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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, sicheri@lunenfeld.ca, 416-586-8471
Mike Tyers, md.tyers@umontreal.ca, 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
Huang et al. Supplementary Information p.2
Nature Chemical Biology: doi:10.1038/nchembio.1412
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
Huang et al. Supplementary Information p.3
Nature Chemical Biology: doi:10.1038/nchembio.1412
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 =
Huang et al. Supplementary Information p.4
Nature Chemical Biology: doi:10.1038/nchembio.1412
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.
Huang et al. Supplementary Information p.5
Nature Chemical Biology: doi:10.1038/nchembio.1412
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
Huang et al. Supplementary Information p.6
Nature Chemical Biology: doi:10.1038/nchembio.1412
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
Huang et al. Supplementary Information p.7
Nature Chemical Biology: doi:10.1038/nchembio.1412
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).
Huang et al. Supplementary Information p.8
Nature Chemical Biology: doi:10.1038/nchembio.1412
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.
Huang et al. Supplementary Information p.9
Nature Chemical Biology: doi:10.1038/nchembio.1412
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.
Huang et al. Supplementary Information p.10
Nature Chemical Biology: doi:10.1038/nchembio.1412
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
� � � � � � �
� � � � � �
� 4
� � � �� �� ���
1 2 1 � 4
� � � � � ��
� �
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.
Huang et al. Supplementary Information p.11
Nature Chemical Biology: doi:10.1038/nchembio.1412
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.
Huang et al. Supplementary Information p.12
Nature Chemical Biology: doi:10.1038/nchembio.1412
[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)
Huang et al. Supplementary Information p.13
Nature Chemical Biology: doi:10.1038/nchembio.1412
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
Huang et al. Supplementary Information p.14
Nature Chemical Biology: doi:10.1038/nchembio.1412
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-� ��������� ������"##�����#?>"���� �� ������ ��������^_ �̀� �����^_������� �����
Huang et al. Supplementary Information p.15
Nature Chemical Biology: doi:10.1038/nchembio.1412
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
log[inhibitor], �M
log[inhibitor], �M
% u
biqu
itina
tion
activ
ity%
ubi
quiti
natio
n ac
tivity
% u
biqu
itina
tion
activ
ity
Huang et al. Supplementary Information p.16
Nature Chemical Biology: doi:10.1038/nchembio.1412
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.
Huang et al. Supplementary Information p.17
Nature Chemical Biology: doi:10.1038/nchembio.1412
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).
Huang et al. Supplementary Information p.18
Nature Chemical Biology: doi:10.1038/nchembio.1412
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