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.