Supplementary Information E2 enzyme inhibition by ... Information E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin Hao Huang1†, Derek F Ceccarelli1†,

Supplementary Information

E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

Hao Huang1†, Derek F Ceccarelli1†, Stephen Orlicky1†, Daniel J. St-Cyr2, Amy Ziemba3, Pankaj

Garg4,5, Serge Plamondon2, Manfred Auer6, Sachdev Sidhu4,5, Anne Marinier2,7, Gary Kleiger3,

Mike Tyers2,8*, Frank Sicheri1,4*

1Centre for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada M5G 1X5 2 Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Québec H3C 3J7, Canada 3 Department of Chemistry, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, NV, 89154 4Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

5 Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada 6School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR United Kingdom 7Department of Chemistry, University of Montreal, Montreal, Québec H3C 3J7, Canada

8Department of Medicine, University of Montreal, Montreal, Québec H3C 3J7, Canada † these authors contributed equally to this work

*To whom correspondence should be addressed:

Frank Sicheri, [email protected], 416-586-8471

Mike Tyers, [email protected], 514-343-6668

Huang et al. Supplementary Information p.1

Nature Chemical Biology: doi:10.1038/nchembio.1412

Supplementary Results

Supplementary Table 1. Data collection and refinement statistics for the CC0651-Cdc34A-

ubiquitin complex. Data was collected from a single frozen crystal. Highest resolution shell is

shown in parenthesis.

Data collection

Space group P1

Cell dimensions

a, b, c (Å) 55.4, 72.2, 75.9

α, β, γ (°) 71.9, 79.6, 81.6

Resolution (Å) 44.6-2.6

Rsym or Rmerge 7.9 (45.7)

I/σI 13.7 (1.2)

Completeness (%) 95.8 (85.5)

Redundancy 2.2 (1.7)

Refinement

Resolution (Å) 44.6-2.6

No. reflections 31377

Rwork/ Rfree 0.204/0.259

No. atoms

Protein 7545

Ligand/ion 166

Water 46

B-factors

Protein 39.2

Ligand/ion 56.9

Water 51.5

RMS deviations

Bond lengths (Å) 0.008

Bond angles (º) 1.26



Supplementary Table 2. Compound Characterization Data Compound Analytical Data

1 (CC0651) 1H NMR (400 MHz, CD3OD): δ (ppm) 7.49-7.59 (m, 4H), 7.30-7.42 (m, 3 H),

4.37 (ddd, J = 9.8, 8.2, 4.3 Hz, 1 H), 4.30 (d, J = 2.0 Hz, 1 H), 3.99 (dd, J =

8.2, 2.0 Hz, 1 H), 3.81 (d, J = 15.3 Hz, 1 H), 3.71 (d, J = 15.3 Hz, 1 H), 3.3

(s, 3 H, overlapping with solvent), 3.24 (dd, J = 14.1, 4.3 Hz, 1 H), 2.87 (dd,

J = 13.9, 10.0 Hz, 1 H). LC (Method B): Ret. time = 2.14 min. HRMS (ESI-

TOF) m/z: [M + H]+ Calcd for C20H22Cl2NO6 442.0819; found: 442.0813.

[α]D20 41 (c 0.18, CHCl3).

2 1H NMR (400 MHz, CD3OD): δ (ppm) 7.49 (pseudo d, J = 8.1 Hz, 2 H), 7.31

(pseudo d, J = 8.2 Hz, 2 H), 7.18 (s, 2H), 6.96 (s, 1 H), 4.41-4.33 (m, 1 H,

CHN), 4.31 (d, J = 1.8 Hz, 1H, CHCO2H), 3.98 (dd, J = 7.9, 1.8 Hz, 1 H,

OCHCHN), 3.83 (d, J = 15.3 Hz, 1 H, CH2OMe), 3.72 (d, J = 15.3 Hz, 1 H,

CH2OMe), 3.30 (s, 3 H, OCH3, overlapping with solvent signal), 3.21 (dd, J =

13.9, 4.5 Hz, 1 H, CH2Ar), 2.86 (dd, J = 14.1, 9.7 Hz, 1 H. CH2Ar), 2.34 (s, 6

H, ArCH3). 13C NMR (100 MHz, CD3OD, CH(1-3) from HSQC): δ (ppm) 130.6

(CH), 129.4 (CH), 127.7 (CH), 125.5 (CH), 74.0 (OCHCN), 72.3 (CH2OMe),

71.9 (CHCO2H), 59.3 (OCH3), 53.9 (CHN), 37.3 (CH2), 21.2 (CH3). HRMS

(ESI-TOF) m/z: [M + H]+ Calcd for C22H28NO6 402.1911; Found 402.1934.

3 1H NMR (400 MHz , CD3OD): δ (ppm) 7.60-7.51 (m, 2 H), 7.42-7.32 (m, 2

H), 7.28-7.15 (m, 2 H), 6.93-6.84 (m, 1 H), 4.43-4.33 (m, 1 H), 4.23 (d, J =

1.9 Hz, 1 H), 3.97 (dd, J = 7.9, 1.9 Hz, 1 H), 3.81 (d, J = 15.6 Hz, 1 H), 3.71

(d, J = 15.3 Hz, 1 H), 3.30 (s, 3 H, overlapping with solvent signal), 3.23 (dd,

J = 13.8, 4.4 Hz, 1 H), 2.86 (dd, J = 13.9, 9.8 Hz, 1 H). 13C NMR (100 MHz,

CD3OD, CH(1-3) from HSQC): δ (ppm) 131.0 (CH), 127.7 (CH), 110.3 (CH),

102.7 (CH), 74.0 (OCHCN), 72.3 (CH2OMe), 72.1 (CHCO2H), 59.3 (OCH3),

53.9 (CHN), 37.5 (CH2Ar). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for

C20H22F2NO6 410.1410; Found 410.1420.

4 1H NMR (400MHz ,cd3od): δ (ppm) 7.50 (pseudo d, J = 8.2 Hz, 2 H), 7.35 (s,

1 H), 7.33-7.26 (m, 3 H), 7.16 (d, J = 7.9 Hz, 1 H), 4.41-4.32 (m, 1 H), 4.31-

4.26 (m, 1 H), 3.98 (d, J = 8.2 Hz, 1 H), 3.82 (d, J = 15.3 Hz, 1 H), 3.72 (d, J

= 15.3 Hz, 1 H), 3.30 (s, 3 H, overlapping with solvent signal), 3.20 (dd, J =

14.1, 4.4 Hz, 1 H), 2.85 (dd, J = 13.8, 9.7 Hz, 1 H), 2.31 (s, 3 H), 2.28 (s, 3



H). 13C NMR (100 MHz, CD3OD, CH(1-3) from HSQC): δ (ppm) 130.8 (CH),

130.5 (CH), 128.7 (CH), 127.4 (CH), 124.9 (CH), 73.9 (CH), 72.3 (CH2), 72.0

(CH), 59.3 (CH3), 53.9 (CH), 37.3 (CH2), 19.7 (CH3), 19.2 (CH3). HRMS

(ESI-TOF) m/z: [M + H]+ Calcd for C22H28NO6 402.1911; found 402.1926.

5 1H NMR (400 MHz , CD3OD): δ (ppm) 7.52 (psudo d, J = 8.2 Hz, 2 H), 7.32

(pseudo d, J = 8.2 Hz, 2 H), 6.93-6.79 (m, 2 H), 4.42-4.34 (m, 1 H), 4.27 (d,

J = 1.5 Hz, 1 H), 3.97 (dd, J = 7.6, 1.4 Hz, 1 H), 3.90 (s, 6 H), 3.83 (d, J =

15.1 Hz, 1 H), 3.79 (s, 3 H), 3.73 (d, J = 15.3 Hz, 1 H), 3.32 (s, 3 H,

overlapping with solvent signal), 3.21 (dd, J = 13.9, 4.3 Hz, 1 H), 2.86 (dd, J

= 13.9, 9.5, 1 H). 13C NMR (100 MHz, CD3OD, CH(1-3) from HSQC): δ (ppm)

130.1 (CH), 127.2 (CH), 104.7 (Ar CHCO), 73.7 (OCHCN), 71.9 (CH2OMe),

71.5 (CHCO2H), 60.8 (p-ArOCH3), 58.9 (CH2OCH3), 56.0 (m-ArOCH3), 53.7

(CHN), 36.8 (CH2Ar). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C23H30NO9

464.1915; found 464.1926.

6 1H NMR (400MHz , CD3OD): δ (ppm) 7.51 (d, J = 8.2 Hz, 2 H), 7.46 (d, J =

7.3 Hz, 2 H), 7.41-7.35 (m, 2 H), 7.35-7.27 (m, 4 H), 7.21-7.14 (m, 2 H), 6.96

(dd, J = 2.1, 7.9 Hz, 1 H), 5.14 (s, 2 H), 4.37 (d, J = 2.9 Hz, 1 H), 4.32-4.24

(m, 1 H), 3.98 (d, J = 8.2 Hz, 1 H), 3.82 (d, J = 15.3 Hz, 1 H), 3.72 (d, J =

15.3 Hz, 1 H), 3.30 (s, 3 H, overlapping with solvent signal), 3.21 (dd, J =

4.4, 13.8 Hz, 1 H), 2.86 (dd, J = 9.8, 13.9 Hz, 1 H). 13C NMR (100 MHz,

CD3OD, CH(1-3) from HSQC): δ (ppm) 130.6 (CH), 129.3 (CH), 128.7 (CH),

128.3 (CH), 127.6 (CH), 120.2 (CH), 114.4 (CH), 114.3 (CH), 73.9

(OCHCN), 72.3 (CH2OMe), 71.9 (CHCO2H), 70.8 (OCH2Ar), 59.1

(CH2OCH3), 54.0 (CHN), 37.2 (CH2Ar). HRMS (ESI-TOF) m/z: [M + H]+

Calcd for C27H30NO7 480.2017; found 480.2044.

7 1H NMR (400 MHz, CDCl3): δ (ppm) 7.43 (pseudo d, J = 8.6 Hz, 2 H), 7.10

(pseudo d, J = 8.2 Hz, 2 H), 4.72 (br. s., 1 H), 3.83 (br. s., 1 H), 3.61-3.72

(m, 1 H), 3.48-3.61 (m, 1 H), 2.81 (d, J = 7.04 Hz, 2 H), 2.19 (br. s., 1 H),

1.42 (s, 9 H). LC (Method A): Ret. time = 2.22 min. LC-MS (APCI) m/z: [M -

Boc + H2]+ Calcd for C9H13BrNO 230.0; found 230.2, 232.0. [α]D20 –24.9 (c

1.07, CHCl3).

8 1H NMR (400 MHz, CDCl3): δ (ppm) 7.49 (d, J = 8.2 Hz, 2 H), 7.41-7.46 (m,

2 H), 7.28-7.36 (m, 3 H), 4.76 (br. s., 1 H), 3.91 (br. s., 1 H), 3.71 (dt, J =



10.3, 4.8 Hz, 1 H), 3.60 (dt, J = 10.8, 5.2 Hz, 1 H), 2.91 (d, J = 7.0 Hz, 2 H),

2.19 (br. s., 1 H), 1.43 (s, 9 H). LC (Method A): Ret. time = 2.69 min. LC-MS

(APCI) m/z: [M - Boc + H2]+ Calcd for C15H16Cl2NO 296.1; found 296.0,

298.2.

9 1H NMR (400 MHz, CDCl3): δ (ppm) 9.67 (s, 1 H), 7.49 (d, J = 7.0 Hz, 2 H),

7.44 (s, 2 H), 7.34 (d, J = 1.6 Hz, 1 H), 7.27 (t, J = 3.5 Hz, 3 H), 5.08 (br. s.,

1 H), 4.48 (d, J = 5.9 Hz, 1 H), 3.03-3.32 (m, 2 H), 1.45 (s, 9 H).

10 1H NMR (400 MHz, CDCl3): δ (ppm) 7.49 (d, J = 8.2 Hz, 2 H), 7.45 (d, J =

2.0 Hz, 2 H), 7.32-7.37 (m, 1 H), 7.23-7.30 (m, 2 H), 6.93 (dd, J = 15.7, 5.1

Hz, 1 H), 5.82-5.96 (m, 1 H), 4.65 (br. s., 1 H), 4.54 (br. s., 1 H), 3.74 (s, 3

H), 2.95 (d, J = 6.7 Hz, 2 H), 1.31-1.49 (m, 9 H). LC (Method A): Ret. time =

2.89 min. LC-MS (APCI) m/z: [M - Boc + H2]+ Calcd for C18H18Cl2NO2 350.1;

found 350.2, 352.0.

11 1H NMR (400 MHz, CD3OD): δ (ppm) 7.48-7.60 (m, 4 H), 7.32-7.43 (m, 3 H),

4.31-4.40 (m, 1 H), 3.93 (td, J = 9.5, 3.7 Hz, 1 H), 3.69-3.88 (m, 4 H), 3.24

(dd, J = 13.7, 3.5 Hz, 1 H), 2.70 (dd, J = 13.7, 10.2 Hz, 1 H), 1.26-1.38 (m, 9

H). LC (Method A): Ret. time = 2.79 min. LC-MS (APCI) m/z: [M - Boc + H2]+

Calcd for C18H20Cl2NO4 384.1; found 384.0, 386.0.

12 1H NMR (400 MHz, CD3OD): δ (ppm) 7.50-7.60 (m, 4 H), 7.33-7.43 (m, 3 H),

4.32-4.42 (m, 2 H), 3.97 (dd, J = 8.0, 2.2 Hz, 1 H), 3.68-3.86 (m, 5 H), 3.33

(s, 3 H, overlapping with solvent signal), 3.23 (dd, J = 13.7, 4.3 Hz, 1 H),

2.87 (dd, J = 13.9, 10.0 Hz, 1H). LC (Method A): Ret. time = 2.22 min. LC-

MS (APCI) m/z: [M + H]+ Calcd for C21H24Cl2NO6 456.1; found 456.2, 458.0.



a

105

110

115

120

125

15N

(ppm

)7.08.09.0 6.0

1H (ppm)

[15N-Ub] : [Cdc34AFL] : [CC0651] 1 : 1 : 0 1 : 1 : 0.5

[15N-Ub] : [CC0651] 1 : 0 1 : 2 105

110

115

120

125

15N

(ppm

)

7.08.09.0 6.01H (ppm)

7.08.09.0 6.01H (ppm)

105

110

115

120

125

15N

(ppm

)

[15N-Ub] : [Cdc34AFL] 1 : 0 1 : 1

7.08.09.0 6.01H (ppm)

105

110

115

120

125

15N

(ppm

)

[15N-Ub] : [Cdc34ACAT] 1 : 0 1 : 1

c

b

d

15N-Ub +/- Cdc34ACAT

15N-Ub +/- Cdc34AFL

15N-Ub +/- CC0651

(15N-Ub+Cdc34AFL) +/- CC0651

Supplementary Figure 1. Interaction analysis of 15N-Ub, Cdc34A and CC0651. (a-d) Superposition of the 1H, 15N-HSQC spectra of (a) 15N-Ub (black) versus 15N-Ub:Cdc34ACAT (red), (b) 15N-Ub (black) versus 15N-Ub:CC0651 (red), (c) 15N-Ub (black) versus 15N-Ub:Cdc34AFL (red) and (d) 15N-Ub:Cdc34AFL (black) versus 15N-Ub:Cdc34AFL:CC0651 (red). Stoichiometric ratios of components in each experiment are indicated. (e-f) Peak intensity change versus residue number for (e) 15N-Ub versus 15N-Ub:Cdc34AFL (panel c) and (f) 15N-Ub:Cdc34AFL versus 15N-Ub:Cdc34AFL:CC0651 (panel d). (g) Interaction surfaces on ubiquitin for Cdc34AFL in the absence and presence of CC0651. Surfaces were defined using peak intensity change cut-offs shown as red dashed lines in panels e and f.

L73Q40

K48F45

L67

Q49Q41

L71

R42V70

K6

T7I44

R72

G47

L8

S65N60Y59

A46 T12

K33

G76

L73

D39

G76

180o

0

0.2

0.4

0.6

0.8

1

3 8 14 22 29 34 41 47 52 58 63 69 74

Nor

mal

ized

Pea

k In

tens

ity

0

0.2

0.4

0.6

0.8

1

3 8 14 22 29 34 41 47 52 58 63 69 74

Nor

mal

ized

Pea

k In

tens

ity

0.1

e f

Cdc34AFL

L73

Q40

K48F45

G47

Q49 T7

L8Q41

L71R42

V70

L43

R72

g

L73

R72180o

Cdc34AFL + CC0651

Ubiquitin ResiduesUbiquitin Residues



c

a

10 5.0

11 0.0

11 5.0

12 0.0

12 5.0

13 0.0

N (

pp

m)

7.08.09.010.01H (ppm)

105

110

115

120

125

130

15

N (

pp

m)

[15N-Cdc34ACAT] : [Ub] : [CC0651] 1 : 0 : 0 1 : 4 : 0

2 4 8 16 31 62 125

250

500

[CC0651], �M

�–Catenin

�-Catenin-(Ub)n

Supplementary Figure 2. CC0651 potentiates the interaction between 15N-Cdc34A and ubiquitin. (a) Super-position of 1H, 15N-HSQC spectra at the indicated molar ratios for 15N-Cdc34ACAT (black) versus 15N-Cdc34ACAT:Ub (red). (b) CC0651 titration analysis of indicated E2 enzyme binding to ubiquitin using a TR-FRET assay. Data presented as mean +/- S.E.M., n=2. (c) Dose response inhibition profile of CC0651 on �� signals were quantified and plotted as mean +/- S.E.M., n=2.

Log [CC0651], M

IC50= 18 ± 1.3 �M

-6 -5 -4 -3

0.02

0.04

0.06

0.08

0

min

-1

b

Cdc34A + Ub : EC50= 14 ± 2 �MCdc34B + Ub : NDCdc34ACAT + Ub : ND

Cdc34A Cdc34B Cdc34ACAT

0 20 40 60 80 1000.0

0.1

0.2

0.3

0.4

[CC0651], �M

Em

issi

on R

atio



a

b

Supplementary Figure 3. Representative electron density maps. (a) Unbiased electron density map of the CC0651-Cdc34A-ubiquitin complex before inclusion of CC0651 coordinates into the refinement model. 2Fo-Fc �� !�� "�#$&�� '� ��!�� <�>$&�� #?>"�� '��Y��#?>"\��^_ �̀� �� {\�� Y��|��!&�� -�{��'� �� #?>"��^_`�� }�� "�#$�� '�as in (a).



a

bUb

Cdc34A

Glu133

Asn132

Gln49Arg42

Arg72

Ser129

Glu133

Asn132

Gln49Arg42

Arg72

Ub

Cdc34A

Ser129

CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

Gly47

Lys48

Cdc34A

UbCC0651

Gln49

Val70

Ile44

Leu8Leu125

Thr122

Ser129

Ile128 Ser126

Ub

Cdc34A

Val70

Ile44

Leu8Leu125

Thr122

Ser129

Ile128 Ser126

Ub

Cdc34A

c

Gly47

Lys48

Cdc34A

UbCC0651

Gln49

Supplementary Figure 4. Notable contact features of the CC0651-Cdc34A-ubiquitin complex. Stereo views are shown for (a) contacts between ubiquitin and CC0651, (b) hydrogen bonding network between ubiquitin and Cdc34A and (c) hydrophobic contacts between ubiquitin and Cdc34A. Color scheme as in Fig. 3.



Cdc34A+Ub+SumoUbc9

41˚

Cdc34A+UbUbc1+Ub

26˚

Supplementary Figure 5. Binding orientation of ubiquitin to Cdc34A induced by CC0651 is similar to a previously characterized E2-SUMO interaction. Overlay of the CC0651-Cdc34A-ubiquitin complex with a UbcH9-SUMO complex (PDB 1Z5S 1) is shown. Superposition was performed using the E2 coordi-nates. Rotation angles relate the orientations of ubiquitin and SUMO subunits. Overlay of the CC0651-Cdc34A-ubiquitin complex with the Ubc1-Ub covalent complex (PDB 1FXT 2) shown in Fig. 3e is included for reference.



Cdc34A M A R P L V P S S Q K A L L L E L K G L Q E E P V E G F R V T L V D E G - - - D L Y N W E V A I F G P - P N T Y Y E G G Y F K A R L K F P I D Y P Y S P P A F R F L T K MW H P N I Y E T G -Cdc34B M A Q Q Q M T S S Q K A L M L E L K S L Q E E P V E G F R I T L V D E S - - - D L Y N W E V A I F G P - P N T L Y E G G Y F K A H I K F P I D Y P Y S P P T F R F L T K MW H P N I Y E N G -Ube2G1 - - - M T E L Q S A L L L R R Q L A E L N K N P V E G F S A G L I D D N - - - D L Y R W E V L I I G P - P D T L Y E G G V F K A H L T F P K D Y P L R P P K M K F I T E I WH P N V D K N G -Ube2G2 - - - - M A G T A L K R L M A E Y K Q L T L N P P E G I V A G P M N E E - - - N F F E W E A L I M G P - E D T C F E F G V F P A I L S F P L D Y P L S P P K M R F T C E M F H P N I Y P D G -Ube2A - - - - M S T P A R R R L M R D F K R L Q E D P P A G V S G A P S E N - - - - N I M V WN A V I F G P - E G T P F E D G T F K L T I E F T E E Y P N K P P T V R F V S K M F H P N V Y A D G -Ube2B - - - - M S T P A R R R L M R D F K R L Q E D P P V G V S G A P S E N - - - - N I M QW N A V I F G P - E G T P F E D G T F K L V I E F S E E Y P N K P P T V R F L S K M F H P N V Y A D G -Ube2C S G G A A R G P V G K R L Q Q E L M T L M M S G D K G I S A F P E S D N - - - - L F K W V G T I H G A - A G T V Y E D L R Y K L S L E F P S G Y P Y N A P T V K F L T P C Y H P N V D T Q G -Ube2D1 - - - - - - - M A L K R I Q K E L S D L Q R D P P A H C S A G P V G D D - - - - L F H W Q A T I M G P - P D S A Y Q G G V F F L T V H F P T D Y P F K P P K I A F T T K I Y H P N I N S N G -Ube2D2 - - - - - - - M A L K R I H K E L N D L A R D P P A Q C S A G P V G D D - - - - M F H W Q A T I M G P - N D S P Y Q G G V F F L T I H F P T D Y P F K P P K V A F T T R I Y H P N I N S N G -Ube2D3 - - - - - - - M A L K R I N K E L S D L A R D P P A Q C S A G P V G D D - - - - M F H W Q A T I M G P - N D S P Y Q G G V F F L T I H F P T D Y P F K P P K V A F T T R I Y H P N I N S N G -Ube2D4 - - - - - - - - A L K R I Q K E L T D L Q R D P P A Q C S A G P V G D D - - - - L F H WQ A T I M G P - N D S P Y Q G G V F F L T I H F P T D Y P F K P P K V A F T T K I Y H P N I N S N G -Ube2E1 K N S K L L S T S A K R I Q K E L A D I T L D P P P N C S A G P K G D N - - - - I Y E WR S T I L G P - P G S V Y E G G V F F L D I T F T P E Y P F K P P K V T F R T R I Y H C N I N S Q G -Ube2E2 K T A A K L S T S A K R I Q K E L A E I T L D P P P N C S A G P K G D N - - - - I Y E WR S T I L G P - P G S V Y E G G V F F L D I T F S P D Y P F K P P K V T F R T R I Y H C N I N S Q G -Ube2E3 K T T A K L S T S A K R I Q K E L A E I T L D P P P N C S A G P K G D N - - - - I Y E W R S T I L G P - P G S V Y E G G V F F L D I T F S S D Y P F K P P K V T F R T R I Y H C N I N S Q G -Ube2F D S T R R V S V R D K L L V K E V A E L E A N L P C T C K V H F P D P N - - - - K L H C F Q L T V T P - D E G Y Y Q G G K F Q F E T E V P D A Y N M V P P K V K C L T K I W H P N I T E T G -Ube2H - - M S S P S P G K R R M D T D V V K L I E S K H E V T I L G G - - - - - - - - L N E F V V K F Y G P - Q G T P Y E G G V W K V R V D L P D K Y P F K S P S I G F M N K I F H P N I D E A S GUbe2I - - - - M S G I A L S R L A Q E R K A W R K D H P F G F V A V P T K N P D G M N L M N W E C A I P G K - K G T P W E G G L F K L R M L F K D D Y P S S P P K C K F E P P L F H P N V Y P S G -Ube2J1 T R Y N L K S P A V K R L M K E A A E L - K D P T D H Y H A Q P L E D N - - - - L F E WH F T V R G P - P D S D F D G G V Y H G R I V L P P E Y P M K P P S I I L L T A N G R F E V G K - - -Ube2J2 S S K R A P T T A T Q R L K Q D Y L R I K K D P V P Y I C A E P L P S N - - - - I L E W F K R F S W L S L L S S WD G G Y Y H G K L I F P R E F P F K P P S I Y M I T P N G R F K C N T - - -Ube2K - M A N I A V Q R I K R E F K E V L K S E E T S K N Q I K V D L V D E N - - - - F T E L R G E I A G P - P D T P Y E G G R Y Q L E I K I P E T Y P F N P P K V R F I T K I WH P N I S S V T GUbe2L3 - - - - - - M A A S R R L M K E L E E I R K C G M K N F R N I Q V D E A - - - - N L L T W Q G L I V P - D N P P Y D K G A F R I E I N F P A E Y P F K P P K I T F K T K I Y H P N I D E K G -Ube2L6 - - - - - - M M A S M R V V K E L E D L Q K K P P P Y L R N L S S D D A - - - - N V L V W H A L L L P - D Q P P Y H L K A F N L R I S F P P E Y P F K P P M I K F T T K I Y H P N V D E N G -Ube2M G S S K K A S A A Q L R I Q K D I N E L N - - L P K T C D I S F S D P D - - - - D L L N F K L V I C P - D E G F Y K S G K F V F S F K V G Q G Y P H D P P K V K C E T M V Y H P N I D L E G -Ube2N - - - - - - - G L P R R I I K E T Q R L L A E P V P G I K A E P D E S N - - - - A R Y F H V V I A G P - Q D S P F E G G T F K L E L F L P E E Y P M A A P K V R F M T K I Y H P N V D K L G -Ube2NL - - - - - M A E L P H R I I K E T Q R L L A E P V P G I K A E P D E S N - - - - A R Y F H V V I A G E S K D S P F E G G T F K R E L L L A E E Y P M A A P K V R F M T K I Y H P N V D K L E -Ube2O1 - - - - - - - - F F S T V R K E M A L L A T S L P E G I M V K T F E D R - - - - M D L F S A L I K G P - T R T P Y E D G L Y L F D I Q L P N I Y P A V P P H F C Y L S Q R L N P N L Y D N G -Ube2Q1 G A V S G S V Q A T D R L M K E L R D I Y R S Q S F K G G N Y A V E L V N D - S L Y D WN V K L L K V D Q D S A L H A D F I L L N F S F K D N F P F D P P F V R V V S P V L S G G Y V L G G GUbe2Q2 G A V S G S V Q A S D R L M K E L R D I Y R S Q S Y K T G I Y S V E L I N D - S L Y D WH V K L Q K V D P D S P L H I E Y I L L N F S F K D N F P F D P P F V R V V L P V L S G G Y V L G G GUbe2QL1 R Q Q H C T Q V R S R R L M K E L Q D I A R L S D R F I S V E L V D - - - - E S L F D W N V K L H Q V D K D S V L MKT E F I L L N L T F P D N F P F S P P F M R V L S P R L E N G Y V L D G GUbe2S N V E N L P P H I I R L V Y K E V T T L T A D P P D G I K V F P N E E D - - - - L T D L Q V T I E G P - E G T P Y A G G L F R M K L L L G K D F P A S P P K G Y F L T K I F H P N V G A N G -Ube2T - - - - - - M Q R A S R L K R E L H M L A T E P P P G I T C W Q D K D Q - - - - M D D L R A Q I L G G - A N T P Y E K G V F K L E V I I P E R Y P F E P P Q I R F L T P I Y H P N I D S A G -Ube2U - - - - M H G R A Y L L L H R D F C D L K E N N Y K G I T A K P V S E D - - - - M M E W E V E I E G L - Q N S V W Q G L V F Q L T I H F T S E Y N Y A P P V V K F I T I P F H P N V D P H T GUbe2V1 - - T G V K V P R N F R L L E E L E E G Q K G V G D G T V S WG L E D D E D M T L T R W T G M I I G P - P R T I Y E N R I Y S L K I E C G P K Y P E A P P F V R F V T K I N M N G V N S S N GUbe2V2 V S T G V K V P R N F R L L E E L E E G Q K G V G D G T V S WG L E D D E D M T L T R W T G M I I G P - P R T N Y E N R I Y S L K V E C G P K Y P E A P P S V R F V T K I N M N G I N N S S GUbe2W - - - - - M A S M Q K R L Q K E L L A L Q N D P P P G M T L N E K S V Q N - - S I T QW I V D M E G A - P G T L Y E G E K F Q L L F K F S S R Y P F D S P Q V M F T G E P V H P H V Y S N G -Ube2Z - - - R T A P Q C L L R I K R D I M S I Y K E P P P G M F V V P D T V D - - - - M T K I H A L I T G P - F D T P Y E G G F F L F V F R C P P D Y P I H P P R V K L M T T R F N P N F Y R N G -BIRC6 A A R A R R L A Q E A V T L S T S L P L S S S S S V F V R C D E E R L D I M K V L I T G P - A D T P Y A N G C F E F D V Y F P Q D Y P S S P P L V N L E T T G F N P N L Y N D G -TSG101 - - V S K Y K Y R D L T V R E T V N V I T L Y K D L K P V L D D F N D G S S R E L M N L T G T I P V P Y R G N T Y - - - N I P I C L W L L D T Y P Y N P P I C F V � T I K T G K H V D A N G -UEVLD - - - - - M E F D C E G L R R L L G K Y K F R D L T V E E L R N - - - - - - - - V N V F F P H F K Y S - M D T Y G N T Y N I P I R F W I L D S H P F A P P I C F L K P T A N M G I L V G K H VFTS P P R T A P K K Q L P S I P K N A L P I T K P T S P A P A A Q S T N G T H A S L L A E F T L V V K Q K L P G V Y V Q D G V F K F T V Y I P D N Y P G D C P R L V F D I P V F H P L V D P T S GScCdc34 - M S S R K S T A S S L L L R Q Y R E L T D P K K A I P S F H E I L E D D - S N I F T WN I G V M V L N E D S I Y H G G F F K A Q M R F P E D F P F S P P Q F R F T P A I Y H P N V Y R D G -

Cdc34A D V C I S I L H P P V D D P Q S G E L P S E R W N P T Q N V R T I L L S V I S L L N E P N T F S P A N V D A S V M Y R K W K E S K G T K D R E Y T D I I R K Q V L G T K V D A E R D G V K VCdc34B D V C I S I L H P P V D D P Q S G E L P S E R W N P T Q N V R T I L L S V I S L L N E P N T F S P A N V D A S V M F R K W R D S K G T K D K E Y A E I I R K Q V S A T K A E A E K D G V K VUbe2G1 D V C I S I L H E P G E D K Y G Y E K P E E R W L P I H T V E T I M I S V I S M L A D P N G D S P A N V D A A K E WR E D R I - - - - - N G E F K R K V A R C V R K S Q E T A F E - - - - -Ube2G2 R V C I S I L H A P G D D P M G Y E S S A E R W S P V Q S V E K I L L S V V S M L A E P N D E S G A N V D A S K MW R D D R V - - - - - - E Q F Y K I A K Q I V Q K S L G L - - - - - - - -Ube2A S I C L D I L Q N - - - - - - - - - - - - - R W S P T Y D V S S I L T S I Q S L L D E P N P N S P A N S Q A A Q L Y Q E N K T - - - - - - R E Y E K R V S A I V E Q S WR D C - - - - - - -Ube2B S I C L D I L Q N - - - - - - - - - - - - - R W S P T Y D V S S I L T S I Q S L L D E P N P N S P A N S Q A A Q L Y Q E N K T - - - - - - R E Y E K R V S A I V E Q S W N D S - - - - - - -Ube2C N I C L D I L K E K - - - - - - - - - - - - - W S A L Y D V R T I L L S I Q S L L G E P N I D S P L N T H A A E L W K N P T L - - - - A F K K Y L Q E T Y S K Q V T S Q E P - - - - - - - -Ube2D1 S I C L D I L R S Q - - - - - - - - - - - - - W S P A L T V S K V L L S I C S L L C D P N P D D P L V P D I A Q I Y K S D K A - - - - - - E K Y N R H A R E W T Q K Y A M - - - - - - - - -Ube2D2 S I C L D I L R S Q - - - - - - - - - - - - - W S P A L T I S K V L L S I C S L L C D P N P D D P L V P E I A R I Y K T D R A - - - - - - E K Y N R I A R E W T Q K Y A M - - - - - - - - -Ube2D3 S I C L D I L R S Q - - - - - - - - - - - - - W S P A L T I S K V L L S I C S L L C D P N P D D P L V P E I A R I Y K T D R A - - - - - - D K Y N R I S R E W T Q K Y A M - - - - - - - - -Ube2D4 S I C L D I L R S Q - - - - - - - - - - - - - W S P A L T V S K V L L S I C S L L C D P N P D D P L V P E I A H T Y K A D R A - - - - - - E K Y N R L A R E W T Q K Y A M - - - - - - - - -Ube2E1 V I C L D I L K D N - - - - - - - - - - - - - W S P A L T I S K V L L S I C S L L T D C N P A D P L V G S I A T Q Y M T N R A - - - - - - A E H D R M A R QW T K R Y A T - - - - - - - - -Ube2E2 V I C L D I L K D N - - - - - - - - - - - - - W S P A L T I S K V L L S I C S L L T D C N P A D P L V G S I A T Q Y M T N R A - - - - - - A E H D R M A R QW T K R Y A T - - - - - - - - -Ube2E3 V I C L D I L K D N - - - - - - - - - - - - - W S P A L T I S K V L L S I C S L L T D C N P A D P L V G S I A T Q Y L T N R A - - - - - - A E H D R I A R QW T K R Y A T - - - - - - - - -Ube2F E I C L S L L R E H S I D G T - - - - - - - GW A P T R T L K D V V W G L N S L F T D L L N F D D P L N I E A A E H H L R D K T - - - - E D F R N K V D D Y I K R Y A R - - - - - - - - - -Ube2H T V C L D V I N Q T - - - - - - - - - - - - - W T A L Y D L T N I F E S F L P L L A Y P N P I D P L N G D A A A M Y L H R P L - - - - - E E Y K Q K I K E Y I Q K Y A T E E A L K E Q E E GUbe2I T V C L S I L E E D - - - - - - - - - - - K D W R P A I T I K Q I L L G I Q E L L N E P N I Q D P A Q A E A Y T I Y C Q N R A - - - - V E Y E K R V R A Q A K K F A P S - - - - - - - - - -Ube2J1 K I C L S I S G H H P E T - - - - - - - - - - W Q P S W S I R T A L L A I I G F M P T K G E G A I G S L D Y T P E E R R A L A K K S Q S D F C C E G C G S A M K D V L L P L K S G S D S S QUbe2J2 R L C L S I T D F H P D T - - - - - - - - - - W N P A W S V S T I L T G L L S F M V E K G P T L G S I E T S D F T K R Q L A V Q S L A A F N L K D K V F C E L F P E V V E E I K Q K Q K A QUbe2K A I C L D I L K D Q - - - - - - - - - - - - - W A A A M T L R T V L L S L Q A L L A A A E P D D P Q D A V V A N Q Y K Q N P A - - - - E M F K Q T A R L W A H V Y A G A P V S S P E Y T K -Ube2L3 Q V C L P V I S A E N - - - - - - - - - - - - W K P A T K T D Q V I Q S L I A L V N D P Q P E H P L R A D L A E E Y S K D R A - - - - K K F C K N A E E F T K K Y G E K R P V D - - - - - -Ube2L6 Q I C L P I I S S E N - - - - - - - - - - - - W K P C T K T C Q V L E A L N V L V N R P N I R E P L R M D L A D L L T Q N P C - - - - E L F R K N A E E F T L R F G V D R P S - - - - - - -Ube2M N V C L N I L R E D - - - - - - - - - - - - - W K P V L T I N S I I Y G L Q Y L F L E P N P E D P L N K E A A E V L Q N N R V - - - - R L F E Q N V Q R S M R G G Y I G S T Y F E R C L K -Ube2N R I C L D I L K D K - - - - - - - - - - - - - W S P A L Q I R T V L L S I Q A L L S A P N P D D P L A N D V A E QW K T N E A - - - - - - A Q A I E T A R A W T R L Y A M N N I - - - - - -Ube2NL R I S L D I L K D K - - - - - - - - - - - - - W S P A L Q I R T V L L S I Q A L L N A P N P D D P L A N D V V E QW K T N E A - - - - - - A Q A I E T A R A W T R L Y A M N S I - - - - - -Ube2O1 K V C V S L L G T W I G K G T - - - - - - E R W T S K S S L L Q V L I S I Q G L I L V N E P Y Y N E A G F D S D R G L Q E G Y K - - - - E N S R C Y N E M A L I R V V Q S M T Q L - - - - -Ube2Q1 A I C M E L L T K Q G - - - - - - - - - - - - W S S A Y S I E S V I M Q I S A T L V K G K A R V Q F G A N K S Q Y S L T R A A - - - - - Q Q S Y K S L V Q I H E K N GW Y T P P K E D G - -Ube2Q2 A L C M E L L T K Q G - - - - - - - - - - - - W S S A Y S I E S V I M Q I N A T L V K G K A R V Q F G A N K N Q Y N L A R A A - - - - - Q Q S Y N S I V Q I H E K N GW Y T P P K E D G - -Ube2QL1 A I C M E L L T P R G - - - - - - - - - - - - W S S A Y T V E A V M R Q F A A S L V K G Q G R I C R K A G K S K K S F S R K E A - - - A E A T F K S L V K T H E K Y GW V T P P V S D G - -Ube2S E I C V N V L K R D - - - - - - - - - - - - - W T A E L G I R H V L L T I K C L L I H P N P E S A L N E E A G R L L L E N Y E - - - - E E Y A A R A R L L T E I H G G A G G P S G - - - - -Ube2T R I C L D V L K L P P K G A - - - - - - - - - W R P S L N I A T V L T S I Q L L M S E P N P D D P L M A D I S S E F K Y N K S - - - - P A F L K N A R QW T E K H A R Q K Q K A - - - - - -Ube2U Q P C I D F L D N P E K - - - - - - - - - - - W N T N Y T L S S I L L A L Q V M L S N P V L E N P V N L E A A R I L V K D E N S L Y R T I L R L F N R P L Q M K D D S Q E L P K D P R K - -Ube2V1 V V D P R A I S V L A K - - - - - - - - - - - W Q N S Y S I K V V L Q E L R R L M M S K E N M K L P Q P P E G Q C Y S N S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Ube2V2 M V D A R S I P V L A K - - - - - - - - - - - W Q N S Y S I K V V L Q E L R R L M M S K E N M K L P Q P P E G Q T Y N N S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Ube2W H I C L S I L T E D - - - - - - - - - - - - - W S P A L S V Q S V C L S I I S M L S S C K E K R R P P D N S F Y V R T C N K N P K K T A K WW Y H D D T C - - - - - - - - - - - - - - - - - !Ube2Z K V C L S I L G T W T G P - - - - - - - - - A W S P A Q S I S S V L I S I Q S L M T E N P Y H N E P G F E Q E R H P G D S K A - - - - N Y N E C I R H E T I R V A V C D M M E G K C P C - -BIRC6 K V C L S I L N T W H G R P E E K - - - - - - W N Q T S S F L Q V L V S V Q S L I L V A E P Y F N E P G Y E R S R G T P S G T Q - - - - - S S R E Y D G N I R Q A T V K W A M L E Q I R N P ! !TSG101 K I Y L P Y L H E - - - - - - - - - - - - - - W K H P Q S D L L G L I Q V M I V V F G D E P P V F S R P I S A S Y P P Y Q A T G P P N P T S Y M P G M P G G I S P Y P S G Y P P N P S G Y PUEVLD D A Q G R I Y L P Y L Q N - - - - - - - - - - W S H P K S V I V G L I K E M I A K F Q E E L P M Y S L S S S D E A R Q V D L L A Y I A P K I T E G V S D T N S K S W A N H E N K T V - - - -FTS E L D V K R A F A K - - - - - - - - - - - - - W R R N H N H I W Q V L M Y A R R V F Y K I D T A S P L N P E A A V L Y E K D I N - - - - Q L F K S K V V D S V K V C T A R L F D Q P K I E DScCdc34 R L C I S I L H Q - S G D P M T D E P D A E T W S P V Q T V E S V L I S I V S L L E D P N I N S P A N V D A A V D Y R K N P E - - - - - - Q Y K Q R V K M E V E R S K Q D I P - K G F I M P

Cdc34A P T T L A E Y C V K T K A P A P D E - - - - - - G S D L F Y D D Y Y E D G - - - - - - E V E E E A D S C F G D D E D D S G T E E SCdc34B P T T L A E Y C I K T K V P S N D N - - - - - - S S D L L Y D D L Y D D D - - - - I D D E D E E E E D A D C Y D D D D S G N E E SScCdc34 P T S E S A Y I S Q S K L D E P E S N K 15 E N G S V I L Q D D D Y D D G N N 14 N D D D D E R I E F E D D D D D D D D S I D N D S V M D R K Q P H K A E D E S E D V E D V E R V S K K I

Ubiquitin M Q I F V K T L T G K T I T L E V E P S D T I E N V K A K I Q D K E G I P P D Q Q R L I F A G K Q L E D G R T L S D Y N I Q K E S T L H L V L R L R G G SUMO-1 I K L K V I G Q D S S E I H F K V K M T T H L K K L K E S Y C Q R Q G V P M N S L R F L F E G Q R I A D N H T P K E L G M E E E D V I E V Y Q E Q T G G

1 1 2 3 4

2 3

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1 2 1 � 4

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Human Ubiquitin/Sumo

Human E2s

b

a

Supplementary Figure 6. Structure-based sequence alignments for human E2 enzymes and for ubiquitin. (a) Residue numbers and secondary structure elements for human Cdc34A are indicated above the aligned sequences. A region of disorder in the Cdc34A structure is indicated by a dashed line. Residues mutated in this study are high-lighted by red stars. The catalytic cysteine and asparagine are in red and conserved hydrophobic residues in yellow. Cdc34A residues comprising the ubiquitin interface are in pink and boxed across all E2 enzymes. Ubiquitin contact residues of UbcH5A and UbcH5B (PDB 4AP4; PDB 4AUQ), and Ubc9 (PDB 1Z5S) are in purple. (b) Residue numbers and secondary structure elements for human ubiquitin are indicated above the aligned sequences of wild type ubiquitin and SUMO-1. Residues that contact Cdc34A and Ubc9 are in cyan and green.



7.08.09.0 6.01H (ppm)

105

110

115

120

125

15N

(ppm

)

[15N-Ub] : [Cdc34ACAT E133R] : [CC0651]1 : 2 : 01 : 2 : 2

[15N-Ub] : [Cdc34ACAT S129R] : [CC0651]1 : 2 : 01 : 2 : 2

7.08.09.0 6.01H (ppm)

105

110

115

120

125

15N

(ppm

)

105

110

115

120

125

15N

(pp

m)

7.08.09.0 6.01H (ppm)

[15N-Ub] : [Cdc34ACAT S129L] : [CC0651]1 : 2 : 01 : 2 : 2

7.08.09.0 6.01H (ppm)

105

110

115

120

125

15N

(pp

m)

[15N-Ub] : [Cdc34ACAT] : [CC0651] 1 : 1 : 0 1 : 1 : 2

a 15N-Ub + Cdc34ACAT Glu133Arg +/- CC0651

15N-Ub + Cdc34ACAT Ser129Arg +/- CC0651 15N-Ub + Cdc34ACAT Ser129Leu +/- CC0651

15N-Ub + Cdc34ACAT +/- CC0651

d c

b

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

3 8 14 22 29 34 41 46 51 57 62 68 73 Nor

mai

lzed

Pea

k In

tens

ity

Ubiquitin Residues

0

0.2

0.4

0.6

0.8

1

1.2

3 8 14 22 29 34 41 46 51 57 62 68 73 Nor

mai

lzed

Pea

k In

tens

ity

Ubiquitin Residues

0 0.33

1 3.3

10 33 100

0 0.33

1 3.3

10 33 100

Cdc34ASer129Arg

+UbWtCdc34ASer129Leu

+UbWt

pSic

1 on

ly

[CC0651]

pSic1

pSic1-(Ub)n

e f

g

Supplementary Figure 7. Effect of structure-guided mutations in Cdc34A on interactions of 15N-Ub, Cdc34A and CC0651 detected by NMR. (a-d) Superposition of the 1H, 15N-HSQC spectra of (a) 15N-Ub:Cdc34ACAT (black) versus 15N-Ub:Cdc34ACAT:CC0651 (red), (b) 15N-Ub:Cdc34ACAT-Glu133Arg (black) versus 15N-Ub:Cdc34ACAT-Glu133Arg:CC0651 (red), (c) 15N-Ub:Cdc34ACAT-Ser129Leu (black) versus 15N-Ub:Cdc34ACAT-

Ser129Leu:CC0651 (red) and (d) 15N-Ub:Cdc34ACAT-Ser129Arg (black) versus 15N-Ub:Cdc34CAT-Ser129Arg:CC0651 (red). Stoichiometric ratios of each component are indicated. (e-f) Peak intensity change versus residue number for the 1H, 15N-HSQC spectra of (e) 15N-ubiquitin:Cdc34ACAT Ser129Leu versus 15N-ubiquitin:Cdc34ACAT

Ser129Leu:CC0651 (panel c) and (f) 15N-ubiquitin:Cdc34ACAT Ser129Arg versus 15N-ubiquitin:Cdc34ACAT

Ser129Arg:CC0651 (panel d). (g) Sensitivity of the SCFCdc4 Sic1 ubiquitination reaction to CC0651 for the indicated mutants of Cdc34A.



[15N-Rbx1] : [Ub] : [CC0651] : [Cdc34AFL] 1 : 0 : 0 : 0 1 : 2 : 2 : 0

H108

7.08.09.010.01H (ppm)

105

110

115

120

125

15N

(ppm

)

130

Q92

I37V38

V39

Q92

Q104

W72

H77

Y106

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1

35 40 45 51 56 61 66 70 75 80 85 89 94 100 105

180o

H108

Y106

a b

c

V39VV39V39V3939393939V39V39V39V39V39V393393933V3

Q929292229222222292292222

Q1Q10Q1Q10Q10Q100Q1040040QQ1Q 0444Q 441

W72W72W72W72W72W72W72W7W72W72W72W72W72W722222W 2WW 2

H77H77H77H77H777H7777H7H77H7H77H7H7H777

Y1066666666666666666666666660606000

HH108H108H108H108H108H108H108000

YY106Y106Y106Y106Y10606Y1066666666Y10666666Y11 666666666666666666666666666666666666

H108

Y106Q92Q92Q92Q92Q92Q92Q92Q92Q929292QQ92Q9Q9QQQQ9292Q9Q922

I37I37I37777I37I3737I373737V3V3V38V38V38V38V388388V38V38V3838V3V38V3V338V3V3

[15N-Rbx1] : [Ub] : [CC0651] : [Cdc34AFL] 1 : 0 : 0 : 0 1 : 0 : 0 : 4

E102

W87

T90

V93

K89

R91

7.08.09.010.01H (ppm)

105

110

115

120

125

15N

(ppm

)

130

[15N-Rbx1] : [Ub] : [CC0651] : [Cdc34AFL] 1 : 6 : 6 : 0 1 : 6 : 6 : 4

E102

W87

V93

K89

R91

T90

L96

N98

7.08.09.010.01H (ppm)

105

110

115

120

125

15N

(ppm

)

130

7.08.09.010.01H (ppm)

105

110

115

120

125

15N

(ppm

)

130

[15N-Rbx1] : [Ub] : [CC0651] : [Cdc34AFL-T117E] 1 : 0 : 0 : 0 1 : 0 : 0 : 4

7.08.09.010.01H (ppm)

105

110

115

120

125

15N

(ppm

)

130

[15N-Rbx1] : [Ub] : [CC0651] : [Cdc34AFL-Y70R] 1 : 0 : 0 : 0 1 : 0 : 0 : 4

f

ed

g

Supplementary Figure 8. Interaction analysis of 15N-Rbx1 with Cdc34A, ubiquitin and CC0651. (a) Superposi-tion of the 1H, 15N-HSQC spectra of 15N-Rbx1 (residues 12-108) (black) versus 15N-Rbx1 (residues 12-108):Ub:CC0651 (red) are shown for the indicated stoichiometric ratios. (b) Chemical shift perturbation for the 1H, 15N-HSQC spectra superpositions in panel a. (c) Front and back surface representations of the Rbx1 RING domain (PDB 2LGV 3) with residues affected by the addition of ubiquitin and CC0651 to 15N-Rbx1 shown in blue. Affected residues were defined by the red line in panel b. (d-g) Superposition of 1H, 15N-HSQC spectra for (d) 15N-Rbx1 (black) versus 15N-Rbx1:Cdc34AFL (red), (e) 15N-Rbx1 (black) versus 15N-Rbx1:Cdc34AFL-Thr117Glu (red), (f) 15N-Rbx1 (black) versus 15N-Rbx1:Cdc34AFL-Tyr70Arg (red), and (g) 15N-Rbx1:CC0651:ubiquitin (black) versus 15N Rbx1:CC0651:Cdc34AFL:ubiquitin (red) at the indicated stoichiometric ratios.

Rbx1 residues

CS

P (p

pm)



a

b

[CC0651]

pSic1

pSic1-(Ub)n

0 -ATP

2.5

5 10 20 2.5

5 10 20 2.5

5 10 20Ub

Ub-Ub

Cdc34A~Ub

0 -ATP

2.5

5 10 20 2.5

5 10 20 2.5

5 10 20

Cdc34Awt Cdc34AY70R Cdc34AT117E Cdc34Awt Cdc34AY70R Cdc34AT117E

-DTT +DTT

0 0.33

1 3.3

10 33 100

Cdc34AY70R

0 0.33

1 3.3

10 33 100

Cdc34AT117E

0 0.33

1 3.3

10 33 100

Cdc34Awt

Supplementary Figure 9. Functional characterization of Cdc34A mutations that alter the Rbx1 interaction. (a) Sensitivity of the SCFCdc4 Sic1 ubiquitination reaction to CC0651 for wild type, Tyr70Arg and Thr117Glu versions of Cdc34A. (b) Charging of wild type, Tyr70Arg and Thr117Glu versions of Cdc34A by E1 enzyme. Cdc34A-ubiquitin thioester was detected under non-reducing conditions (-DTT).

(Ub)3



Cdc34B (�M) Cdc34A (�M) CC0651 (100 �M)

80+

832+

816+

88+

84+

8-

+8-

-

80+

832+

816+

88+

84+

8-

+8-

-

10 �M ubiquitin 50 �M ubiquitin

Cdc34B (�M) Cdc34A (�M) CC0651 (100 �M)

80+

832+

816+

88+

84+

8-

+8-

-

80+

832+

816+

88+

84+

8-

+8-

-

25 nM E3 150 nM E3

Cdc34B (�M) Cdc34A (�M) CC0651 (100 �M)

20+

28+

24+

22+

21+

8-

+8-

-

80+

832+

816+

88+

84+

8-

+8-

-

2 �M Cdc34B 8 �M Cdc34B

Cdc34B (�M) Cdc34A (�M) CC0651 (100 �M)

80+

832+

816+

88+

84+

8-

+8-

-

80+

832+

816+

88+

84+

8-

+8-

-

0.2 �M E1 4 �M E1a

b

c

d

pSic1

pSic1-(Ub)n

pSic1

pSic1-(Ub)n

pSic1

pSic1-(Ub)n

pSic1

pSic1-(Ub)n

% u

biqu

itina

tion

% u

biqu

itina

tion

% u

biqu

itina

tion

% u

biqu

itina

tion

0 10 20 30 400

20

40

60

80

100

low E1high E1

[Cdc34], �M

0 10 20 30 400

20

40

60

80

100

low E3high E3

[Cdc34A], �M

0 10 20 30 400

20

40

60

80

100

low ubhigh ub

[Cdc34A], �M

0 1 2 3 4 50

20

40

60

80

100

low E2high E2

ratio [Cdc34A]/[Cdc34B]

Supplementary Figure 10. Dominant interference of the CC0651-Cdc34A-ubiquitin complex on Cdc34B-SCFCdc4 activity. The CC0651-Cdc34A complex was titrated into Sic1 ubiquitination reactions in the presence of a fixed concentration of Cdc34B in the presence of the indicated concentrations of E1, E2, E3 and ubiquitin, in the pres-� �� "##��#?>"�� ^_ �̀� ��^_��



1 (CC0651)

2

3

4

5

6

0 100 200 300 400

0.0

0.5

1.0

0 100 200 300 400

0.0

0.5

1.0

CC0651: IC50 = 2.5±1�M 2: IC50 = 4.4±2�M

0 100 200 300 400

0.0

0.5

1.0

2: EC50 = 170±20�MCC0651: EC50 = 51±2�M

-7 -6 -5 -40

50

100

150

-7 -6 -5 -40

50

100

150

-7 -6 -5 -40

50

100

150

HN

OH

HO

O

HO

O OMe

Cl

Cl

HN

OH

HO

O

HO

O OMe

Me

Me

HN

OH

HO

O

HO

O OMe

F

F

HN

OH

HO

O

HO

O OMe

Me

Me

HN

OH

HO

O

HO

O OMe

OMe

OMe

OMe

HN

OH

HO

O

HO

O OMe

OBn

ID and structure Inhibition activity Binding activity

Supplementary Figure 11. CC0651 structure-activity relationship analysis. (left) Structure of CC0651 analogs. Benzyl moiety is indicated by Bn. (center) Inhibition profiles of CC0651 analog titration on ubiquitination of phospho-Sic1 by SCFCdc4 in vitro, as monitored by the addition of FAM-ubiquitin conjugates to phospho-Sic1. Data represented mean +/- S.E.M., n=2. (right) Effect of CC0651 analogs on Cdc34A-ubiquitin interaction by TR-FRET assay. Data represented as mean +/- S.E.M., n=2.

Em

issi

on R

atio

Em

issi

on R

atio

Em

issi

on R

atio

[inhibitor], �M

[inhibitor], �M

[inhibitor], �M

log[inhibitor], �M



% u

biqu

itina

tion

activ

ity%

ubi

quiti

natio

n ac

tivity

% u

biqu

itina

tion

activ

ity



0 0.1

0.2

0.4

0.8

1.6

3.2

6.4

12.5

25 50 100

0 0.1

0.2

0.4

0.8

1.6

3.2

6.4

12.5

25 50 100

0 0.1

0.2

0.4

0.8

1.6

3.2

6.4

12.5

25 50 100

Preparation # 1 Preparation # 3Preparation # 2

Preparation # 1 5.8 ± 1 μM

Preparation # 3 8.4 ± 4 μMPreparation # 2 2.4 ± 0.2 μM

(Ub)n

μM

IC50

-7 -6 -5 -40

50

100

log [inhibitor], M

% u

biqu

itina

tion

activ

ity

b

a

NHR

OH

MeO2C

HO Cl

Cl

NBoc

MeO2C

Cl

Cl

NBoc Cl

Cl

O

H

HNBocHO

H

a. 3,5-Cl2C6H3B(OH)2, Pd(PPh3)4, Na2CO3, MeCN/H2O (1:1), 80 °C, 93%; b. Dess-Martin reagent, CH2Cl2, 23 °C, 92%; c. (MeO)2P(O)CH2CO2Me, DBU, LiCl, MeCN, 78%; d. K2OsO2(OH)4, (DHQ)2Pyr, K4Fe(CN)4, MeSO2NH2, K2CO3, tBuOH/H2O (1:1), 54%; e. TFA, CH2Cl2; f. HO2CCH2OMe, HATU, DIPEA, MeCN, 81 % (for two steps); g. LiOH, THF, H2O, 5 °C; then HCl 1 N, 5 °C, 63%.

aCl

Cl8

NBocBr

HOH

7

b

d 1(CC0651)

g

c

9

1011: R = Boc12: R = C(O)CH2OMe e, f

H

Supplementary Figure 12. Synthetic route for CC0651, and reproducibility of potency in vitro. (a) CC0651 synthesis. Chiral amino alcohol 7 and (3,5-dichlorophenyl)boronic acid substrates were subjected to a Suzuki cross-coupling reaction to afford biaryl intermediate 8, which was selectively oxidized under non-racemizing conditions furnishing aldehyde 9. Wadsworth-Emmons olefination gave an a,b-unsaturated ester 10 which was diastereoselectively syn-dihydroxylated by Sharpless oxidation to give 11. Boc deprotection followed by HATU-mediated acylation of the resulting amine with 2-methoxyacetic acid gave amide 12. Saponification of 12 followed by acidification to pH 2 yielded CC0651. Chiral SFC analysis yielded an enantiomeric ratio of >98%. (b) Sensitivity of the SCFCdc4 Sic1 ubiquitination reaction to different preparations of CC0651. Representative gels are shown for each preparation. IC50 values +/- S.E.M. were calculated for two replicate experiments.



Supplementary References

1. Reverter, D. & Lima, C. D. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature 435, 687-692, (2005).

2. Hamilton, K. S. et al. Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail. Structure 9, 897-904, (2001).

3. Spratt, D. E., Wu, K., Kovacev, J., Pan, Z. Q. & Shaw, G. S. Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase. J Biol Chem 287, 17374-17385, (2012).



Documents

Supplementary Information E2 enzyme inhibition by ... Information E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin Hao Huang1†, Derek F Ceccarelli1†,