SUPPLEMENTARY INFORMATION with E34 and G35 of ubiquitin in the structure of the RNF4...

W W W. N A T U R E . C O M / N A T U R E | 1

SUPPLEMENTARY INFORMATIONdoi:10.1038/nature11376SUPPLEMENTARY TABLE

Supplementary Table 1 Data collection and refinement statistics Data collection Space group P212121 Cell dimensions a, b, c (Å) 50.5, 65.1, 189.7 α, β, γ (°) 90, 90, 90 Resolution (Å) 99 – 2.21 (2.26 – 2.21)* Rmerge 0.033 (0.172) I/σI 44 (7) Completeness (%) 99 (92) Redundancy 10.6 (3.5) Refinement Resolution (Å) 95 – 2.21 (2.26 – 2.21) No. reflections 30,468 Rwork / Rfree 20.8 (26.2) / 24.2 (31.0) No. atoms Protein 4607 Zn2+ 4 Water 137 B-factors Protein 49 Zn2+ 33 Water 41 R.m.s deviations Bond lengths (Å) 0.008 Bond angles (º) 1.2

*Highest resolution shell is shown in parenthesis. One crystal was used to collect the data set.

SUPPLEMENTARY INFORMATION

2 | W W W. N A T U R E . C O M / N A T U R E

RESEARCH

350

0

50

100

150

200

250

300

5040 60 70 80 90 100 110

Abs

orba

nce

at 2

80 n

m (m

AU

)

Elution volume (ml)

Ubiquitin

UbcH5A(S22R/C85K)–Ub126 -

63 -

35 -25 -17 -12 -

7.5 -

kDa

UbcH5A(S22R/C85K)–Ub

Ubiquitin

Load

Fractions

a

b c

26

126 -63 -

35 -25 -

17 -12 -

7.5 -

kDa

Time (h):

Conjugationreaction

2280 Time (h): 0

Cleavagewith TEV

18

UbcH5A–HisUbUbcH5A–Ub

UbiquitinHisUbiquitin

Ni-NTAchromatography

Load

FTLoad

UbcH5A–HisUbUbcH5A(S22R/C85K)

HisUbiquitin

Ni-NTAchromatography

FT Elution

Supplementary Figure 1. Preparation of a ubiquitin-loaded E2 with an isopeptide linkage between the active site of the E2 and the C terminus of ubiquitin. The active site cysteine (C85) in E2 UbcH5A was mutated to a lysine to allow formation of a stable isopeptide linkage between the C-terminal glycine of ubiquitin and the active site of the E2. For structural analysis, we also introduced the S22R mutation into UbcH5A to eliminate the “backside” non-covalent interaction with ubiquitin28. This interaction is not required for initial E3-mediated ubiquitin transfer, but is required for processive ubiquitin addition and is therefore absent from E2s that only carry out monoubiquitination. a, Purified E2 (UbcH5A(S22R/C85K)) and His6-tagged ubiquitin were incubated at 37° for 26 hours in the presence of ubiquitin activating enzyme and ATP. Subsequently, Ni-NTA affinity chromatography was carried out to remove free E2. Elution fractions were pooled and treated overnight with TEV protease to remove the His6-tag from ubiquitin. The sample was then again applied onto a Ni-NTA column to remove any uncleaved E2–HisUb, the free His6-tag, and the TEV protease (also His6-tagged). A flow-through fraction was applied onto a gel filtration column to separate the ubiquitin-loaded E2 from free ubiquitin. Coomassie blue-stained SDS-PAGE gels are shown. FT, flow-through fraction. b, Chromatogram from gel filtration chromatography showing separation of the ubiquitin-loaded E2 and free ubiquitin. c, SDS-PAGE gel showing peak fractions from gel filtration chromatography containing the ubiquitin-loaded E2.

SUPPLEMENTARY FIGURES


SUPPLEMENTARY INFORMATION RESEARCH

_

y₁₇

I

y₁₆

a₂

Y

y₁₅

b₃

H

y₁₄

b₄

P

y₁₃

b₅

N

y₁₂

b₆

I

y₁₁

b₇

N

y₁₀

b₈

S

y₉

b₉

N

y₈

b₁₀

G

y₇

b₁₁

S

y₆

b₁₂

I

y₅

b₁₃

gl

K

y₄

b₁₄

I

y₃

b₁₅

D

y₂

b₁₆

I

y₁

b₁₇

I R _

y₁175.119

a₂249.1598

y₂-NH₃271.1765

y₂288.203

y₃-NH₃384.2605

y₃401.2871

b₃414.2136

y₄-NH₃499.2875

b₄511.2663

y₄516.314

b₅625.3093

y₅629.3981 b₆-NH₃

721.3668

b₆738.3933

b₁₃²⁺776.897

y₁₃²⁺778.9414

b₇852.4363

y₆-NH₃854.5094

y₆871.536

b₈-H₂O921.4577

b₈939.4683

y₇984.62

b₁₇²⁺1004.037

b₉1053.511

y₈1071.652

b₁₀1110.533

y₉1128.674

b₁₁-H₂O1179.554

b₁₁1197.565

y₁₀-NH₃1225.69

y₁₀1242.716

b₁₂-H₂O1292.638

b₁₂1310.649

y₁₁-NH₃1312.722

y₁₁1329.748

y₁₂-NH₃1426.765

y₁₂1443.791

y₁₃-NH₃1539.849

b₁₃1552.787

y₁₃1556.875

y₁₄-NH₃1653.892

b₁₄1665.871

y₁₄1670.918

y₁₅-NH₃1750.945

b₁₅-NH₃1763.871

y₁₅1767.971

b₁₅1780.898

b₁₆1893.982

y₁₆1905.03

b₁₇2007.066

y₁₇-NH₃2051.067

y₁₇2068.093

020

4060

8010

012

0

Rel

ativ

e Ab

unda

nce

01

23

4

Intensity [10e7]

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

m/z [amu]

UbcH5A(S22R/C85K)

UbcH5A(S22R/C85K)–Ub

12.5-

17.0-

25.0-kDa

-Ub +Ub

UbcH5A(S22R/C85K)

0

5E+09

1E+10

1.5E+10

2E+10

GG-K85 LRGG-K85

Pep

tide

inte

nsity

-Ub+Ub

a

c

d

b

Supplementary Figure 2. Mass spectrometric evidence for an isopeptide bond between ubiquitin G76 and K85 in UbcH5A(S22R/C85K). a, 5 μg of non-ubiquitinated recombinant UbcH5A(S22R/C85K) (left) or the ubiquitinated form (right) was fractionated by SDS-PAGE. The indicated Coomassie stained bands were excised and tryptic peptides extracted. b, Peptide samples were analysed by LC-MS/MS. Both Gly-Gly and Leu-Arg-Gly-Gly variable modifications to lysine were detected at K85 of ubiquitin-loaded

_

y₁₇-NH₃

I

a₂

Y

y₁₅

b₃

H

y₁₄

P

y₁₃²⁺

b₅

N

y₁₂

b₆

I

y₁₁

b₇

N

y₁₀

b₈

S

y₉

N

y₈

b₁₀

G

y₇

b₁₁-NH₃

S

y₆

I

y₅

lr

K

y₄

b₁₄²⁺

I

y₃

D

y₂

I

y₁

I R _

y₁175.119

a₂249.1598

y₂288.203

y₃401.2871

b₃414.2136

y₄516.314

b₅-NH₃608.2827

b₅625.3093

y₅629.3981

b₆738.3933

b₇-NH₃835.4097

b₇852.4363

y₁₃²⁺913.534

b₈-H₂O921.4577

b₈939.4683

b₁₄²⁺968.0316

b₁₀1110.533

y₆-NH₃1123.695

y₆1140.721

b₁₁-NH₃1180.538

y₇1253.805

y₈1340.837

y₉1397.859

y₁₀-NH₃1494.875

y₁₀1511.902

y₁₁-NH₃1581.907

y₁₁1598.934

y₁₂-NH₃1695.95

y₁₂1712.977

y₁₃-NH₃1809.034

y₁₄-NH₃1923.077

y₁₄1940.104

y₁₅-NH₃2020.13

y₁₅2037.156

y₁₇-NH₃2320.252

020

4060

8010

012

0

Rel

ativ

e Ab

unda

nce

00.5

11.5

22.5

3

Intensity [10e6]

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

m/z [amu]



RESEARCH

WB: α-UbcH5A

Inpu

t (4%

)M

BP

RN

F4

RN

F4(M

140A

/R18

1A)

C85K

Inpu

t (4%

)M

BP

RN

F4

RN

F4( M

140A

/R18

1A)

C85SUbcH5A:

17 -12 -

kDa

63 -

45 -

kDa

MBP

MBP–RNF4

Coomassie blue-stainedSDS-PAGE gel

UbcH5A–Ub

WB: α-UbcH5A

C85K S22R/C85K

Inpu

t (15

%)

MB

P

C85SUbcH5A:

E2 siteY193A

RING1–RING2linear fusion

E2 siteY193AIn

put (

15%

)M

BP


–– – ––– – –

––– –

–

UbcH5A–Ub

MBP–RING1–RING2MBP

17 -12 -

45 -63 -

–– – ––– – –

––– –

–

Inpu

t (15

%)

MB

P


–– – ––– – –

––– –

–

Mutations in:

RIN

G1–R

ING

2

Coomassie blue-stained SDS-PAGE gels

kDa

17 -

25 -

45 -

128 -

8 -

kDa

UbcH5A(C85K)–Ub

RNF4

RNF4:Time (h):

– +0 5 24 48 72 10

015

00 5 24 48 72 10

015

0

a

cd

b

UbcH5A(S22R/C85K). c, MS/MS spectrum of the peptide 73-IYHPNINSNGSIKLDILR-90 containing K85 showing the Gly-Gly adduct characteristic of ubiquitination. The peptide was confidently identified with a high score (326.53) and low mass error (-0.33965). d, MS/MS spectrum of the peptide 73-IYHPNINSNGSIKLDILR-90 containing K85 with the Leu-Arg-Gly-Gly adduct characteristic of ubiquitination with a single missed tryptic cleavage at the C terminus. Again high score (176.23) and low mass error (0.3127 ppm) are evident. Together, these identify an isopeptide linkage between G76 of ubiquitin and K85 of the UbcH5A mutant.

Supplementary Figure 3. Isopeptide bond-linked UbcH5A–Ub is a stable structural mimic for the UbcH5A–Ub thioester. a, Isopeptide bond-linked UbcH5A–Ub is completely stable in the presence of RNF4. UbcH5A(C85K)–Ub (30 μM) was incubated at room temperature either in the presence or absence of RNF4 (30 μM). Samples were taken at indicated time points and analyzed by SDS-PAGE, followed by staining with Coomassie blue. b, Isopeptide-linked UbcH5A–Ub binds to RNF4 with a similar efficiency as UbcH5A–Ub linked with an oxyester bond. Pull-down experiment showing interaction between MBP-tagged RNF4 and a ubiquitin-loaded E2 linked either via an oxyester bond (UbcH5A(C85S)–Ub) or via an isopeptide bond (UbcH5A(C85K)–Ub). No binding was detected using RNF4 mutant with disrupted E2-binding site (RNF4(M140A/R181A))3. WB, Western blot. c, Residue Y193 in one RING protomer and a functional E2-binding site in the other

0 10 20 30

UbcH5A(C85K)–Ub

free UbcH5A + Ub

Inhibitor (μM)

Rea

ctio

n ra

te (n

M s

-1)

0

5

10

15



Supplementary Figure 4. Electron density at the isopeptide bond between UbcH5A(C85K) and ubiquitin. Difference electron density (Fo-Fc) contoured at 3σ is shown. The phases are derived from a molecular replacement solution that had K85 from UbcH5A and G76 from ubiquitin removed prior to solution to eliminate bias. UbcH5A is shown in green and ubiquitin is coloured orange.

G75

G76

UbiquitinUbcH5A

K85

protomer of the dimeric RNF4 RING domain are both required for efficient binding of a ubiquitin-loaded E2. Y193 is located at the dimerization interface of the RNF4 RING domain and interacts with E34 and G35 of ubiquitin in the structure of the RNF4 RING–UbcH5A–Ub complex presented herein. The importance of this interaction was confirmed by a pull-down experiment with a RING domain containing a Y193A mutation. A linear fusion of two RNF4 RING domains was used as different mutations can be introduced into the two RING protomers, which is not feasible in an unfused, homodimeric RING domain. Mutation Y193A is marked with and E2-binding site mutations (M140A/R181A) are marked with . Isopeptide-linked UbcH5A(C85K)–Ub and UbcH5A(S22R/C85K)–Ub showed the same behaviour in this binding assay as the UbcH5A(C85S)–Ub oxyester. d, Isopeptide bond-linked UbcH5A–Ub is a potent inhibitor of RNF4-mediated ubiquitination. UbcH5A(C85K)–Ub was added at indicated concentrations to single-turnover substrate ubiquitination reactions containing ~3 μM UbcH5A–Ub thioester, 0.275 μM RNF4, and 5.5 μM substrate (4 × SUMO-2) radiolabelled with iodine-125. Substrate ubiquitination activity was determined as described in Methods. An equimolar mixture of free UbcH5A (wild-type) and ubiquitin did not show any inhibition at concentrations tested.



RESEARCH

UBE2D1 (UbcH5A)UBE2D2 (UbcH5B)UBE2D3 (UbcH5C)UBE2D4UBE2A (Rad6a)UBE2B (Rad6b)UBE2CUBE2E1UBE2E2UBE2E3UBE2G1UBE2G2UBE2H (E2-20K)UBE2J1UBE2J2UBE2K (E2-25K)UBE2L3 (UbcH7)UBE2L6UBE2N (Ubc13)UBE2O (E2-230K)UBE2Q1UBE2Q2UBE2R1 (Cdc34)UBE2R2 (Cdc34b)UBE2SUBE2TUBE2UUBE2WUBE2ZUBE2I (Ubc9)UBE2FUBE2M (Ubc12)UBE2V1 (Uev1a)UBE2V2 (Uev2)

59595959626288

105113119646360678665606061

1011324277676769606274

1576790859772

1191191191191221221481651731791371361221281471261211211211081386339140140129124125136227129157145161136

D Y P F K P P K I A F T T K I - - - - - YH PN I N S - N G S I C L D I L R - - - - - - - - - - - - - SQWS P A L T V S K V L L S I C - S L L C D P - N PD D P LD Y P F K P P K V A F T T R I - - - - - YH PN I N S - N G S I C L D I L R - - - - - - - - - - - - - SQWS P A L T I S K V L L S I C - S L L C D P - N PD D P LD Y P F K P P K V A F T T R I - - - - - YH PN I N S - N G S I C L D I L R - - - - - - - - - - - - - SQWS P A L T I S K V L L S I C - S L L C D P - N PD D P LD Y P F K P P K V A F T T K I - - - - - YH PN I N S - N G S I C L D I L R - - - - - - - - - - - - - SQWS P A L T V S K V L L S I C - S L L C D P - N PD D P LE Y PN K P P T VR F V S KM - - - - - F H PN V Y A - D G S I C L D I L Q - - - - - - - - - - - - - N R WS P T YD V S S I L T S I Q - S L L D E P - N PN S P AE Y PN K P P T VR F L S KM - - - - - F H PN V Y A - D G S I C L D I L Q - - - - - - - - - - - - - N R WS P T YD V S S I L T S I Q - S L L D E P - N PN S P AG Y P YN A P T V K F L T PC - - - - - YH PN VD T - QGN I C L D I L K - - - - - - - - - - - - - E KWS A L YD VR T I L L S I Q - S L L G E P - N I D S P LE Y P F K P P K V T F R T R I - - - - - YH C N I N S - QG V I C L D I L K - - - - - - - - - - - - - D N WS P A L T I S K V L L S I C - S L L TD C - N P AD P LD Y P F K P P K V T F R T R I - - - - - YH C N I N S - QG V I C L D I L K - - - - - - - - - - - - - D N WS P A L T I S K V L L S I C - S L L TD C - N P AD P LD Y P F K P P K V T F R T R I - - - - - YH C N I N S - QG V I C L D I L K - - - - - - - - - - - - - D N WS P A L T I S K V L L S I C - S L L TD C - N P AD P LD Y P L R P P KM K F I T E I - - - - - WH PN VD K - N GD VC I S I L H E PG ED K YG Y E K P E ER WL P I H T V E T I M I S V I - SM L AD P - N GD S P AD Y P L S P P KMR F T C EM - - - - - F H PN I Y P - D GR VC I S I L H A PGD D PMG Y E S S A ER WS P VQ S V E K I L L S V V - SM L A E P - N D E SG AK Y P F K S P S I G F MN K I - - - - - F H PN I D E A SG T VC L D V I N - - - - - - - - - - - - - Q TWT A L YD L TN I F E S F L PQ L L A Y P - N P I D P LE Y PM K P P S I I L L T AN - - - - - - - GR F E V - G K K I C L S I SGH - - - - - - - - - - H P E TWQ P SWS I R T A L L A I I - G FM P T K - G EG A I GE F P F K P P S I YM I T PN - - - - - - - GR F KC - N TR L C L S I TD F - - - - - - - - - - H PD TWN P AWS V S T I L TG L L - S FM V E K - G P T L G ST Y P F N P P K VR F I T K I - - - - - WH PN I S S V T G A I C L D I L K - - - - - - - - - - - - - D QWA A AM T L R T V L L S L Q - A L L A A A - E PD D PQE Y P F K P P K I T F K T K I - - - - - YH PN I D E - KGQ VC L P V I S - - - - - - - - - - - - A EN WK P A T K TD Q V I Q S L I - A L VN D P - Q P EH P LE Y P F K P PM I K F T T K I - - - - - YH PN VD E - N GQ I C L P I I S - - - - - - - - - - - - S EN WK PC T K TC Q V L E A L N - V L VN R P - N I R E P LE Y PM A A P K VR F M T K I - - - - - YH PN VD K - L GR I C L D I L K - - - - - - - - - - - - - D KWS P A L Q I R T V L L S I Q - A L L S A P - N PD D P LI Y P A V P PH F C Y L SQC - - SGR L N PN L YD - N G K VC V S L L G TW I G - - - - - - KG T ER WT S K S S L L Q V L I S I Q - G L I L VN - E P Y Y E AN F P F D P P F VR V V S P V - - - - L SGG Y V L G - GG A I C M E L L T - - - - - - - - - - - - KQGWS S A Y S I E S V I MQ I S - A T L V KG - K AR VQ FN F P F D P P F VR V V L P V - - - - L SGG Y V L G - GG A L C M E L L T - - - - - - - - - - - - KQGWS S A Y S I E S V I MQ I N - A T L V KG - K AR VQ FD Y P Y S P P A F R F L T KM - - - - - WH PN I Y E - T GD VC I S I L H P P VD D PQ SG E L P S ER WN P TQN VR T I L L S V I - S L L N E P - N T F S P AD Y P Y S P P T F R F L T KM - - - - - WH PN I Y E - N GD VC I S I L H P P VD D PQ SG E L P S ER WN P TQN VR T I L L S V I - S L L N E P - N T F S P AD F P A S P P KG Y F L T K I - - - - - F H PN VG A - N G E I C VN V L K - - - - - - - - - - - - - R D WT A E L G I R H V L L T I K - C L L I H P - N P E S A LR Y P F E P PQ I R F L T P I - - - - - YH PN I D S - AGR I C L D V L K L P - - - - - - - - - P KG AWR P S L N I A T V L T S I Q - L L M S E P - N PD D P LE YN Y A P P V V K F I T I P - - - - - F H PN VD PH T GQ PC I D F L D N - - - - - - - - - - - P E KWN TN Y T L S S I L L A L Q - VM L SN P - V L EN P VR Y P F D S PQ VM F T G EN - - - I P VH PH V Y S - N GH I C L S I L T - - - - - - - - - - - - - ED WS P A L S VQ S VC L S I I - SM L S SC - K E KR R PD Y P I H P PR V K L M T T GN N T VR F N PN F YR - N G K VC L S I L G TW - - - - - - - - - TG P AWS P AQ S I S S V L I S I Q - S L M T EN P YH N E PGD Y P S S P P KC K F E P P L - - - - - F H PN V Y P - SG T VC L S I L E E - - - - - - - - - - - D KD WR P A I T I KQ I L L G I Q - E L L N E P - N I QD P AA YN M V P P K V KC L T K I - - - - - WH PN I T E - T G E I C L S L L R EH S - - - - - - - I D G TGWA P TR T L KD V VWG L N - S L F TD L L N FD D P LG Y PH D P P K V KC E T M V - - - - - YH PN I D L - EGN VC L N I L R - - - - - - - - - - - - - ED WK P V L T I N S I I YG L Q - Y L F L E P - N P ED P LK Y P E A P P F VR F V T K I - - - - - N MN G VN S SN G V VD PR A I S V - - - - - - - - - - - L A KWQN S Y S I K V V L Q E L R - R L MM S K EN M K L PQK Y P E A P P S VR F V T K I - - - - - N MN G I N N S SGM VD AR S I P V - - - - - - - - - - - L A KWQN S Y S I K V V L Q E L R - R L MM S K EN M K L PQ

Supplementary Figure 5. Sequence alignment of human E2 enzymes. Sequences of human ubiquitin E2 enzymes, the SUMO E2 UBE2I, the Nedd8 E2s UBE2F and UBE2M, and the pseudo E2s UBE2V1 and UBE2V2 were aligned. The active site cysteines are shown in red. Residues in UbcH5A that were mutated in this study are marked with a circle (mutations that do not affect RNF4-mediated substrate ubiquitination activity), a diamond (moderate decrease in ubiquitination activity), or a triangle (>10-fold reduction in activity). Symbols are coloured blue for residues interacting with the RING domain in the RNF4 RING–UbcH5A–Ub structure, orange for ubiquitin-interacting residues, yellow for residues interacting with both the RING domain and ubiquitin, and green for residues interacting with neither the RING nor ubiquitin. Residue N77 (UbcH5A numbering), that is proposed to

100Sequence identity (%): 80 60 50 0



stabilize the tetrahedral intermediate during the nucleophilic attack on the activated thioester bond, is highly conserved in active E2 enzymes. Residue D87 in UbcH5A hydogen bonds to the main chain of ubiquitin R74 in the RNF4 RING–UbcH5A–Ub structure. Mutation D87A abolished activity, indicating that this interaction is critical for locking the C-terminal tail of ubiquitin in the E2 active site groove. Aspartate or serine is found in this position in most of the E2 enzymes that were shown to be active with RING-type E3 ligases. Serine can be engaged in a similar hydrogen bonding interaction as aspartate. This was indeed observed in the SUMO–RanGAP1–Ubc9–RanBP2 complex23,25 (see Supplementary Fig. 17c). D117, a residue proposed to position and activate the incoming substrate lysine, is often aspartate or serine in other E2s. It was shown that D127S Ubc9 (corresponding to D117 in UbcH5A) retains activity, but a decrease in catalytic efficiency was observed21, suggesting that E2s with a serine in this position might be less efficient in activating the incoming substrate lysine. In the RING–UbcH5A–Ub complex, ubiquitin is folded back onto the E2, contacting the α2 helix of UbcH5A. This interaction is centered on L104 in UbcH5A and I44 in ubiquitin. A hydrophobic residue is found in the position of UbcH5A L104 in most of the E2 enzymes that were shown to be active with RING E3s.

Supplementary Figure 6. The active site groove shows higher conservation than other surfaces in E2 enzymes. Conserved residues present in active human E2 enzymes (Supplementary Fig. 5) were mapped onto the surface of UbcH5A in the RNF4 RING–UbcH5A–Ub complex. Identical residues are indicated in red, highly conserved residues in pink and conserved residues in green. Ubiquitin and the RNF4 RING domain are shown in ribbon representation, with ubiquitin coloured orange and the two protomers in the dimeric RING domain coloured cyan and blue. Conserved residues surround the E2 active site groove, the region that makes extensive contacts with the C-terminal tail of ubiquitin in the RNF4 RING–UbcH5A–Ub structure. A patch of conserved residues is also located at the RING interaction interface.

UbcH5A

Ubiquitin

RNF4 RING

RNF4 RING



RESEARCH

UbcH5A

RING

RING

Ubiquitin

UbcH5A

RING

RINGK4

M1

P61

E9

R5

F62

L97P95

S94

Q92

M140

D141

I138

R181

P137F162

T179

P178

C139

C163

C158 C180

C177H160

A96

a b

Supplementary Figure 7. Interaction interface between the RING domain of RNF4 and UbcH5A. a, Overall view of the complex of the dimeric RNF4 RING domain bound to ubiquitin-loaded UbcH5A (top panel) and the same complex with ubiquitin removed to show the interaction interface between the RING domain and UbcH5A (bottom panel). The two monomers in the dimeric RING domain are coloured cyan and blue, UbcH5A is coloured green, and ubiquitin is orange. Zinc ions are shown as grey spheres. b, Detailed view of the interaction interface between one monomer of the RNF4 RING (blue) and UbcH5A (green). Residues at the interface and zinc-coordinating residues in the RING domain are shown as sticks, hydrogen bonds are indicated as black dashes. In UbcH5A, the interaction interface is formed by the α1 helix (residues K4 to D12) and by two loops spanning residues P61 to K63 and Q92 to T98, respectively. The latter loop contains an SPA motif (S94, P95 and A96) that is conserved in many E2 enzymes. UbcH5A residues P61 and F62 together with the SPA motif insert into a shallow hydrophobic groove on the surface of the RING domain delineated by residues P137 and I138 from the first zinc-binding loop, F162, the α2 helix residues (D169 to N173), and P178 from the loop chelating the second zinc ion.

α1

α2

α1

α2



HumanRatOpossumChickenXenopus laevisXenopus tropicalisDanio rerioDrosophila melanogasterAedes aegyptiArabidopsis thaliana

Slx8 (S. pombe)Slx8 (S. cerevisiae)Rfp1 (S. pombe)Rfp2 (S. pombe)

12713112913198

125121254150122

201201184142

190194192194161188184312209182

269274254205

SG T V SC P I C MD G Y S E I VQN - GR L I V S T EC GH V F C SQC L R D S L KN - - - - - - AN TC P - - TC R K K I N H K - R YH P I Y I - - - - - - - - - - -SG T V SC P I C MD G Y S E I VQN - GR L I V S T EC GH V F C SQC L R D S L KN - - - - - - AN TC P - - TC R K K I N H K - R YH P I Y I - - - - - - - - - - -SG T V SC P I C MD G Y S E I L QN - GR L I V S T KC GH V F C SQC L R D A L R N - - - - - - AN TC P - - TC R K K L N Q K - Q YH P I Y I - - - - - - - - - - -SG T V SC P I C MD G Y S E I VQN - GR L I V S T KC GH V F C SQC L R G S L R N - - - - - - AN TC P - - TC R K K L TH R - Q YH P I Y I - - - - - - - - - - -SG K V SC P I C MD C Y S E I VQN - GR L I V S T KC GH I FC SQC L R D A L KN - - - - - - AN TC P - - TC R K K L N H K - Q YH P I Y I - - - - - - - - - - -SG K V SC P I C MD S Y S E I VQN - GR L I V S T KC GH I FC SQC L R D A L KN - - - - - - AN TC P - - TC R K K L N H K - Q YH P I Y V - - - - - - - - - - -PG A I SC P VC MD V Y S E I MQN - GR L M V S T KC GH L F C SQC I R D S L SR - - - - - - AN TC P - - TC R K K L TH K - Q YH P I Y I - - - - - - - - - - -E E L Y KC P I C MD S V S - - - - - - KR E P V S T KC GH V F C R EC I E T A I R A - - - - - - AN TC P - - I C N K K L T AR - Q F FR I Y L - - - - - - - - - - -P A S VN C P I C F E S V Y - - - - - - R R Q A A S T I C GH L F C N AC I T A EMR I - - - - - - AN TC P - - L C KH P L KWQ - Q VH P I Y FN - - - - - - - - - -E P K F SC P I C L C P F T Q - - - - - - - - E V S T KC GH I FC K KC I KN A L S L - - - - - - AN TC P - - TC R K K I T V K - D L I R V F L P T TR - - - - - - -

L AD Y KC V I C L D S P - - - - - - - - EN L SC T PC GH I FC N FC I L S A L G T T A A - - - TQ KC P - - VC R R K VH PN - K V I C L EMM - - L G SQ K K K SA KD YR C P I C F E P P - - - - - - - - E T A L M T L C GH V F C C PC L FQM VN S SR TC R Q FGH C A - - L C R S K V Y L K - D VR L I I L R K KQ V K K K V K SD T L M VC PR C Q E P L G T S K S K E K S A L WA T KC GH V YC G SC A K V L K T S KR S - - - Q S KC L VN D C GR Y L N T KN AMWE L F Y - - - - - - - - - - -H N N I AC A KC GN E L - - - V SD E K K S I F A A KC GH L FC S TC A K E L R K K - - - - - - T V PC P VQH C R KR I T K K - F I F P L Y L - - - - - - - - - - -

Supplementary Figure 8. Sequence conservation in dimeric RING-type ubiquitin E3 ligases. a, Sequence alignment of RNF4 homologues from various species. b, Sequence alignment of RING and U-box domains of well-studied dimeric ubiquitin E3 ligases. Zinc co-ordinating residues are coloured red. Residue H160 in RNF4 (numbering corresponds to rat RNF4) serves the dual purpose of ligating one of the Zn atoms and hydrogen bonding to the main chain carbonyl of ubiquitin E34 in the RNF4 RING–UbcH5A–Ub structure. A histidine at this position is a characteristic feature of the RING domain, suggesting there is a preference for a His rather than a Cys at this position, expressly to carry out this dual function. A conserved hydrophobic residue in the first zinc co-ordinating loop (I138 in RNF4) that is required for the interaction with E2 enzymes, is coloured green. A positively charged residue (Arg or Lys) in the loop that chelates the second zinc ion is highlighted in yellow. This residue (R181) interacts with both ubiquitin and UbcH5A in the RNF4 RING–UbcH5A–Ub structure. A number of the dimeric RINGs have Tyr or Phe at the equivalent of position 193 in RNF4 (highlighted in orange) and in this case, the planar side chain makes a stacking interaction with the ubiquitin main chain between E34 and G35. Previous mutational

100Sequence identity (%): 80 60 50 0

RNF4 (rat)cIAP1cIAP2MDM2MDMXIDOLTRAF6BRCA1CHIPPrp19

13456955544215

3856822

2313

194618604497704341237928658

V SC P I C MD G Y S E I VQN GR L I V S T E - - - C GH V - F C SQC L R D S L K - - - N AN TC P TC R K K I N H KR YH P I Y I -R T C K VC MD K E V - - - - - - - - S V V F I - - PC GH L V VC Q EC A P S L - - - - - - - R KC P I C R G I I K - - G T VR T F L SR T C K VC MD K E V - - - - - - - - S I V F I - - PC GH L V VC KD C A P S L - - - - - - - R KC P I C R S T I K - - G T VR T F L SE PC V I C QGR P KN G - - - - - - - - C I VH G K TGH L M AC F TC A K K L K K - - - R N K PC P VC R Q P I Q - - M I V L T Y F PK PC S L C E KR PR D G - - - - - - - - N I I H GR TGH L V TC FH C AR R L K K - - - AG A SC P I C K K E I Q - - L V I K V F I AM L C M VC C E E E I - - - - - - - - N S T FC - - PC GH T VC C E SC A AQ L - - - - - - - Q SC P VC R SR V E - - H VQH V Y L PY EC P I C L M A L - - - - - - - R E A VQ T P - - - C GH R - F C K AC I I K S I R D - - AGH KC P VD N E I L L EN Q L F PD N F AL EC P I C L E L I - - - - - - - K E P V S T K - - - C D H I - FC K F C M L K L L N Q K KG P SQC P L C KN D I T KR S L Q E S TR FL C G K I S F E L M - - - - - - - R E PC I T P - - - SG I T - YD R KD I E EH L QR - - VGH FD P V TR S P L TQ EQ L I PN L AML I C S I SN E V P EH P - - - - - - - C V S P - - V SN H V - Y ER R L I E K Y I A - - - EN G TD P I N N Q P L S E EQ L I D I K V A

a

b


1 0 | W W W. N A T U R E . C O M / N A T U R E

RESEARCH

analysis indicated that mutation of conserved residues R181 and Y193 dramatically reduced the ability of RNF4 to catalyze ubiquitin transfer and mediate hydrolysis of a UbcH5A(C85S)–Ub oxyester3. We presume that for those RING and U-box domains that lack the equivalent of Y193 in RNF4 (e.g. TRAF6 and CHIP), the relative orientation of ubiquitin and the RING/U-box in the complex will be similar, with the I36 hydrophobic patch of ubiquitin (L8, I36, and L71) buried upon complex formation. However, additional interactions not seen in the RNF4 RING–UbcH5A–Ub complex are likely to stabilize the RING-ubiquitin interface in these E3 ligases.Yeast RNF4 homologues Rfp1 and Rfp2 are inactive as ubiquitin E3 ligases but form a heterodimer with Slx8 (ref. 42,43). Rfp1 and Rfp2 lack key E2-binding residues but have a Tyr/Phe in a position equivalent to Y193 in RNF4. Thus, in the heterodimer, ubiquitin-loaded E2 would be recruited by Slx8, while the C terminus of Rfp1/Rfp2 would engage in an interaction with ubiquitin, stabilizing the RING–E2–Ub complex.

a

c d

b

α2

α3

α4

α1

D117

K85 D117

K85

G76UbcH5A–ubiquitinUbcH5A (2C4P)UbcH5A (3OJ4)

UbcH5A (2YHO)UbcH5A (3PTF)

M140

I138

R181

M140

I138

R181

Y193

Y193

NN

NCC

C

N

C

CC

C

N

Supplementary Figure 9. Structural changes induced by complex formation in the RNF4 RING–UbcH5A–Ub structure. a, The dimeric RNF4 RING domain does not undergo any major structural change as a result of complex formation. The fused RING dimer from the RNF4 RING–UbcH5A–Ub structure was superimposed onto a structure of free RNF4 RING (Protein Data Bank accession 2XEU)3. The two RING domains in the fused dimer are coloured cyan and blue, with

W W W. N A T U R E . C O M / N A T U R E | 1 1


the linker region (a single glycine residue) coloured yellow. The uncomplexed RNF4 RING is shown in orange. Key residues involved in interactions with E2 (I138 and M140), ubiquitin (Y193), and both E2 and ubiquitin (R181) are shown as sticks. Zinc ions are shown as spheres and N- and C-termini are labelled with N and C, respectively. b, Superposition of ubiquitin from the RNF4 RING–UbcH5A–Ub complex (orange) with free ubiquitin structures 1UBQ32 (blue) and 3ONS44 (residues 1-72, cyan). Residue L8 is highlighted in green (complexed ubiquitin) or yellow (structures of free ubiquitin). The loop at L8 moves over 4 Å to contact both UbcH5A and the RING in the RNF4 RING–UbcH5A–Ub complex. c, Superposition of UbcH5A from the present structure (green) with structures of free UbcH5A (Protein Data Bank accession 2C4P, cyan) and UbcH5A in a variety of non-covalent complexes: UbcH5A bound to the IDOL RING domain14 (Protein Data Bank accession 2YHO, yellow), UbcH5A in complex with ubiquitin12 (Protein Data Bank accession 3PTF, grey), and UbcH5A–ubiquitin–A20 zinc finger 4 complex13 (Protein Data Bank accession 3OJ4, blue). In the RING–UbcH5A–Ub complex, the α1 helix of UbcH5A and the loop which connects to the β1 strand adjust their position to avoid clashing with K6, G47 and H68 of ubiquitin. This loop, however, varies in other structures of the E2, suggesting it is a mobile region. The D117 loop moves as a consequence of isopeptide bond formation between the C terminus of ubiquitin and K85 in UbcH5A (shown in sticks representation and coloured violet). The unconjugated structures all have the side chain of D117 (shown as sticks) pointing towards C85 in a position that would clash with the isopeptide (thioester) bond. d, Detailed view of the D117 loop in the UbcH5A structures shown in c. The isopeptide bond that connects the C-terminal tail of ubiquitin (orange) with UbcH5A residue K85 (violet) in the RNF4 RING–UbcH5A–Ub structure, is shown as sticks.



RESEARCH

Ub:Time:

WT (Sigma) WT L8A K11A K33A G35A I36A Q40A R42A I44A V70A L71A R72A R74AL73A

UbcH5A UbcH5A–Ub

Ubiquitin

4 × SUMO-2

Ubiquitinated4 × SUMO-2

UbcH5A UbcH5A–Ub

Ubiquitin

4 × SUMO-2

I36A Q40A R42A1 2 3 4 1 2 3 4 1 2 3 4 1

I44A V70A L71A2 3 4 1 2 3 4 1 2 3 4

R72A R74AL73A1 2 3 4 1 2 3 4 1 2 3 4

Ub:Lane: 1

WT (Sigma) WT L8A K11A K33A G35A2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 41 2 3 4

a

b

Supplementary Figure 10. Substrate ubiquitination assay with ubiquitin mutant proteins.a, Single-turnover, RNF4-dependent substrate ubiquitination assay with mutant forms of ubiquitin. A linear fusion of four SUMO-2 molecules (4 × SUMO-2) was used as a substrate for RNF4. Reactions contained ~20 μM UbcH5A–Ub thioester, 5.5 μM 4 × SUMO-2, and 0.275 μM RNF4. Samples were incubated at room temperature and reaction time points were taken at 0.5 min, 1.5 min, 5 min, and 20 min. Reactions were stopped by non-reducing SDS-PAGE loading buffer to preserve the UbcH5A–Ub thioester, followed by SDS-PAGE analysis and staining with Coomassie blue. The first lane shows unmodified substrate (4 × SUMO-2; marked with *). Ubiquitin mutant proteins used in this assay contained four extra residues from the expression vector at the N terminus. Ubiquitination reaction containing wild-type ubiquitin obtained from Sigma (with no extra residues at the N-terminus) is also shown for comparison. b, Formation and stability of UbcH5A–Ub thioesters with mutations in ubiquitin. Mutant forms of ubiquitin were incubated at 37° for 12 min with UbcH5A, E1, and ATP to generate the UbcH5A–Ub thioester (lane 1: time 0; lane 2: after 12 min at 37°). Subsequently, apyrase was added to deplete ATP and thus to stop E1-mediated loading of E2. Stability of the UbcH5A–Ub thioesters in the substrate ubiquitination assay in the absence of RNF4 was then tested. Reactions were performed as in a, but without RNF4. Time points were taken at 0.5 min (lane 3) and 20 min (lane 4). Non-reducing SDS-PAGE gels stained with Coomassie blue are shown.

*



Supplementary Figure 11. Mutational analysis of the RNF4 RING–UbcH5A–Ub complex: mutations in ubiquitin. a, Ability of mutant forms of ubiquitin to form thioesters with UbcH5A. Ubiquitin mutants were incubated with UbcH5A, E1 and ATP at 37° for 12 min and the samples were analyzed by non-reducing SDS-PAGE, followed by staining with Coomassie blue. b, Single-turnover, RNF4-dependent substrate ubiquitination assays with mutant forms of ubiquitin. 125I-labelled substrate was used to allow quantification of ubiquitination activity. Phosphorimager scans of SDS-PAGE gels are shown. The first lane (*) shows unmodified substrate. Percentage of substrate modified with ubiquitin was quantified for each time point and reaction rates were calculated using data points within the linear range of the reaction. Quantification of reaction rates is shown in Figure 3c.

UbcH5A–Ub UbcH5A

Ubiquitin

Ubiquitin:

120

WT

120

L8A

Time (min): 120

K11A

120

K33A

120

G35A

120

I36A

120

Q40A

120

R42A

120

I44A

120

V70A

120

L71A

120

R72A

120

L73A

120

R74A

17 -kDa

12 -

8 -

a

bUb:

Time (min): 0.5 1 1.5

WT

0.5 1 1.5 0.5 1 1.5

K11A

0.5 1 1.5

K33A

10 20 30

L8A

10 20 30

I36A Q40A

5 10 15

G35A

4 × SUMO-2


* * * * * * *

Ub:

Time (min): 0.5 1 1.5

R42A

0.5 1 1.5

L73A

10 20 30

I44A

2 4 6

V70A

5 10 15

L71A

1 2 3

R72A

1 2 3

R74A

4 × SUMO-2


* * * * * * *



RESEARCH

a

b

UbcH5A:Time:

WT N77A D87A R90A Q92A L97A K101A D112A N114A D117A

UbcH5A UbcH5A–Ub

Ubiquitin

4 × SUMO-2


UbcH5A:Lane:

WT N77A

UbcH5A UbcH5A–Ub

Ubiquitin

4 × SUMO-2

1 2 3 4 1 2 3 4 1

D87A R90A Q92A2 3 4 1 2 3 4 1 2 3 4

L97A K101A D112A1 2 3 4 1 2 3 4 1 2 3 4

N114A D117A1 2 3 4 1 2 3 4

Supplementary Figure 12. Substrate ubiquitination assay with UbcH5A mutant proteins.a, Single-turnover, RNF4-dependent substrate ubiquitination assay with mutant forms of UbcH5A. Reactions were performed as described in Supplementary Figure 10a. Reactions were stopped by non-reducing SDS-PAGE loading buffer, followed by SDS-PAGE analysis. Coomassie blue-stained gels are shown.b, Formation and stability of UbcH5A–Ub thioesters with mutations in UbcH5A. The experiment was performed as described in Supplementary Figure 10b. Lanes 1 and 2: formation of UbcH5A–Ub thioesters. Mutant forms of UbcH5A were incubated with ubiquitin, E1, and ATP to generate the UbcH5A–Ub thioester (lane 1: time 0; lane 2: after 12 min at 37°). Lanes 3 and 4: stability of the UbcH5A–Ub thioesters in the substrate ubiquitination assay in the absence of RNF4. Time points were taken at 0.5 min (lane 3) and 20 min (lane 4). Non-reducing SDS-PAGE gels stained with Coomassie blue are shown.



Supplementary Figure 13. Mutational analysis of the RNF4 RING–UbcH5A–Ub complex: mutations in UbcH5A. a, Ability of UbcH5A mutants to form thioesters with ubiquitin. UbcH5A mutant proteins (His6-tagged) were incubated with ubiquitin, E1 and ATP at 37° for 12 min and the samples were analyzed by non-reducing SDS-PAGE, followed by staining with Coomassie blue. b, Single-turnover, RNF4-dependent substrate ubiquitination assays with mutant forms of UbcH5A. 125I-labelled substrate was used to allow quantification of ubiquitination activity. Phosphorimager scans of SDS-PAGE gels are shown. The first lane (*) shows unmodified substrate. Percentage of substrate modified with ubiquitin was quantified for each time point and reaction rates were calculated using data points within the linear range of the reaction. Quantification of reaction rates is shown in Figure 3d.

a

b

UbcH5A:

120

WT

120

N77A

Time (min): 120

D87A

120

R90A

120

Q92A

120

L97A

120

K101A

120

L104

A120

S108A

120

D112A

120

N114A

120

D117A

120

F62A

120

A96D

UbcH5A–Ub UbcH5A

Ubiquitin

17 -

kDa

12 -

8 -

UbcH5A:Time (min): 1.5 3 4.5

S108A

*10 20 30

L104A

* 1 2 3

D112A

* 5 10 15

D117A

*10 20 30

L97A

* 0.5 1 1.5

K101A

*

4 × SUMO-2


1 2 3

N114A

*

UbcH5A:Time (min): 0.5 1 1.5

WT

* 0.5 1 1.5

R90A

* 0.5 1 1.5

Q92A

*10 20 30

N77A

*10 20 30

F62A

* 10 20 30

A96D

*10 20 30

D87A

*

4 × SUMO-2




RESEARCH

126 -

35 -25 -17 -12 -

8 -

kDa

126 -

35 -25 -17 -12 -

8 -

kDa

UbcH5A(C85S) UbcH5A–Ub

Ubiquitin

RNF4

E1


Ubiquitin

RNF4

E1

WT+– – – –

L8A+–

K33A+

G35A+

I36A+

Ub:RNF4:Time:

R42A+–

I44A+–

L71A+– –

R74A+

Ub:RNF4:Time:

Supplementary Figure 14. UbcH5A–Ub oxyester hydrolysis assay with mutant forms of ubiquitin. To generate UbcH5A(C85S)–Ub oxyesters, UbcH5A(C85S) was incubated with ubiquitin (wild-type or mutant) and E1 in the presence of ATP, followed by addition of apyrase to deplete ATP and thus to stop the E1-mediated charging reaction. This material was then incubated at room temperature either in the presence or absence of RNF4. Time points were taken as follows: reactions without RNF4: 2.5 min and 180 min; reactions with RNF4: 2.5 min, 30 min, 60 min, 90 min, and 180 min. Reactions were stopped with SDS-PAGE loading buffer and analyzed by SDS-PAGE. Coomassie blue-stained gels are shown. Quantification of reaction rates is shown in Figure 3e.



UbcH5A(C85S) UbcH5A–Ub RNF4

E1


Ubiquitin

Ubiquitin

RNF4

E1

WT+–

N77A+–

D87A+–

UbcH5A(C85S):RNF4:Time:

A96D+–

L97A+–

K101A+–

L104A+–

S108A+–

N114A+–

D117A+–

UbcH5A(C85S):RNF4:Time:

126 -kDa

126 -

35 -25 -17 -12 -

8 -

kDa

Supplementary Figure 15. UbcH5A–Ub oxyester hydrolysis assay with mutant forms of UbcH5A. His6-tagged UbcH5A(C85S) mutants were loaded with ubiquitin in E1-mediated reaction, followed by depletion of ATP. The yield of the charging reaction was low for UbcH5A(N77A/C85S) and UbcH5A(C85S/L104A). The UbcH5A(C85S)–Ub oxyesters were then incubated at room temperature either in the presence or absence of RNF4. Time points were taken as follows: reactions without RNF4: 2.5 min and 180 min; reactions with RNF4: 2.5 min, 15 min, 30 min, 60 min, and 180 min. Reactions were stopped with SDS-PAGE loading buffer and analyzed by SDS-PAGE. Coomassie blue-stained gels are shown. Quantification of reaction rates is shown in Figure 3f.

35 -25 -17 -12 -

8 -



RESEARCH

UbiquitinUbiquitin

UbcH5AUbc1

G76

G76

C85N77

D117

C88N79

D120

Supplementary Figure 16. The C-terminal tail of ubiquitin adopts a different conformation in the model of free Ubc1–Ub thioester compared to UbcH5A–Ub bound to the RNF4 RING. a, Ubc1 in the NMR model of the Ubc1–Ub thioester15 (Protein Data Bank accession 1FXT) was superimposed onto UbcH5A in the model of the UbcH5A–Ub thioester bound to the RNF4 RING described herein. Ubc1 and ubiquitin in the Ubc1–Ub thioester are coloured grey and bluish grey, respectively. UbcH5A and ubiquitin from the RNF4 RING–UbcH5A–Ub complex are coloured green and orange, respectively. The RNF4 RING domain is not shown. b, Detailed view of the E2 active sites. The active site cysteines of the E2s and the C-terminal G76 of ubiquitin are shown in sticks representation. Proposed catalytic residues N77 and D117 in UbcH5A and the corresponding residues N79 and D120 in Ubc1 are also shown as sticks. Colour scheme is as in a.

a b

UbcH5A–ubiquitin

Ubc1–ubiquitin (1FXT)



N124N114

N124N114

D117 D117

Y87 Y87

L119 L119A129 A129

G76 G76G97 G97

G96 G96G75 G75

R74 R74

L73 L73

UbiquitinUbcH5A

SUMO-1RanGAP1

Ubc9UbiquitinUbcH5A

SUMO-1RanGAP1

Ubc9 D127 D127

N77

N85 N85

N77

R72 R72

L71 L71

E93

T95 T95

E93

Q92 Q92

K524 K524

C93

S95 S95

C93

K85 K85

D87 D87Q94 Q94

Ubc9UbcH5A

SUMO-1

RanBP2Ubiquitin

RNF4 RING

RNF4 RING

RanGAP1

Ubc9UbcH5A

SUMO-1

RanBP2

Ubiquitin

RNF4 RING

RNF4 RINGRanGAP1

a

b

c

Supplementary Figure 17 (continued)

150°



RESEARCH

UbcH5A

Ubiquitin

RNF4 RING

RNF4 RING RanGAP1

Supplementary Figure 17. Structural comparison of the RNF4 RING–UbcH5A–ubiquitin complex with a complex of SUMO-modified RanGAP1 bound to Ubc9 and the SUMO E3 ligase RanBP2. a, Ubc9 in the SUMO–RanGAP1–Ubc9–RanBP2 complex25 (Protein Data Bank accession 3UIP) was superimposed onto UbcH5A in the RNF4 RING–UbcH5A(S22R/C85K)–Ub complex. The two protomers of the dimeric RNF4 RING domain are coloured cyan and blue, UbcH5A is green, ubiquitin is orange, SUMO-1 is yellow, RanGAP1 is violet, Ubc9 is grey, and RanBP2 is red. Zinc ions are shown as grey spheres. b, As in a, but the structures are rotated by 150° as indicated. c, Stereo view of the active sites of the E2s. Colour scheme is as in a. Hydrogen bonds are indicated by dashed lines coloured either black (RNF4 RING–UbcH5A–Ub complex) or blue (SUMO–RanGAP1–Ubc9–RanBP2 complex). d, Model for a RING–substrate–UbcH5A–Ub thioester complex. The SUMO–RanGAP1–Ubc9–RanBP2 structure provides a model for how a substrate might interact with the RING-activated UbcH5A–Ub thioester. To generate this model, UbcH5A in our model of the RING–UbcH5A–Ub thioester complex was superimposed onto Ubc9 in the SUMO–RanGAP1–Ubc9– RanBP2 complex. RanGAP1 was then moved by 1.9 Å to stop all interpenetration with UbcH5A. In this position, the side chain of K524 in RanGAP1 is poised to attack the activated thioester bond in the UbcH5A–Ub thioester. Proteins are coloured as in a. e, Detailed view of the thioester bond with the incoming substrate lysine. In our model of a hypothetical substrate (RanGAP1) approaching the activated thioester bond, D117 in UbcH5A positions and activates the incoming substrate lysine for a nucleophilic attack. The resulting tetrahedral intermediate would be stabilized by a hydrogen bond with the side chain of N77. An intricate network of hydrogen bonding interactions (grey dashes) between the C-terminal tail of ubiquitin and the active site groove of UbcH5A lock the thioester bond in the activated configuration.

UbcH5AUbiquitin

RanGAP1

d

e

G76G75R74

L73C85

D117N114

D87

L86

K524

N77



SUPPLEMENTARY REFERENCES 42 Kosoy, A., Calonge, T. M., Outwin, E. A. & O'Connell, M. J. Fission yeast Rnf4 homologs

are required for DNA repair. J. Biol. Chem. 282, 20388–20394 (2007).

43 Sun, H., Leverson, J. D. & Hunter, T. Conserved function of RNF4 family proteins in

eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J. 26, 4102–4112

(2007).

44 Huang, K. Y., Amodeo, G. A., Tong, L. & McDermott, A. The structure of human ubiquitin

in 2-methyl-2,4-pentanediol: a new conformational switch. Protein Sci. 20, 630-639 (2011).

SUPPLEMENTARY INFORMATION with E34 and G35 of ubiquitin in the structure of the RNF4...

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Transcript of SUPPLEMENTARY INFORMATION with E34 and G35 of ubiquitin in the structure of the RNF4...