SUPPLEMENTARY INFORMATION

Supplementary Table S1 Kinetic Analyses of the AMSH-LP mutants AMSH-LP K M (μm) k cat 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 proximal S358A 75.1 ± 8.2 82.8 ± 5.3 binding C402S 33.9 ± 2.4 2.14 ± 0.12 F407A 30.2 ± 4.6 24.9 ± 1.7 E329A 209 ± 49.6 440 ± 19.7 distal F332A 897 ±.8 528 ± 60.9 binding M370A 1277 ± 110.4 487 ±.8 E292A N.D. N.D. active S357A 71.2 ± 6.7 24.6 ± 0.89 site D360A N.D. N.D. N.D., not detectable; the mean values±s.e.m. in three independent experiments are shown. www.nature.com/nature 1

Supplementary Table S2 Data collection, phasing, and refinement statistics AMSH-LP E292A K63-Ub 2 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 P2 1 P6 5 P6 5 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).0-1.60 (1.62-1.60).0-1.20 (1.21-1.20).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 (.0) I / σ(i) 16.9 (3.20) 34.3 (14.1) 162 (147) R sym 0.068 (0.267) 0.046 (0.158) 0.106 (0.153) Phasing Statistics Number of zinc sites 2 Phasing power 2.953 R culis 0.438 Mean overall figure of merit (Centric/Acentric) 0.118/0.565 Refinement Statistics Number of atoms: protein; ligand/ion 72; 619 1432; 328 Rmsd bond length (Å) 0.010 0.008 Rmsd bond angle ( ) 1.265 1.261 Average B factors (Å 2 ) :protein; liand/ion 13.9;.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 R work, R free 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). www.nature.com/nature 2

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. Zn 2+ and the coordinating water molecule in the active site are shown as grey and red spheres, respectively. Residues involved in Zn 2+ 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. www.nature.com/nature 3

Supplementary Figure S4 The closed active site of AMSH-LP. AMSH-LP and K63-Ub 2 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. % 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 Zn 2+ 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. www.nature.com/nature 5

a b linkage Lys48 Lys63 linkage Lys48 Lys63 AMSH-LP DUB (kda) 66.4-44.3 - - + - + AMSH-LP DUB - AMSH-LP UBPY - AMSH-LP UBPY UBPY AMSH-LP 29.0-20.1 - polyubiquitin chain polyubiquitin chain 14.3-6.5 - ubiquitin monomer ubiquitin monomer Fukai, S. Supplementary Figure S1 www.nature.com/nature 6

a β1 α1 β2 β3 β4 β5 α2 β6 H.sapiens_AMSH-LP-ss A.fulgidus_JAMM-ss 2 6 4 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0 3 4 0 3 H.sapiens_AMSH-LP E G L R C V V L P E D L C H K F L Q L A E S N T V R G I E T C G I L C G K L T H N E F T I T H V I V P K Q S A G P D Y C D M E N V E E L F N V Q D Q H D L L T L G W I H T H P T A.fulgidus_JAMM.. G S S M K I S R G L L K T I L E A A K S A H.. P D E F I A L L S G S K.... D V M D E L I F L P F V S G S V S A V I H L D M L P I G...... M K V F G T V H S H P S α3 β7 β8 α4 β9 β10 β11 H.sapiens_AMSH-LP-ss A.fulgidus_JAMM-ss 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 4 1 0 4 2 0 4 3 0 4 3 6 H.sapiens_AMSH-LP Q T A F L S S V D L H T H C S Y Q L M L P E A I A I V C S.. P K H K D T G I F R L T N A G M L E V S A C K K K G F H P H T K E P R L F S I C K H V L V K. D I K I I V L D L R A.fulgidus_JAMM P S C R P S E E D L S L F T R F G..... K Y H I I V C Y P Y D E N S W K C Y N R........................ K G E E V E L E V V E K D......... b Ins-2 Ins-1 disordered region H.sapiens AMSH-LP A.fulgidus JAMM Fukai, S. Supplementary Figure S2 www.nature.com/nature 7

a β1 α1 β2 β3 β4 β5 α2 β6 2 6 4 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 H.sapiens_AMSH-LP E G L R C V V L P E D L C H K F L Q L A E S N T V R G I E T C G I L C G K L T H N E F T I T H V I V P K Q S A G P D Y C D M E N V E E L F N V Q D Q H D L L T L G W I H T H P T Q T M.musculus_AMSH-LP E G L R C V V L S R D L C H K F L L L A D S N T V R G I E T C G I L C G K L T H N E F T I T H V V V P K Q S A G P D Y C D V E N V E E L F N V Q D Q H G L L T L G W I H T H P T Q T X.tropicalis_AMSH-LP D G L R P V V L P R D L S Q R F L Q L A E A N T S R G I E T C G I L C G K L T H D E F T I T H V I V P K Q S A G P D Y C D M E N V E E L F N V Q D Q H N L L T L G W I H T H P T Q T H.sapiens_AMSH D G L R H V V V P G R L C P Q F L Q L A S A N T A R G V E T C G I L C G K L M R N E F T I T H V L I P K Q S A G S D Y C N T E N E E E L F L I Q D Q Q G L I T L G W I H T H P T Q T D.melanogaster_AMSH G S L R L V Y V P G D T M E V F L K L A L A N T S K N I E T C G V L A G H L S Q N Q L Y I T H I I T P Q Q Q G T P D S C N T M H E E Q I F D V Q D Q M Q L I T L G W I H T H P T Q T S.pombe_AMSH K P L R T I Y L P K L L K K V F L D V V K P N T K K N L E T C G I L C G K L R Q N A F F I T H L V I P L Q E A T S D T C G T T D E A S L F E F Q D K H N L L T L G W I H T H P T Q T α3 β7 β8 α4 β9 β10 β11 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 4 1 0 4 2 0 4 3 0 4 3 6 H.sapiens_AMSH-LP A F L S S V D L H T H C S Y Q L M L P E A I A I V C S P K H K D T G I F R L T. N A G M L E V S A C K K K G. F H P H T. K E P R L F S I C K H V L V K. D I K I I V L D L R.. M.musculus_AMSH-LP A F L S S V D L H T H C S Y Q L M L P E A I A I V C S P K H K D T G I F R L T. N A G M L E V S T C K K K G. F H P H T. K D P K L F S I C S H V L V K. D I K T T V L D L R.. X.tropicalis_AMSH-LP A F L S S V D L H T H C S Y Q L M L P E A I A I V C S P K H N D T G I F R L T. S A G M L E V S A C K K K G. F H P H S. K E P R Q F N T C R H V T V Q. D A G I T V L D L R.. H.sapiens_AMSH A F L S S V D L H T H C S Y Q M M L P E S V A I V C S P K F Q E T G F F K L T. D H G L E E I S S C R Q K G. F H P H S. K D P P L F C S C S H V T V V. D R A V T I T D L R.. D.melanogaster_AMSH A F L S S V D L H T H C S Y Q I M M P E A L A I V C A P K Y N T T G F F I L T P H Y G L D Y I A Q C R Q S G. F H P H P. N D P P L F M E A Q H I R M D N Q A K I K V I D L R R. S.pombe_AMSH C F M S S V D L H T H C S Y Q L M L P E A I A I V M A P S K N T S G I F R L L D P E G L Q T I V K C R K P G L F H P H E G K V Y T M V A Q P G H V R E I. N S K L Q V V D L R V K b Ins-1 active site Ins-1 active site proximal ubiquitin 90 proximal ubiquitin distal ubiquitin Ins-2 distal ubiquitin binding site distal ubiquitin Ins-2 proximal ubiquitin binding site Fukai, S. Supplementary Figure S3 www.nature.com/nature 8

Phe407 Asp321 Lys63 Gly76 Fukai, S. Supplementary Figure S4 www.nature.com/nature 9

(kda) 66.4-44.3-29.0-20.1 - T353A Proximal binding F355A S358A AMSH-LP + K48-polyUb Distal binding C402S F407A E329A F332A M370A E292A S357A D360A wt K48-polyUb only Active site - AMSH-LP K48- polyub 14.3-6.5 - - Ub Fukai, S. Supplementary Figure S5 www.nature.com/nature 10

β1 α1 β2 264 EXXG φ φ XG 303 AMSH-LP EGLRCVVLP---EDLCHKFLQLAESNTVRGI---ETCGILCGKLTH AMSH DGLRHVVVP---GRLCPQFLQLASANTARGV---ETCGILCGKLMR BRCC36 MAVQVVQAVQAVHLESDAFLVCLNHALSTEKE--EVMGLCIGELND Rpn11 VDTAEQVY-----ISSLALLKMLKHGRAGVPM--EVMGLMLGEFVD MYSM1 EEKQEPFQV---KVASEALLIMDLHAHVSMA---EVIGLLGGRYSE CSN5 PWTKDHHYFKYCKISALALLKMVMHARSGGNL--EVMGLMLGKVDG eif3γ eif3η Rpn8 CSN6 SAVKQVQID---GLVVLKIIKHYQEEGQGTE---VVQGVLLGLVVE PGGRVVRLH---PVILASIVDSYERRNEGAA---RVIGTLLGTVDK LAVQKVVVHP---LVLLSVVDHFNRIGKVGNQK-RVVGVLLGSWQK TGSVSVALHP---LVILNISDHWIRMRSQEGRPVQVIGALIGKQEG β6 α3 β7 β8 φgwihthp SXφD FRL 340 Y S YKT 395 AMSH-LP AMSH BRCC36 Rpn11 MYSM1 CSN5 eif3γ eif3η Rpn8 CSN6 LLTLGWIHTHPTQTAFLSSVDLHTHCSYQLMLP----EAIAIVCSPKHK------DTGIFRLTNAG LITLGWIHTHPTQTAFLSSVDLHTHCSYQMMLP----ESVAIVCSPKFQ------ETGFFKLTDHG MRVVGWYHSHPHITVWPSHVDVRTQAMYQMMDQGFVGLIFSCFIEDKNT-KTGRVLYTCFQSIQAQ EMVVGWYHSHPGFGCWLSGVDINTQQSFEALSE----RAVAVVVDPIQS-VKGKVVIDAFRLINAN FSVIGWYHSHPAFDPNPSLRDIDTQAKYQSYFS-23-QITCLVISEEIS------PDGSYRLPYKF ENAIGWYHSHPGYGCWLSGIDVSTQMLNQQFQE----PFVAVVIDPTRTISAGKVNLGAFRTYPKG HLHVGWYQSTYYG-SFVTRALLDSQFSYQHAIE... ELILGWYATGHDITEHSVLIHEYYSREAPNPIH... ERIVGWYHTGP----KLHKNDIAINELMKRYCP... LEFLGWYTTGG----PPDPSDIHVHKQVCEIIE... Fukai, S. Supplementary Figure S6 www.nature.com/nature 11

β1 α1 β2 β3 β4 β5 α2 β6....... 2 6 4 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 H.sapiens_AMSH-LP E G L R C V V L P E D L C H K F L Q L A E S N T V R G I E T C G I L C G K L T H N E F T I T H V I V P K Q S A G P D Y C D M E N V E E L F N....... V Q D Q H D L L T L G W I H T H P T Q T M.musculus_AMSH-LP E G L R C V V L S R D L C H K F L L L A D S N T V R G I E T C G I L C G K L T H N E F T I T H V V V P K Q S A G P D Y C D V E N V E E L F N....... V Q D Q H G L L T L G W I H T H P T Q T X.tropicalis_AMSH-LP D G L R P V V L P R D L S Q R F L Q L A E A N T S R G I E T C G I L C G K L T H D E F T I T H V I V P K Q S A G P D Y C D M E N V E E L F N....... V Q D Q H N L L T L G W I H T H P T Q T H.sapiens_AMSH D G L R H V V V P G R L C P Q F L Q L A S A N T A R G V E T C G I L C G K L M R N E F T I T H V L I P K Q S A G S D Y C N T E N E E E L F L....... I Q D Q Q G L I T L G W I H T H P T Q T D.melanogaster_AMSH G S L R L V Y V P G D T M E V F L K L A L A N T S K N I E T C G V L A G H L S Q N Q L Y I T H I I T P Q Q Q G T P D S C N T M H E E Q I F D....... V Q D Q M Q L I T L G W I H T H P T Q T S.pombe_AMSH K P L R T I Y L P K L L K K V F L D V V K P N T K K N L E T C G I L C G K L R Q N A F F I T H L V I P L Q E A T S D T C G T T D E A S L F E....... F Q D K H N L L T L G W I H T H P T Q T H.sapiens_POH1 D T A E Q V Y I S S L A L L K M L K H G R A G V P.. M E V M G L M L G E F V D. D Y T V R V I D V F A M P Q S G T G V S V E A V D P V F Q A K M L D M L K Q T G R P E M V V G W Y H S H P G F G D.melanogaster_Rpn11 D T A E Q V Y I S S L A L L K M L K H G R A G V P.. M E V M G L M L G E F V D. D Y T V Q V I D V F A M P Q T G T G V S V E A V D P V F Q A K M L D M L K Q T G R P E M V V G W Y H S H P G F G S.pombe_Rpn11 D N S E C V Y I S S L A L L K M L R H G R H G T P.. M E V M G L M L G E F V D. D F T V R V V D V F A M P Q S G T G V S V E A V D P V F Q K N M M D M L K Q T G R P E M V V G W Y H S H P G F G α3 β7 β8 α4 β9 β10 β11....... 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 4 1 0 4 2 0 4 3 0 4 3 6 H.sapiens_AMSH-LP A F L S S V D L H T H C S Y Q L M L P E A I A I V C S P K H K D T G..... I F R L T. N A G M L E V S.. A C K K K G. F H P H T. K E P R L F S I C K H V L V K. D I K I I V L D L R M.musculus_AMSH-LP A F L S S V D L H T H C S Y Q L M L P E A I A I V C S P K H K D T G..... I F R L T. N A G M L E V S.. T C K K K G. F H P H T. K D P K L F S I C S H V L V K. D I K T T V L D L R X.tropicalis_AMSH-LP A F L S S V D L H T H C S Y Q L M L P E A I A I V C S P K H N D T G..... I F R L T. S A G M L E V S.. A C K K K G. F H P H S. K E P R Q F N T C R H V T V Q. D A G I T V L D L R H.sapiens_AMSH A F L S S V D L H T H C S Y Q M M L P E S V A I V C S P K F Q E T G..... F F K L T. D H G L E E I S.. S C R Q K G. F H P H S. K D P P L F C S C S H V T V V. D R A V T I T D L R.. D.melanogaster_AMSH A F L S S V D L H T H C S Y Q I M M P E A L A I V C A P K Y N T T G..... F F I L T P H Y G L D Y I A.. Q C R Q S G. F H P H P. N D P P L F M E A Q H I R M D N Q A K I K V I D L R R. S.pombe_AMSH C F M S S V D L H T H C S Y Q L M L P E A I A I V M A P S K N T S G..... I F R L L D P E G L Q T I V.. K C R K P G L F H P H E G K V Y T M V A Q P G H V R E I. N S K L Q V V D L R V K H.sapiens_POH1 C W L S G V D I N T Q Q S F E A L S E R A V A V V V D P I Q S V K G K V V I D A F R L I. N A N M M V L G H E P R Q T T S. N L G H L. N K P S I Q A L I H G L N R H. Y Y S I T I N Y R K D.melanogaster_Rpn11 C W L S G V D I N T Q Q S F E A L S E R A V A V V V D P I Q S V K G K V V I D A F R L I. N P N M L V L G Q E P R Q T T S. N L G H L. Q K P S V Q A L I H G L N R H. Y Y S I S I N Y R K S.pombe_Rpn11 C W L S S V D I N T Q Q S F E Q L T P R A V A V V V D P I Q S V K G K V V I D A F R L I. N P S T L M M G Q E P R Q T T S. N L G H I. N K P S I Q A L I H G L G R H. Y Y S L R I N Y K K Fukai, S. Supplementary Figure S7 www.nature.com/nature 12

AMSH-LP (nm) time (min) K63-Ub 2 (µm) wt 10 Ub(µg) 4.3 8.6 12.8 AMSH-LP (nm) time (min) K63-Ub 2 (µm) 400 E329A 10 Ub(µg) 800 400 800 4.3 8.6 12.8 Ub Ub AMSH-LP (nm) time (min) K63-Ub 2 (µm) T353A 0 10 S358A 0 15 Ub(µg) 4.3 8.6 12.8 AMSH-LP (nm) time (min) K63-Ub 2 (µm) F407A 0 15 S357A 0 15 Ub(µg) 4.3 8.6 12.8 Ub Ub AMSH-LP (nm) time (min) K63-Ub 2 (µm) F355A 0 60 C402S 0 Ub(µg) 4.3 8.6 12.8 AMSH-LP (nm) time (min) K63-Ub 2 (µm) F332A 15 M370A 15 1 300 600 1 1 300 600 1 1 300 600 1 1 300 600 1 Ub(µg) 4.3 8.6 12.8 Ub Ub Fukai, S. Supplementary Figure S8 www.nature.com/nature 13