stke.sciencemag.org/cgi/content/full/13/641/eabb6707/DC1
Supplementary Materials for
Redox priming promotes Aurora A activation during mitosis
Daniel C. Lim*, Vladimir Joukov, T. Justin Rettenmaier, Akiko K
umagai, William G. Dunphy,
James A. Wells, Michael B. Yaffe*
*Corresponding author. Email: da
nlim@mit.edu (D.C.L.); myaffe@m
it.edu (M.B.Y.)
Published 21 July 2020,
Sci. Signal.
13
, eabb6707 (2020)
DOI: 10.1126/scisignal.abb6707
The PDF file includes:
Fig. S1. Mass spectrum of t–Aurora A C247V + C319V construct co
nsistent with ~
2
/
3
of the
CoAlated protein.
Table S1. Crystallization and cryoprotection conditions for t–A
urora A constructs.
Table S2. Crystallographic data collection and refinement stati
stics.
Legend for data file S1
Other Supplementary Material for this manuscript includes the f
ollowing:
(available at stke.sciencemag.org/cgi/content/full/13/641/eabb6
707/DC1)
Data file S1 (Microsoft Excel format). Data from tethering scre
en.
Fig. S1. Mass spectrum of t
–
Aurora A C247V + C319V construct consistent with ~
2
/
3
of the
CoAlated protein.
t
-
Aurora A C247V + C319V protein containing C290 as the single cysteine was
CoAlated by thiol
-
disulfide exchange prior to crystallization. An aliquot of the sample was analyzed on
an Applied Biosystems QStar Elite attached to a nanoLC (C4 column) to check
the extent of CoAlation.
The raw data has been deposited in the MassIVE respository
as dataset MSV000085641
(ftp://massive.ucsd.edu/MSV000085641/)
.
Table S1. Crystallization and cryoprotection conditions for t
–
Aurora A constructs.
unmodified
wild
type
cacodylate
-
modified wild
type
7
-
80 modified
wild type
8
-
34 modified
wild type
CoAlated,
unphosphorylated
wild type
CoAlated
unphosphorylated
C247V, C319V
disulfide
homodimer,
C247V, D256N,
C319V
protein stock
concentration
(mg/mL)
21.3
21.3
10
10
10
10
12.6
protein buffer
10 mM Tris pH
8, 0.2 M NaCl,
1 mM AMP
-
PNP, 2 mM
MgSO
4
, 20 mM
DTT
10 mM Tris pH
8, 0.2 M NaCl,
1 mM AMP
-
PNP, 2 mM
MgSO
4
, 40 mM
DTT
10 mM Tris pH
8, 0.5 M NaCl,
0.5 mM AMP
-
PNP, 2 mM
MgCL
2
, 2%
DMSO, 1 mM
7
-
80
10 mM Tris pH
8, 0.5 M NaC
l,
0.5 mM AMP
-
PNP, 2 mM
MgCL
2
, 2%
DMSO, 1 mM
8
-
34
10 mM Tris pH 8,
0.5 M NaCl,
5
mM
AMP
-
PNP, 10 mM
MgCl
2
, 1 mM CoA
-
TNB disulfide
6 mM Tris pH 8, 0.5
M NaCl, 1 mM AMP
-
PNP, 2 mM MgSO
4
, 5
mM AMP
-
PNP, 10
mM MgCl
2
, 1 mM
CoA
-
TNB disulfide
10 mM Tris pH
8, 0.5 M
NaCl,
1 mM AMP
-
PNP, 4 mM
MgCl
2
reservoir buffer
0.1 M HEPES
pH 7, 0.9 M
KCl, 1.08 M
ammonium
sulfate, 12%
glycerol
0.1 M sodium
cacodylate pH
6, 2 M NaCl,
9% PEG 3350
0.1 M sodium
malonate pH
5.5, 12% PEG
3350, 17%
glycerol
0.1 M sodium
malonate pH
5.5, 7
.5% PEG
3350, 11%
glycerol
0.2 M sodium
citrate pH 4, 0.1 M
sodium citrate pH
5.5, 18 mM sodium
malonate pH 6, 4
mM MgCl
2
0.2 M sodium citrate
pH 4, 0.8 M sodium
citrate pH 5.5, 6%
PEG 3350, 8%
trehalose
0.336 M sodium
malonate pH
7.5, 0.348 M
sodium
malon
ate pH
6.5
stabilization
buffer
0.1 M HEPES
pH 7, 1 M KCl,
1.05 M
ammonium
sulfate, 8%
glycerol, 1 mM
AMP
-
PNP, 2
mM MgSO
4
, 20
mM DTT
0.1 M sodium
cacodylate pH
6, 1.5 M NaCl,
9% PEG 3350,
1 mM AMP
-
PNP, 2 mM
MgSO
4
, 20 mM
DTT
0.1 M sodium
malonate pH
5.5,
15% PEG
3350, 18%
glycerol, 0.5
mM AMP
-
PNP, 2 mM
MgSO
4
0.1 M sodium
malonate pH
5.5, 15% PEG
3350, 18%
glycerol, 0.5
mM AMP
-
PNP, 2 mM
MgSO
4
0.2 M sodium
citrate pH 4, 0.1 M
sodium citrate pH
5.5, 5 mM AMP
-
PNP, 10 mM
MgCl
2
0.2 M sodium citrate
pH 4, 0.8 M s
odium
citrate pH 5.5, 8%
PEG 3350, 9%
trehalose, 5 mM AMP
-
PNP, 10 mM MgCl
2
0.72 M sodium
malonate pH
6.5, 1 mM
AMP
-
PNP, 4
mM MgCl
2
cryoprotectant
buffer
0.1 M HEPES
pH 7, 1 M KCl,
1.05 M
ammonium
sulfate, 24%
glycerol, 1 mM
AMP
-
PNP, 2
mM MgSO
4
, 20
mM DTT
60 mM sodium
cacodylate pH
6, 1.5 M NaCl,
15% PEG
3350, 1 mM
AMP
-
PNP, 2
mM MgSO
4
, 20
mM DTT, 15%
glycerol
same as
stabilization
buffer
same as
stabilization
buffer
0.2 M sodium
citrate pH 4, 50
mM sodium citrate
pH 5.5, 5 mM
AMP
-
PNP, 10 mM
MgCl
2
, 32%
treha
lose
0.2 M sodium citrate
pH 4, 0.8 M sodium
citrate pH 5.5, 10%
PEG 3350, 29.5%
trehalose, 5 mM AMP
-
PNP, 10 mM MgCl
2
0.96 M sodium
malonate pH
6.5, 1 mM
AMP
-
PNP, 4
mM MgCl
2
,
32% sucrose
Table S2. Crystallographic data collection and refinement
statistics.
fully reduced
wild type
cacodylate
-
modified wild
type
7
-
80 modified
wild type
8
-
34 modified
wild type
CoAlated,
unphosphorylated
wild type
CoAlated
unphosphorylated
C247V, C319V
disulfide
homodimer,
C247V, D256N,
C319V
Data collection
statistis
X
-
ray source
Rigaku RU
-
300
Rigaku RU
-
300
Bruker AXS
Microstar
Rigaku RU
-
300
Advanced Photon
Source NE
-
CAT
24
-
ID
-
E
Advanced Photon
Source NE
-
CAT
24
-
ID
-
E
Rigaku RU
-
300
Wavelength (Å)
1.54178
1.54178
1.54178
1.54178
0.97918
0.97918
1.54178
Resolution
range (Å)
30
-
2.65 (2.74
-
2.65)
30
-
2.14 (2.22
-
2.14)
50
-
1.86 (1.93
-
1.86)
50
-
1.58 (1.64
-
1.58)
50
-
2.65 (2.74
-
2.65)
50
-
2.10 (2.18
-
2.10)
23.33
-
2.00 (2.03
-
2.00)
Space group
P4
1
2
1
2
P6
1
22
P2
1
2
1
2
1
P2
1
2
1
2
1
P4
1
2
1
2
P4
1
2
1
2
P4
1
2
1
2
Unit cell (a, b,
c) (Å)
83,562, 83,562,
114.516
89.081, 89.081,
183.806
50.637, 85.562,
152.934
50.730, 85.769,
152.492
86.31, 86.31,
76.834
90.941, 90.941,
118.882
85.292, 85.292,
128.246
Unique
reflections
12380 (1206)
24064 (1876)
56724 (5501)
72049 (5427)
8795 (840)
29617
(2900)
31479 (1522)
Multiplicity
4.9 (4.8)
9.6 (3.5)
10.9 (8.4)
11.3 (3.8)
6.0 (5.5)
12.3 (12.2)
3.9 (3.7)
Completeness
(%)
99.8 (99.6)
98.8 (89.1)
99.9 (99.3)
89.2 (80.0)
99.18 (98.01)
100.0 (100.0)
98.1 (97.5)
Mean I/
(I)
18.7 (1.8)
24.1 (3.2)
30.8
(6.4)
31.4 (1.6)
23.5 (1.0)
48.2 (0.9)
25.8 (2.1)
CC
1/2
0.993 (0.746)
1.000 (0.891)
1.000 (0.966)
1.000 (0.699)
0.998 (0.880)
1.001 (0.521)
ND (0.790)
R
sym
0.074 (0.904)
0.062 (0.286)
0.062 (0.271)
0.059 (0.612)
0.064 (0.807)
0.052 (1.8)
0.047 (0.617)
R
pim
0.037 (0.456)
0.019 (0.170)
0.019 (0.097)
0.014 (0.350)
0.028 (0.368)
0.015 (0.545)
0.026 (0.355)
Refinement statistics
Resolution
limits (Å)
27.1
-
2.64 (2.74
-
2.64)
29.5
-
2.144 (2.22
-
2.14)
48.1
-
1.86 (1.93
-
1.86)
22.9
-
1.58 (1.64
-
1.58)
47.79
-
2.65 (2.7
5
-
2.65)
45.5
-
2.10 (2.18
-
2.10)
23.263
-
2.00
(2.07
-
2.00)
Number of
reflections
12369 (1207)
24081 (2261)
56647 (5507)
81814 (7228)
8795 (840)
29572 (2894)
32025 (3138)
R
work
0.199 (0.314)
0.187 (0.240)
0.168 (0.270)
0.170 (0.234)
0.220 (0.350)
0.190 (0.310)
0.186 (0.258)
R
free
0.245 (0.354)
0.216 (0.275)
0.202 (0.339)
0.202 (0.243)
0.279 (0.364)
0.214 (0.316)
0.215 (0.302)
Number of
nonhydrogen
atoms
2436
2596
5281
5279
2148
2577
2608
Macromolecule
2333
2356
4695
4761
2074
2410
2335
Ligand
40
65
86
16
48
54
40
Water
63
175
500
502
26
113
233
Protein residues
282
300
572
584
252
285
278
RMS bonds (Å)
0.0016
0.0035
0.0067
0.0143
0.002
0.0019
0.0026
RMS angles ( ̊)
0.46
0.72
0.89
1.39
0.51
0.54
0.52
Ramachandran
favored (%)
95.3
95.56
98.13
98.13
95.56
95.6
97.45
Ramachandran
outliers (%)
0.0
0.37
0.0
0.0
0.0
0.37
0.0
MolProbity
Clashscore
3.60
1.70
4.75
5.37
3.08
3.86
3.57
Average B
-
factor
83.7
44.6
28.7
33.1
96.8
72.0
41.7
Macromolecule
83.0
44.3
27.4
32.1
96.5
72.1
40.4
Ligand
140.1
48.5
51.3
50.9
117.6
66.9
72.4
Water
71.3
47.0
37.5
42.6
82.0
72.1
49.5
PDB ID code
6VPG
6VPH
6VPL
6VPM
6XKA
6VPJ
6VPI
deposition
code
D_1000246794
D_1000246798
D_1000246826
D_1000246830
D_1000250309
D_1000246824
D_1000246823
Data file S1. Data from tethering screen.
Excel spreadsheet containing
a
list of
disulfide
compounds
from the tethering screen with the single cysteine Aurora A kinase domain construct (C290A + C319V).
Listed for each compound are the molecular mass of th
e compound in the disulfide capped state, the
expected mass of the disulfide adduct formed upon thiol
-
disulfide exchange with a cysteine thiol
(uncapped reduced mass), the native mass of the Aurora A kinase domain protein,
and
the expected mass
of singly l
abeled protein and of doubly labeled protein. Double labeling may have occurred due
to
side
reactions resulting in compound dimerization in the DMSO stocks during storage or non
-
disulfide
covalent labeling of the protein. Side reactions also likely account for distinct peaks in the mass spectra
that differ from the expected masses for sever
al hits, that likely resulted from modification(s) of the
compounds. Masses of peaks in the mass spectra
equal
to
or greater than the native protein mass and with
at least 20% of the maximum intensity of the spectrum
are listed in the
right most
columns.
The structure
of each compound is provided in SMILES notation with the chemical structural formula also shown for
the hit compounds. The raw data for all mass spectra have been deposited in the MassIVE
repository
as
dataset MSV000085641 (ftp://massive.uc
sd.edu/MSV000085641/)
.