green and the CARP domain b-strand from interface 2 in teal. (c) A single experiment of nucleotide exchange of 0.5 μM subtilisin cleaved ADP-actin. Exchange ...
Structural basis of actin monomer re-charging by cyclase-associated protein
Kotila et al.
Supplementary Figure 1 a
b
PDB
Protein
2btf 1eqy 3daw 1sqk 6fm2
Profilin Gelsolin Twinfilin WH2 CARP
Interface (Å2)
Tot. actin surface (Å2)
1039 1078 1157 854 1939
16778 16132 17136 16271 17099
CD1
c
d
e
Leu16 CD2
CG
CB
O
Gly15
Gly156
O
N
C
Lys18
C
CA
Gly13
N
CA
C
O CB
N N
Mg
CD
90°
CA
0.1
MG
O2B
NZ CG
OG
O1B
0.19 0.06
CE
CB
0.06
0.15 PB
O3B
Ser14
CA
C
0.15
O2A
O O3A
0.31 0.02
PA
Gly301
C
N
CA
O5’
Asp157
OD2
CB
O3’
0.060.04
Gly302
N
C5’
0.02
ADP
C CA
O
C4’
CG OD1
C3’ C2’
O4’ C1’
C
Tyr59
Gln59
0.22
OE2
Arg210
-0.3
CA
-0.03
O
C4
N3
CB
Thr303
CD OE1
N9 C8
CG
NZ
C5 N7
C2
N
CB OG1
Arg21 0
Gly182
O2’
N
f
O
O1A
N1 C6
CE
CG2 N6
0.11
Glu214
CA
CD
C NZ
Tyr306
His73
O
CG
CD
Met305
CB
Tyr69
CG
CE O
CA
CB
N C
ADP
N
CA
Lys336
Ile75
Lys213
C
O
g
h
ADP
TMR Tyr169
Cys374 Lys373 Phe375
Supplementary Fig. 1. Crystal structure of the CARP domain-ADP-actin complex. (a) Electron density of ADP in actin in our crystal structure (2F0 FC, contoured at s = 1.0). (b) Comparison of binding surface areas of different actin-binding proteins calculated with PISA80. (c) Comparison of crystallized CARP domains displayed as a ribbon model (for clarity) using Pymol: 2b0r (yellow) Cryptosporidium parvum, 1k4z (magenta) Saccharomyces cerevisiae, 1k8f (green) Homo sapiens, and Mus musculus CARP domain model from our actin complex (blue). (d) Comparison of ADP-actin crystal structures superimposed on subdomain 3 (residues 145-180 and 270-337). CARP-actin (green), un-complexed actin rabbit muscle actin (1j6z, red), and nonpolymerizable Drosophila melanogaster actin (2hf3, blue). A major conformational change is observed mainly in the D-loop located in subdomain 2. (e) Analysis of ADP ligand interaction and bond distances in the nucleotide binding site with Ligplot+ v1.4.581. Distances are shown as a difference to uncomplexed ADP-actin (1j6z). Negative values indicate more distant than in 1j6z; most apparent differences are indicated as red circles. Please note that our structure has Mg2+ ion, and 1j6z has Ca2+ ion. (f) Comparison of side chain movements in actin subdomains 2 and 4 displayed in electron density (2F0 - FC, contoured at s = 1.0). Structures are aligned on the subdomain 3. Shown here are un-complexed actin (1j6z, red) and actin from the CARP domain complex (green). (g) On the left, steric clash (red) between actin-bound twinfilin’s ADF-H domain (yellow) (3daw) and the CARP domain (blue). On the right, profilin (orange) (2btf) overlaid with the CARP domain (blue) on actin (green). (h) C-terminal residues of actin (Lys373, Cys374 and Phe375) are visible in our CARP domain/actin complex (2F0 - FC, contoured at s = 1.0). A shift in Tyr169 is observed when compared to un-complexed actin (red), most likely due to absence of TMR in our structure. Thus, formation of p-p stack with Phe375 is plausible. In addition, a clear movement in a-helix359-365 is visible (pointed with an arrow).
Supplementary Figure 2
a
33° 25°
b
c 90° ADP-actin-CARP domain complex
10
RMSD (Å)
8
D-loop
6 4 2
d
80
Fluorescence (A.U)
ADP-actin
78 76 ADP-actin
74
Subtilisin cleaved ADP-actin
72 0
52
2 10
RMSD (Å)
8
102 152 202 ADP-actin-CARP 252 302 domain 352 complex ADP-actin Actin residue number
0
50
100 Time (s)
150
200
D-loop
6 4 2 0
2
52
102 152 202 252 302 Actin residue number
352
Supplementary Fig. 2. Effects of CARP domain on the conformation of the D-loop of actin. (a) Comparison of different D-loop conformations in 26 actin structures (Supplementary Table 2) where the D-loop is resolved (the CARP domain complexed with actin is depicted in red). (b) Electron density map of the D-loop conformation in the CARP-actin complex in our crystal structure (2F0 - FC, contoured at s = 1.0). Actin molecule from the neighbouring asymmetric unit is shown in green and the CARP domain b-strand from interface 2 in teal. (c) A single experiment of nucleotide exchange of 0.5 µM subtilisin cleaved ADP-actin. Exchange of ADP to e-ATP is ~3-fold faster than in untreated ADP-actin. (d) RMSD (root mean square deviation) per residue of the actin molecule in ADP-actin—CARP domain complex (System 1, Supplementary Table 3) and ADP-actin isolated from it (System 4, Supplementary Table 3). The calculations were performed over each 1.2 μs simulation separately. RMSD per residue with respect to the average actin structure was calculated after superposing the trajectories based on the actin alpha-carbons. Average RMSD per residue over the five trajectories and the standard deviation (band thickness in the plot) is reported.
Supplementary Figure 3 a
b
1
2
3
4
5
6
7
8
9
10
CCA
P
Mutants
C-CAP protein C-CAP 1 2 3 4 5 6 7 8 9 10
kDa ~55 ~35 ~25 ~15
c
Retention volume (ml) 12.4 12.4 12.8 12.4 12.6 12.6 12.3 12.6 12.4 10.6 12.2 *weak signal
Tm (°C) 56 56 56 56 56 54 52 59 57 N.A* 49
d
50 40 30 20 10
ct
in W M T u M t1 u M t2 u M t3 u M t4 u M t5 u M t6 u M t7 u M t8 M ut9 ut 10
0 A
1 2 3 4 5 6 7 8 9 10
Protein C-CAP(217-474) R253A L256A I260A D266ITHA269 270 LKHV273 -> 270AAAA273 279 THKN282 -> 279AAAA282 K347A Y351A Y353A K365A N367A D372A D446A E449A D471EIAG474 Y418A F447A L339A Q399A D446A *estimated from raw data **N.A = unmeasurable
Half-time (s)
#
Kd ADPKd ATPactin (µM) actin (µM) 0.055 0.958 0.218 N.A 0.244 N.A 0.289 N.A 0.087 4.94 > 1.0* 0.618 N.A** 0.881 > 1.0* 0.887 0.009 0.801 N.A** 1.537 0.345 0.934
Supplementary Fig. 3. Biochemical properties of the C-CAP mutants. (a) SDS-PAGE analysis of the purified wild-type and mutant C-CAPs. (b) A table showing their corresponding elution volumes on gel filtration with SD200 Increase 10/300 from protein purification, and their representative unfolding temperatures measured (Tm) with Thermofluor. All mutant proteins, with the exception of mutant 9 (Y418A,F447A; indicated in red), were fully soluble and eluted in gel filtration at an identical volume compared to the wild-type C-CAP. Furthermore, thermal stability of these mutants confirmed that proteins are properly folded. (c) A summary of the affinities of C-CAP proteins for ADP- and ATP-G-actin from NBD-actin binding measurements. (d) Nucleotide exchange assay measuring the incorporation of 25 µM eATP to ADP-actin using 0.1 µM CCAP proteins and 0.5 µM actin. In orange, actin nucleotide exchange rate in the absence of CAPs and in green the rate of nucleotide exchange with 0.1 µM wild-type (WT) C-CAP present. Each data point represents a value from an independent measurement. Half-times were calculated from the fit assuming one-phase association.
Kd =0.087 µM
0.4 0.3
C-CAP - Mut 4
0.5 0.2
Kd =0.087 µM
0.4 0.1 10 0.3
100
1000
10000
C-CAP (nM)
0.2 100
1000
10000
C-CAP- (nM) C-CAP Mut 8
0.5
Kd =0.009 µM
0.4 0.3
C-CAP - Mut 8
0.5 0.2
Kd =0.009 µM
0.4 0.1 10 0.3
100
1000
10000
C-CAP (nM)
0.5
Kd > 1.0 µM
0.4 0.3
C-CAP - Mut 5
0.5 0.2
Kd > 1.0 µM
0.4 0.1 10 0.3
0.1 10
100
1000
10000
1000
10000
C-CAP (nM)
100 1000 10000 C-CAP Mut 9 C-CAP- (nM)
0.5 0.4
Kd = N.A
0.3
C-CAP - Mut 9
0.4 0.1 10 0.3
Kd =0.958 µM
0.2 0.4 10
100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP- (nM) C-CAP Mut 4
0.5
Kd = 4.94 µM
0.4 C-CAP - Mut 4
0.3 0.5
Kd = 4.94 µM
K = N.A 1000 d 10000
100
C-CAP (nM)
0.2 0.4 10
100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP (nM) C-CAP - Mut 8
0.5
Kd = 0.801 µM
0.4 C-CAP - Mut 8
100
1000
1000
10000
10000
C-CAP (nM)
Kd =N.A C-CAP - Mut 1
0.3 0.5 0.2 0.4 10
Kd =N.A
100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP-(nM) C-CAP Mut 5
0.5
Kd =0.618 µM
0.4 C-CAP - Mut 5
100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP-(nM) C-CAP Mut 9
0.5
Kd =1.537 µM
0.4 C-CAP - Mut 9
100 1000 10000 C-CAP Mut 6 C-CAP -(nM)
0.5 0.4
Kd = N.A
0.3
C-CAP - Mut 6
C-CAP - Mut 3 Kd = 0.289 µM
0.1 0.4 10 0.3
100
1000
10000
C-CAP (nM)
0.1 10
100 1000 10000 C-CAP Mut 7 C-CAP -(nM)
0.5
Kd > 1.0 µM
0.4 0.3
C-CAP - Mut 7
0.5 0.2
0.5 0.2 0.4 0.1 10 0.3
K = N.A 1000d 10000
100
C-CAP (nM)
Kd > 1.0 µM
0.4 0.1 10 0.3
100
1000
10000
C-CAP (nM)
0.2
0.1 10
100 1000 10000 C-CAP Mut 10 C-CAP- (nM)
0.5
0.1 10
100
1000
10000
C-CAP (nM)
Kd =0.345 µM
0.4 0.3
C-CAP - Mut 10 Kd =0.345 µM
0.4 0.1 10 0.3
0.1 10
100
1000
10000
C-CAP (nM)
100
1000
100
1000
10000
C-CAP - Mut 2 0.5
10000
C-CAP (nM)
Kd = N.A
0.4 C-CAP - Mut 2
0.3 0.5
Kd = N.A 0.2 0.4 10 100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP -(nM) C-CAP Mut 6
0.5
Kd =0.881 µM
0.4 C-CAP - Mut 6
100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP (nM) C-CAP - Mut 10
0.5
C-CAP - Mut 3 0.5
Kd = N.A
0.4 C-CAP - Mut 3
0.3 0.5
Kd = N.A 0.2 0.4 10 100
1000
10000
C-CAP (nM)
0.3 0.2 10
100
1000
10000
C-CAP C-CAP -(nM) Mut 7
0.5
Kd =0.887 µM
0.4
0.3 0.5
Kd =0.881 µM
0.2 0.4 10
C-CAP - Mut 7 Kd =0.887 µM
0.2 0.4 10 0.3 0.2 10
100
1000
10000
C-CAP (nM) 100
1000
10000
C-CAP (nM)
Kd =0.934 µM
0.4
0.3 0.5
Kd =1.537 µM
0.2 0.4 10
0.2
0.3
0.2 0.5
0.2
0.1 10
0.3 0.5
Kd =0.618 µM
0.2 0.4 10
0.3
Kd = 0.289 µM
0.4
C-CAP (nM)
0.4
0.3 0.5
Kd = 0.801 µM
0.4 0.2 10
100
C-CAP - Mut 1 0.5
0.3 0.5
10000
C-CAP - Mut 3 0.5
0.2
0.1 10
Fluorescence (A.U) Fluorescence (A.U)
C-CAP(217-474)
1000
C-CAP (nM)
0.5 0.2
0.5 0.2
Fluorescence (A.U) Fluorescence (A.U)
0.4
100
0.2
0.1 10
Fluorescence (A.U) Fluorescence (A.U)
Fluorescence (A.U) Fluorescence (A.U) Fluorescence (A.U) Fluorescence (A.U)
Fluorescence (A.U) Fluorescence (A.U)
Kd =0.958 µM
0.3 0.5
0.1 0.4 10 0.3
C-CAP (nM)
C-CAP(217-474) 0.5
0.2
100
0.2
0.2
0.3
100 1000 10000 C-CAP Mut 5 C-CAP-(nM)
0.2
0.1 10
Kd = 0.244 µM
0.2
0.1 10
Fluorescence (A.U) Fluorescence (A.U)
100 1000 10000 C-CAP Mut 4 C-CAP- (nM)
0.5
0.3 0.5
10000
0.2
0.1 10
C-CAP (nM)
b
1000
C-CAP (nM)
C-CAP - Mut 2
Fluorescence (A.U) Fluorescence (A.U)
0.2
100
0.3
Fluorescence (A.U) Fluorescence (A.U)
10000
Kd = 0.218 µM
0.4
Fluorescence (A.U) Fluorescence (A.U)
1000
C-CAP (nM)
0.1 0.4 10 0.3
Kd = 0.244 µM
0.2 0.5
Fluorescence (A.U) Fluorescence (A.U)
100
C-CAP - Mut 1
Fluorescence (A.U) Fluorescence (A.U)
0.1 0.4 10 0.3
0.3
0.2 0.5
C-CAP - Mut 2 0.5
Fluorescence (A.U) Fluorescence (A.U)
Kd = 0.055 µM
Fluorescence (A.U) Fluorescence (A.U)
C-CAP(217-474)
Fluorescence (A.U) Fluorescence (A.U)
0.3 0.2 0.5
Kd = 0.218 µM
0.4
Fluorescence (A.U) Fluorescence (A.U)
0.4
C-CAP - Mut 1 0.5
Fluorescence (A.U) Fluorescence (A.U)
Kd = 0.055 µM
Fluorescence (A.U) Fluorescence (A.U)
0.5
Fluorescence (A.U) Fluorescence (A.U)
Fluorescence (A.U) Fluorescence (A.U)
Fluorescence (A.U) Fluorescence (A.U)
a
Fluorescence (A.U) Fluorescence (A.U)
C-CAP Supplementary Figure (217-474) 4
C-CAP - Mut 10 Kd =0.934 µM
0.2 0.4 10 0.3 0.2
100
1000
10000
C-CAP (nM)
100 1000 10000 10 100 1000 10000 10 100 1000 10000 Supplementary Fig. 4. Representative raw binding data from the 10 NBD-actin assays for wild-type and mutant C-CAPs. C-CAP (nM) C-CAP (nM) C-CAP (nM) (a) NBD-assay with ADP-G-actin. Concentration of C-CAP was varied from 0 to 15 µM and the concentrations of actin and twinfilin169-350 were 0.18 µM and 0.44 µM, respectively. Data were fitted in GraphPad Prism 7 assuming one-site competition with 0.03 µM affinity for twinfilin. Data are representative from a single titration experiment. (b) Direct NBD-assay for ATP-G-actin binding assuming one site binding in fit. C-CAP concentrations were varied from 0 to 15 µM, and the concentration of actin was 0.18 µM. Data are representative from a single titration experiment.
Supplementary Figure 5 a
b
291
c
90°
RMSD (Å)
248
6 4 2 0
alpha-helix LKHV
248
253
258
263
268
273
THKN
278
283
288 291
Residue number of CAP1
6 4 2 0
RMSD (Å)
RMSD (Å)
d
6 4 2 0
LKHV
THKN
alpha-helix
248
253 258 alpha-helix
263
268
273 LKHV
248
253
258
263
268
273
278 283 288 293 298 Residue number of CAP1
303
308
313
318
323
THKN
278
283
90° 288 291
Residue number of CAP1
RMSD (Å)
248
6 4 2 0
LKHV
THKN
alpha-helix
248
253
258
263
268
273
278 283 288 293 298 Residue number of CAP1
303
308
313
318
323
Supplementary Fig. 5. Molecular dynamics simulations of ADP-actin-CAP1248-474 and ATP-actin-WH2 domain complexes. (a) Dynamics of the WH2 domain in the ATP-actin—WH2 domain complex (System 3, Supplementary Table 3). WH2 domain (residues 248-291) is shown in tube representation, where the tube thickness and the color correlate with the RMSD per residue. The calculations were performed over the last 200 ns of five 1.2 μs ATP-actin/WH2 domain simulations. RMSD per residue with respect to the average WH2 structure was calculated after superposing the trajectories based on the actin alpha-carbons. Average RMSD per residue over the trajectories and the standard deviation (band thickness in the plot) are reported. (b-c) Molecular modeling of the WH2 domain connection through PP2 to the CARP domain. The cis (b) and the trans (c) configurations of the WH2 - CARP domain connections are shown. (d) Dynamics of the WH2 domain and PP2 in the ADP-actin—CAP1248-474 domain complex (System 2, Supplementary Table 3). The WH2 domain (residues 248-323) is shown in tube representation, where the tube thickness and the color correlate with the RMSD per residue. The calculations were performed over the last 200 ns of five 1.2 μs simulations. RMSD per residue with respect to the average WH2 structure was calculated after superposing the trajectories based on the actin alpha-carbons.
Supplementary Figure 6 b
Contact frequency
Supplementary Figure 6 a 1.0
b
0.6 1.0
0.4
Contact frequency
a
0.8
0.8
0.2
0.6
rg
17
Ile 75
is
Aradopsis thaliana (O65902) Saccharomyces cerevisiae (P17555)
A
H
0.2
73
0.4
7
0.0
463
465
474
415
463
449
474
456
424
174
457
466
464
466
457 464 183 476
183
517
424
526
476
517
17
7
526
4-5
18 - 21
kDa
70
55
Actin
ADP-actin
Fractions 4-6
60
GST-srv2 + ADP-actin GST-srv2-Δ4C + ADP-actin
50
A.U
454
ADP-actin fractions
A
H
c
rg
is
Ile 75
73
0.0
c c
Danio rerio (Q6YBS2) Mus musculus (P40124) Drosophila melanogaster (Q9VPX6) 454 Danio rerio (Q6YBS2) Caenorhabditis elegans (O02096) 465 Mus musculus (P40124) 415 Dictyostelium discoideum (P54654) Drosophila melanogaster (Q9VPX6) 449 Cryptosporidium parvum (Q5CS32) Caenorhabditis elegans (O02096) Dictyostelium (P54654) 456 Aradopsisdiscoideum thaliana (O65902) 174 Cryptosporidium parvum (Q5CS32) Saccharomyces cerevisiae (P17555)
35
GST-srv2 + ADP-actin fractions
40
kDa
30
17 - 20
GST-srv2
100 70 55
ADP
20
4-6
Fractions 17-21
Actin
35
10 GST-srv2-Δ4C + ADP-actin fractions
0 kDa
0
5
10
15
Retention volume (mL)
20
25 100 70 55
4-6
18 - 21 GST-srv2-Δ4C
Actin
35
Supplementary Fig. 6. Interaction of the C-terminal tail of CAP with actin. (a) The contacts between the four C-terminal CARP domain residues and actin in ADP-actin—CARP domain complex simulations (System 1, Supplementary Table 3). Bar graph showsFig. the6.average frequency contacts (cutoff 3.5 Å) thatactin. any CARP residue the (471-474) Supplementary Interaction of theofC-terminal tail ofofCAP with (a) TheC-terminal contacts between four makes with actin residues over the five repeats; and error bars represent standard deviation of mean. See Fig. 4b, for the position of these C-terminal CARP domain residues and actin in ADP-actin—CARP domain complex simulations (System 1, residues in the nucleotide sensing region of actin. (b) Sequence alignment of the C-terminal regions Supplementary Table 3). Bar graph shows the average frequency of contacts (cutoff of 3.5 Å) that any CARP C- of CAPs from representative Uniprot numbers arefive indicated in brackets. Different standard colors represent sequence terminal residue organisms. (471-474) makes withaccession actin residues over the repeats; and error bars represent conservation for the specific residues among aligned organisms. (c) Binding of GST-tagged full-length deviation of mean. See Fig. 4A, for the position of these residues in the nucleotide sensing region of actin. (b)wild-type Srv2 and Srv2D4Calignment mutant toof ADP-actin monomers was byrepresentative size-exclusionorganisms. chromatography. 300 μl of 10 μM of Sequence the C-terminal regions of analysed CAPs from UniprotRetention accession of numbers ADP-actin alone (in magenta), 10 μM of ADP-actin + 10 μM GST-Srv2 wild type (in black), and 10 μM of ADP-actin + 10 are indicated in brackets. Different colors represent sequence conservation for the specific residues among aligned μM GST-Srv2-D4C (in blue) were analysed using Superdex 200 Increase 10/300 GL gel filtration column organisms. (c) Binding of GST-tagged full-length wild-type Srv2 and Srv2D4C mutant to ADP-actin monomersequilibrated in 5 mManalysed HEPES,by 100 mM NaCl, 0.05 mM ADP, 0.05 mM MgCl mM b-mercaptoethanol. Elution profiles are on the left, 2 and was size-exclusion chromatography. Retention of 300 µl0.5 of 10 µM of ADP-actin alone (in magenta), and SDS-PAGE analysis of peak fractions is on the right. ADP-G-actin alone shows retention volume ~ 15.7 ml. When 10 µM of ADP-actin + 10 µM GST-Srv2 wild type (in black), and 10 µM of ADP-actin + 10 µM GST-Srv2-ofD4C GST-Srv2 polypeptides and ADP-actin are mixed together, ~ 70% of ADP-actin shifts to where GST-Srv2 elutes (~ 8.5 ml) (in blue) were analysed using Superdex 200 Increase 10/300 GL gel filtration column equilibrated in 5 mM and ~ 95% of ADP-actin to where GST-Srv2-D4C elutes (~ 8.5 ml). HEPES, 100 mM NaCl, 0.05 mM ADP, 0.05 mM MgCl2 and 0.5 mM b-mercaptoethanol. Elution profiles are on the left, and SDS-PAGE analysis of peak fractions is on the right. ADP-G-actin alone shows retention volume of ~ 15.7 ml. When GST-Srv2 polypeptides and ADP-actin are mixed together, ~ 70% of ADP-actin shifts to where GST-Srv2 elutes (~ 8.5 ml) and ~ 95% of ADP-actin to where GST-Srv2-D4C elutes (~ 8.5 ml).
Supplementary Figure 7 b
a
Ponceau
Tubulin (1:1000)
Srv2 (1:500)
c
ADP-actin
GST-srv2 + ADP-actin
GST-srv2-d4C + ADP-actin
Supplementary Fig. 7. Uncropped images. (a) Raw image file for SDS-PAGE analysis of C-CAP mutants for Supplementary Figure 3a. (b) The raw image files for Western blot analysis of Figure 5a. (c) The raw image files for gel filtration fraction analysis from Supplementary Fig. 6c.
Supplementary Figure 8 a Thr203
Gly63
b
ADP-actin (System 4) 25 20 15 10 5 ADP-actin–CARP domain complex (System 1)
Distance (Å)
25
c
Distance (Å)
25
20 15 10
20 5 15 ADP-actin-CAP1248-474 complex (System 2) 10
25
5
20 ADP-actin (System 4)
ADP-actin CARP domain complex (System 1)
ADP-actin CAP1 248-474 complex (System 2)
15 10 5 0
200
400
600
800
1000
1200
Time (ns) Supplementary Fig. 8. Analysis of the opening of the nucleotide-binding cleft. (a) Representation of the distances between Thr203 Ca in subdomain 4 and Gly63 Ca in subdomain 2 as a measure to examine opening of the nucleotide-binding cleft upon interaction with CAP. (b) Distances between Thr203 Ca and Gly63 Ca from molecular dynamics simulations of ADPactin (System 4, Supplementary Table 3), ADP-actin—CARP complex (System 1, Supplementary Table 3), and ADP-actin— CAP1248-474 (System 2, Supplementary Table 3) complexes. The distances are calculated for each actin in each simulation separately, resulting in 5 separate time traces for ADP-actin, and 10 for ADP-actin-CARP domain and ADP-actin-CAP1248-474 complexes (shown in different colours). (c) Violin plots displaying the distribution of the distances, the means (lower lines), the medians (upper lines), as well as the minima and extrema over the last 1.0 µs of all simulations. The system was allowed to equilibrate for the first 200 ns.
Supplementary Table 1. Data collection and refinement statistics. Data collection
CARP-ADP-actin* (PDB 6fm2)
Space group Cell dimensions a, b, c (Å)
P 61 2 2
() Resolution (I / I > 1.3) l axis (CC1/2 > 0.3)
90.00 90.00 120.00 39.57-2.8 Å (2.95-2.8 Å)
hk plane (CC1/2 > 0.3)
73.83 73.83 453.37
2.3 Å 3.2 Å
Number of unique reflections
19109 (2749)
Multiplicity
6.0 (6.4)
Completeness (%)
98.2 (99.93)
Rmerge
0.08 (1.371)
Rpim
0.035 (0.585)
I/ I CC1/2
11.6 (1.3) 0.999 (0.777)
Refinement Rwork / Rfree (%)
18.6/23.4 (23.7/33.8)
Protein residues No. atoms Protein
528
Ligand/ion Water B-factors Wilson B-factor Protein Ligand/ion Water Anisotropic B-factor (B11, B22, B33)
39 79 128.7 85.8 129.5 102.1 98.7 -21.7511, -21.7511, 43.5022
4110
R.m.s. deviations Bond lengths (Å) Bond angles ( ) Ramachandran plot Outliers Allowed Favored Clashscore (all-atoms) *highest resolution shell in brackets
0.010 1.22 2 (0%) 38 (7%) 483 (93%) 6
Supplementary Table 2. The crystal structures of actin with D-loop modelled.
PDB ID (reference) 3daw7 3w3d9 1atn10 1j6z4 2a3z11 4jhd12 4pkg13 4pki13 2a4211 2zwh14 4rwt12c 1h1v15 2btf16 3j8a17 1hlu18 4cbw19 4z9420 3eks21 3jbk22 2q9723 2a4011 2a4111 2d1k24 2vcp25 3ffk26 3tu527
Name/Description
Actin state
Structure of the ADF homology domain in complex with Actin Crystal structure of smooth muscle G Actin DNase I complex Atomic structure of the Actin:DNAse I complex Uncomplexed Actin Ternary complex of the WH2 domain of WASP with Actin-DNAse I Crystal structure of an Actin dimer in complex with the Actin nucleator CordonBleu Complex of ATP-Actin with the N-terminal Actin-binding domain of tropomodulin Complex of ATP-Actin with the C-terminal Actin-binding domain of tropomodulin Actin-DNAse I Complex Model for the F-Actin structure Structure of Actin-Lmod complex Structure of the G4-G6/Actin Complex The structure of crystalline profilin- -Actin F-Actin-Tropomyosin-EM structure The structure of an open state of -Actin at 2.65 A resolution Crystal structure of Plasmodium berghei Actin I with D-loop from muscle Actin Actin Complex With a Chimera of Tropomodulin-1 and Leiomodin-1 ActinBinding Site 2 Crystal Structure of monomeric Actin bound to Cytochalasin D Cryo-EM reconstruction of the metavinculin-Actin interface Complex of mammalian Actin with toxofilin from toxoplasma gondii Ternary complex of the WH2 domain of WAVE with Actin-DNAse I Ternary complex of the WH2 Domain of WIP with Actin-DNAse I Ternary complex of the WH2 domain of MIM with Actin-DNAse I Crystal structure of N-Wasp VC domain in complex with skeletal Actin Crystal structure of human Gelsolin domains G1-G3 bound to Actin Actin complex with Gelsolin segment 1 fused to Cobl segment 26 structures in total
ATP ATP ATP ADP ATP ANP ATP ATP ATP ADP ANP ATP ATP ADP ATP ATP ATP ATP ADP ATP ATP ATP ATP ATP ATP ATP
Supplementary Table 3. Summary of the atomistic simulation systems. System no.
System name
Notes on the initial protein configurations
No. of repeats
1
ADP-actin—CARP domain complex
Crystal structure
5
2
ADP-actin— CAP1248-474
3
ATP-actin—WH2 domain complex
4
ADP-actin
WH2 and PP2 domains are modeled in complex with System 1 in trans configuration. 1) CARP and the PP2 domains are removed from System 2. 2) ADP is replaced with ATP. CARP is removed from System 1.
Simulation time (μs)
No. of waters
Box volume (nm3)
×
1.2
59,000
1922
5
×
1.2
59,000
1940
5
×
1.2
25,000
809
5
×
1.2
25,000
803
Supplementary Table 4. Primer sequences used in this study. Plasmid pPL973
pPL974 pPL1064
pPL1065 pPL1066
pPL1067 pPL1068
pPL1069 pPL1071
pPL1072 pPL1073
pPL1075
Explanation pCoofy18-10xHIS-3Cmouse C-CAP(217474) for biochemistry pCoofy18-10xHIS-3Cmouse CAP1(242-474) for crystallizations pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut1
Forward primer(s) CTGGAAGTTCTGTTCCAGGGGCCC AGTGGATTGCCATCTGGACCCTC
pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut2 pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut3 pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut4 pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut5 pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut6 pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut7 pCoofy18-10xHIS-3Cmouse C-CAP(217470)/mut8 pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut9
GGGGAAAGCCTGAAACATGTATCT GATGACATGAAGACTCACAAGAA CC GCAGCGGCAGCTGCATCTGATGAC ATGAAGACTCACAAGAACCCTGCC CTG CATGAAGGCTGCTGCAGCCCCTGC CCTGAAAGCTCAGAGCGG
pCoofy18-10xHIS-3Cmouse C-CAP(217474)-mut10
CTGGAAGTTCTGTTCCAGGGGCCC ACCAGTTCTGGTTCTGACGACTCT G GCTTCAGCAGCTTTTGCACAAGCA AATCAGGGGGAAAGCATCACACA TGCCCTG
GCTCAAGTTGCTGCAATTGCAAAG TGTGTCAACACAACATTGCAAATC AAGGGC GCAATTGCCTCCATTACAGTAGCT AACTGTAAGAAGCTTGGCCTGGTG TTTGATG GGCGGTGCTTTTAACGCGTTCCCA GTCCCCGAGCAGTTCAAGAC CAGTGACATAGCGCCATTAACCTG ATGTTCTGGGGAATATAAGCTTGC F447A: AGGCGGTGATGCTAACGAGTTCCC AGTCCCCGAGCAG Y418A: GCCATGCTGCCCTGAGCAAGAACT CCCTGGACTGTGAGATAG L339A: GAGAATGTTTCTAACGCGGTGATT GATGACACTGAGCTGAAGCAGGTG Q399A: GATGTCAAAGTTGCGGTGATGGGA AAAGTGCCAACCATTTCCATTAAC D446A: AAGGCGGTCGCTTTAACGAGTTCC CAGTCCCCGAGC
Reverse primer(s) CCCCAGAACATCAGGTTAATG GCGCTATCCAGCGATTTCTGT CACTGTGG CCCCAGAACATCAGGTTAATG GCGCTATCCAGCGATTTCTGT CACTGTGG TGCTTGTGCAAAAGCTGCTGA AGCTGATGCAGAGTCGTCAG AACCAGAACTGGT CATGTTTCAGGCTTTCCCCCT GATTAATCTGTGCAAACAGTG CTGAG TGCAGCTGCCGCTGCATGTGT GATGCTTTCCCCCTGATTAAT CTGTGC GGCTGCAGCAGCCTTCATGTC ATCAGATACATGTTTCAGGGC ATGTGTGATGC TGCAATTGCAGCAACTTGAGC CAGCTCAGTGTCATCAATCAC CAGGTTAGAAAC AGCTACTGTAATGGAGGCAA TTGCGCCCTTGATTTGCAATG TTGTGTTGACACAC GGAACGCGTTAAAAGCACCG CCTTCGGTAGGAATGAGGAC ATTCATCTCAG GTTAATGGCGCTATGTCACTG TGGTGACCAACTTCTGTCCGT TC F447A: GGAACTCGTTAGCATCACCGC CTTCGGTAGGAATGAGGC Y418A: CTTGCTCAGGGCAGCATGGCA GCCATCTGTTTTGTTAATGG L339A: CATCAATCACCGCGTTAGAAA CATTCTCCTGGTTTTCCACTCT CC Q399A: TTTTCCCATCACCGCAACTTT GACATCCCTACTATTGATTAT CTCCACAATG D446A: GGAACTCGTTAAAGCGACCG CCTTCGGTAGGAATGAGGAC ATTC
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