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Structure, Volume 22
Supplemental Information
Structural and Mechanistic Insights
into the Recruitment of Talin by RIAM
in Integrin Signaling
Yu-Chung Chang, Hao Zhang, Janusz Franco-Barraza, Mark L. Brennan, Tejash Patel, Edna Cukierman, and Jinhua Wu
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Inte
nsit
y
0 20 40 60 80 100 120[R2R3] μM
TBS1/R2R3
R2R3IB:Anti-His(Pull-down)
25
0 0.5 1.5 4.5 13.5 40.5 100.0 (μM)
Kd = 3.0±0.4 μM
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Inte
nsit
y
0 20 40 60 80 100 120[R2R3] μM
TBS1-2/R2R3
R2R3IB:Anti-His(Pull-down)
25
0 0.5 1.5 4.5 13.5 40.5 100.0 (μM)
Kd = 2.6±0.5 μM
B C
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Inte
nsit
y
0 2 4 6 8 10 12 14 16 18[R7R8] μM
TBS1/R7R8
R7R8IB:Anti-His(Pull-down)
37
0 0.5 1.0 2.0 4.0 8.0 16.0 (μM)
Kd = 1.7±0.2 μM
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Inte
nsit
y
0 2 4 6 8 10 12 14 16 18[R7R8] μM
TBS1-2/R7R8
R7R8IB:Anti-His(Pull-down)
37
0 0.5 1.0 2.0 4.0 8.0 16.0 (μM)
Kd = 2.0±0.4 μM
D E
FIGURE S1
-80
-60
-40
-20
0
20
40
60
200 210 220 230 240 250
R2R3R7R8
(nm)
A
A
S13 S13
TBS1 TBS1
R8 R8
B
R8
TBS1
R7’
R7S25
R1638N1416 D20
FIGURE S2
TBS1 TBS1
Talin R7R8 Vinculin Vd1
B-factor20 50
C
R7R8R9
Input
TBS1Lpd-TBS
GST
RA PHPP
CCLpd-TBS
50 60
RIAM 50 -KDLNESLNALEDQDLDALMADLVADISEAEQRTIQA--- 85Lpd 72 -YNLNEALNQGETVDLDALMADLCS--IEQELSSIGSGN- 107
Lpd-TBS2
70 80
GST-taggedProteins31
45
Figure S3
A
C
-25
-20
-15
-10
-5
0
5
10
200 210 220 230 240 250
TBS1TBS1(S13G)
B
(nm)
10% Input GST-TBS1Pull down
0 5 10 20 20
R9 R7R8 (μM)
GFP-F0R9
GST-TBS1 pull-down
GFP-F0R9
GFP-Talin
GFP-Talin
-E17
70A
Input
Pull down GFP-Talin
GFP-Talin
A B
C
Figure S4
GFP-F0R8
0 0.5
2.5
12.5
0 0.5
2.5
12.5
WC
L
+ R9 (μM)+R7R8 (μM)
GS
T co
ntro
l
GFP-F0R8
D
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-TB
S1-L
15Y-
CA
AX
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-TB
S1-C
AA
X0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-TB
S1-S
13G
-CA
AX
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-TB
S1-E
18A
-CA
AX
A
Talin
TBS1-CAAX +
Talin
TBS1-S13
G-CAAX +
Talin
TBS1-L15
Y-CAAX +
Talin
TBS1-E18
A-CAAX +
Talin
Talin250-
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M-L
15Y
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M-E
18A
B
Talin
RIAM + Ta
lin
RIAM-S13
G + Ta
lin
RIAM-L15Y
+ Ta
lin
RIAM-E18
A + Ta
lin
Talin250-
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M-d
TBS2
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M-d
TBS1
-2
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
C
Talin
RIAM + Ta
lin
RIAM-∆TBS1 + Ta
lin
RIAM-∆TBS2 + Ta
lin
RIAM-∆TBS1-2 +
Talin
250- Talin
D
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+GFP
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+TB
S1-C
AA
X
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+TB
S2-C
AA
X
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+TB
S1-2
-CA
AX
Talin
TBS1-CAAX +
Talin
TBS2-CAAX +
Talin
TBS1-2-C
AAX + Ta
lin
250-Talin
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+GFP
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+L60
-CA
AX
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+L60
-CA
AX-
G29
S
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+TB
S1-C
AA
X
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+TB
S1-C
AA
X+ED
TA
0 102 103 104 105
PAC-1
0102
103
104
105
Talin
+TB
S1-C
AA
X+Ep
ti
E
Talin
L60-C
AAX + Ta
lin
TBS1-CAAX +
Talin
TBS1-CAAX +
Talin
+ EDTA
L60-G
29S-C
AAX + Ta
lin
TBS1-CAAX +
Talin
+ Epti
250- Talin
Figure S5
1 5.33±0.14 1.97±0.06 2.31±0.08 2.88±0.06
1
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M-S
13G
1.80±0.04 1.15±0.02 1.87±0.09
1
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M
2.42±0.01
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M-d
TBS1
1.69±0.01 2.13±0.06 1.73±0.04
1 1.89±0.04 0.91±0.02 1.98±0.02
1 1.81±0.08 2.46±0.07 2.64±0.05
1.53±0.03 1.17±0.10
0 102 103 104 105
PAC-1
0102
103
104
105
GFP
-RIA
M
2.42±0.01
Figure S1, related to Figure 1. (A) CD spectra trace of purified R2R3 (red) and R7R8 (blue)
proteins at 200-250 nM. (B-E) Quantitative pull-down analyses of TBS regions with R2R3 or
R7R8 proteins. Pull-down intensities were quantified by ImageJ. Binding curves were fit to a
single-site (saturating) binding model using SigmaPlot. (B) Pull-down of GST-TBS1 and His-
R2R3. (C) Pull-down of GST-TBS1-2 and His-R2R3. (D) Pull-down of GST-TBS1 and His-
R7R8. (E) Pull-down of GST-TBS1-2 and His-R7R8.
Figure S2, related to Figure 2. (A) |Fo|-|Fc| electronic density map of RIAM TBS1 is shown in
stereo view at 3.0 σ contour represented by blue mesh. (B) Crystallographic symmetry-related
R7 domain (R7’, navy color) and RIAM TBS1 are shown in cartoon diagram, and TBS1-
interacting residues in the R7’ domain are shown in stick. (C) B-factor comparison for main
chain atoms of TBS1 from R7R8:TBS1 (PDB entry 4W8P) and Vd1:TBS1 (PDB entry 3ZDL)
structures. B-factors that range from 20-50 Å2 are represented by the color bar.
Figure S3, related to Figure 5. (A) In vitro binding assay of talin R7R8R9 triple domains with
RIAM TBS1 and Lpd-TBS. (B) CD spectra trace of TBS1 peptide (green) and TBS1-S13G
peptide (yellow) at 200-250 nM. (C) Schematic representation of the domain organization of
Lpd (TBS, talin-binding region; CC, coiled-coil region; PP, poly-proline region; RA, Ras-
association domain; PH, Pleckstrin homology domain). Sequence alignment of Lpd and RIAM
at the TBS2 region reveals another putative talin-binding site in Lpd (Lpd-TBS2). Identical
residues were highlighted in yellow.
Figure S4, related to experiment procedure of GST pull-down and Western blotting and Figure
2. (A) GST-tagged TBS1 was used as bait to pull down GFP-talin F0R8 (residues 1-1653) and
F0R9 (residues 1-1848) expressed in HEK293 cells. 10% of input lysate and bound F0R8 and
F0R9 were analyzed by Western blotting. (B) GFP-talin F0R8 (residues 1-1653) expressed in
HEK293 cells was mixed with purified His-tagged R7R8 or R9 proteins of indicated
concentration and pulled down by GST-tagged TBS1. 10% of input lysate and bound F0R8
was analyzed by Western blotting. (C) GFP-talin F0R9 (residues 1-1848) expressed in
HEK293 cells was mixed with purified His-tagged R7R8 or R9 proteins of indicated
concentration and pulled down by GST-tagged TBS1. 10% of input lysate and bound F0R9
was analyzed by Western blotting. (D) GST-tagged TBS1 was used as bait to pull down GFP-
talin (wild type or E1770A) expressed in HEK293. Input lysate and bound talin proteins were
analyzed by Western blotting.
Figure S5, related to experimental procedure of integrin activation assay. (A-E) FACS data
and talin expression levels from one set of the integrin activation assays shown in Figure 4A-
E, respectively. The fluorescence intensity of GFP (Y-axis) was plotted against that of PAC1
signal (X-axis). The geometric mean fluorescence intensity (GMFI) of PAC1 binding was
calculated by FlowJo software only within the cells expressing high level GFP (103-105
fluorescence units, indicated by the rectangular boxes). A5 cells without GFP transfection were
used to define the base line and GFP positive cell populations. Relative MFIs are indicated.
Talin expression determined by Western blot using anti-HA antibody were shown next to the
FACS data.
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