C G FHPH H DLR L L AI I R K K V K VC M MA SKNTS LDPE LQTIVK R P L EG VYTMVAQPG REI.NS LQ V VK

β1 α1 β2 β3 β4 β5 α2 β6

α3 β7 β8 α4 β9 β10 β11

264 270 280 290 300 310 320 330 340 350

360 370 380 390 400 410 420 430 436


#2008-05-04441A




#2008-05-04441A

Phe407 Asp321

Lys63 Gly76



Proximal

T353

A

bindingDistal

bindingActive

site

66.4 -44.3 -

29.0 -

20.1 -

14.3 -

6.5 -

(kDa) F355

A

E29

2AM

370A

F332

AE

329A

F407

AC

402S

S35

8A

wt

D36

0AS

357A

K48

-pol

yUb

onlyAMSH-LP + K48-polyUb

- AMSH-LP

- Ub

K48- polyUb


#2008-05-04441A



β1 α1 β2

α3 β7 β8

AMSH-LP EGLRCVVLP---EDLCHKFLQLAESNTVRGI---ETCGILCGKLTH

AMSH DGLRHVVVP---GRLCPQFLQLASANTARGV---ETCGILCGKLMR

Rpn11 VDTAEQVY-----ISSLALLKMLKHGRAGVPM--EVMGLMLGEFVD

CSN5 PWTKDHHYFKYCKISALALLKMVMHARSGGNL--EVMGLMLGKVDG

BRCC36 MAVQVVQAVQAVHLESDAFLVCLNHALSTEKE--EVMGLCIGELND

AMSH-LP LLTLGWIHTHPTQTAFLSSVDLHTHCSYQLMLP----EAIAIVCSPKHK------DTGIFRLTNAG

AMSH LITLGWIHTHPTQTAFLSSVDLHTHCSYQMMLP----ESVAIVCSPKFQ------ETGFFKLTDHG

BRCC36 MRVVGWYHSHPHITVWPSHVDVRTQAMYQMMDQGFVGLIFSCFIEDKNT-KTGRVLYTCFQSIQAQ

Rpn11 EMVVGWYHSHPGFGCWLSGVDINTQQSFEALSE----RAVAVVVDPIQS-VKGKVVIDAFRLINAN

CSN5 ENAIGWYHSHPGYGCWLSGIDVSTQMLNQQFQE----PFVAVVIDPTRTISAGKVNLGAFRTYPKG

264 303

340 395

EXXG φ φ XG

φGWIHTHP Y S

SXφD FRLYKT

β6

Rpn8 LAVQKVVVHP---LVLLSVVDHFNRIGKVGNQK-RVVGVLLGSWQK

CSN6 TGSVSVALHP---LVILNISDHWIRMRSQEGRPVQVIGALIGKQEG

eIF3γ SAVKQVQID---GLVVLKIIKHYQEEGQGTE---VVQGVLLGLVVE

eIF3η PGGRVVRLH---PVILASIVDSYERRNEGAA---RVIGTLLGTVDK

eIF3γ HLHVGWYQSTYYG-SFVTRALLDSQFSYQHAIE...

eIF3η ELILGWYATGHDITEHSVLIHEYYSREAPNPIH...

Rpn8 ERIVGWYHTGP----KLHKNDIAINELMKRYCP...

CSN6 LEFLGWYTTGG----PPDPSDIHVHKQVCEIIE...


#2008-05-04441A

MYSM1 EEKQEPFQV---KVASEALLIMDLHAHVSMA---EVIGLLGGRYSE

MYSM1 FSVIGWYHSHPAFDPNPSLRDIDTQAKYQSYFS-23-QITCLVISEEIS------PDGSYRLPYKF



.......

H.sapiens_AMSH-LP L E G G F Q GW H HP LR V F NT TC L L FTITH VP Q D C E V D L TL I T TQTEG C VLPEDLCHK QLAES VRGI I C K THNE VI K SAGP Y DM N EEL N.......V QHD LM.musculus_AMSH-LP L E G G F Q GW H HP LR V F NT TC L L FTITH VP Q D C E V D L TL I T TQTEG C VLSRDLCHK LLADS VRGI I C K THNE VV K SAGP Y DV N EEL N.......V QHG LX.tropicalis_AMSH-LP L E G G F Q GW H HP LR V F NT TC L L FTITH VP Q D C E V D L TL I T TQTDG P VLPRDLSQR QLAEA SRGI I C K THDE VI K SAGP Y DM N EEL N.......V QHN LH.sapiens_AMSH L E G G F Q GW H HP LR V F NT TC L L FTITH P Q D C E D L TL I T TQTDG H VVPGRLCPQ QLASA ARGV I C K MRNE VLI K SAGS Y NT NEEEL L.......I QQG ID.melanogaster_AMSH L E G G F Q GW H HP LR V F NT TC L L ITH P Q D C D L TL I T TQTGS L YVPGDTMEV KLALA SKNI V A H SQNQLY IIT Q QGTP S NTMHEEQI D.......V QMQ IS.pombe_AMSH L E G G F Q GW H HP LR F NT TC L L F ITH P Q D C D L TL I T TQTKP TIYLPKLLKKV DVVKP KKNL I C K RQNA F LVI L EATS T GTTDEASL E.......F KHN LH.sapiens_POH1 L E G G F Q GW H HP V T V E VDTAEQ YISSLALLKM KHGRAGVP..M VM LML EFVD.DY VRVID FAMPQSGTGVSV A DPV QAKMLDMLK TGRPEMVV Y S GFGD.melanogaster_Rpn11 L E G G F Q GW H HP V T V E VDTAEQ YISSLALLKM KHGRAGVP..M VM LML EFVD.DY VQVID FAMPQTGTGVSV A DPV QAKMLDMLK TGRPEMVV Y S GFGS.pombe_Rpn11 L E G G F Q GW H HP V T FT V E VDNSEC YISSLALLKM RHGRHG P..M VM LML EFVD.D VRVVD FAMPQSGTGVSV A DPV QKNMMDMLK TGRPEMVV Y S GFG

..... ..

H.sapiens_AMSH-LP S VD T S A V P G F L H FL S LH HC YQ M PEA I R G C G FHP P H DLRA L L I CS KHKDT .....I T.NA MLEVS..A KKK . T.KE RLFSICK VLVK.DIKIIVLM.musculus_AMSH-LP S VD T S A V P G F L H FL S LH HC YQ M PEA I R G C G FHP P H DLRA L L I CS KHKDT .....I T.NA MLEVS..T KKK . T.KD KLFSICS VLVK.DIKTTVLX.tropicalis_AMSH-LP S VD T S A V P G F L H FL S LH HC YQ M PEA I R G C G FHP P H DLRA L L I CS KHNDT .....I T.SA MLEVS..A KKK . S.KE RQFNTCR VTVQ.DAGITVLH.sapiens_AMSH S VD T S A V P G F L H FL S LH HC YQ M PE I G C G FHP P H DLRA M L SV CS KFQET .....F K T.DH LEEIS..S RQK . S.KD PLFCSCS VTVV.DRAVTIT ..D.melanogaster_AMSH S VD T S A V P G F L H FL S LH HC YQ M PEA I G C G FHP P H DLRA I M L CA KYNTT .....F I TPHY LDYIA..Q RQS . P.ND PLFMEAQ IRMDNQAKIKVI R.S.pombe_AMSH S VD T S A V P G F L H F S LH HC YQ M PEA I R G C G FHP H DLRC M L L I MA SKNTS .....I LDPE LQTIV..K RKP L EGKVYTMVAQPG VREI.NSKLQVV VKH.sapiens_POH1 S VD T S A V P G F L H L A R PCW G IN QQ FEALSER V V VD IQSVK KVVIDA I.NANMMVLGHEPRQTTS.NLG L.NK SIQALIHGLNRH.YYSITINYRKD.melanogaster_Rpn11 S VD T S A V P G F L H L A R PCW G IN QQ FEALSER V V VD IQSVK KVVIDA I.NPNMLVLGQEPRQTTS.NLG L.QK SVQALIHGLNRH.YYSISINYRKS.pombe_Rpn11 S VD T S A V P G F L H L S P A R PCW IN QQ FEQLT R V V VD IQSVK KVVIDA I.NPSTLMMGQEPRQTTS.NLG I.NK SIQALIHGLGRH.YYSLRINYKK

β1 α1 β2 β3 β4 β5 α2 β6

α3 β7 β8 α4 β9 β10 β11

264 270 280 290 300 310 320 330 340 350

360 370 380 390 400 410 420 430 436


#2008-05-04441A



25

200

100

50

25

200

100

50

4.3

12.88.6

time (min)AMSH-LP (nM)

wt

K63-Ub2 (µM)

2510 Ub(µg)

100

800

400

200 100

800

400

200

4.3

12.88.6


E329A

K63-Ub2 (µM)

5010 Ub(µg)

T353A S358A50010

25015

25

200

100

50

25

200

100

50

25

200

100

50

25

200

100

50


K63-Ub2 (µM) 4.3

12.88.6

Ub(µg)

F407A50015

S357A50015

25

200

100

50

25

200

100

50

25

200

100

50

25

200

100

50

4.3

12.88.6

Ub(µg)time (min) AMSH-LP (nM)

K63-Ub2 (µM)

F355A C402S1000

601000100

25

200

100

50

25

200

100

50

25

200

100

50

25

200

100

50

4.3

12.88.6

Ub(µg)time (min)AMSH-LP (nM)

K63-Ub2 (µM)

F332A5015

150

1200

600

300

150

1200

600

300

M370A5015

150

1200

600

300

150

1200

600

300 4.

3

12.88.6

Ub(µg)time (min)AMSH-LP (nM)

K63-Ub2 (µM)


#2008-05-04441A

Ub

Ub

Ub Ub

Ub

Ub



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