Molecular Cell

Article

Structural Basis for the Autoinhibitionof Talin in Regulating Integrin ActivationEsen Goksoy,1,3,4 Yan-Qing Ma,1,2,4 Xiaoxia Wang,1 Xiangming Kong,1 Dhanuja Perera,1

Edward F. Plow,1,2 and Jun Qin1,3,*1Department of Molecular Cardiology2Joseph J. Jacobs Center for Thrombosis and Vascular BiologyLerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA3Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, OH 44195, USA4These authors contributed equally to this work

*Correspondence: qinj@ccf.orgDOI 10.1016/j.molcel.2008.06.011

SUMMARY

Activation of heterodimeric (a/b) integrin transmem-brane receptors by the 270 kDa cytoskeletal proteintalin is essential for many important cell adhesiveand physiological responses. A key step in thisprocess involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin(talin-H) with integrin b membrane-proximal cyto-plasmic tails (b-MP-CTs). Compared to talin-H, intacttalin exhibits low potency in inducing integrin activa-tion. Using NMR spectroscopy, we show that thelarge C-terminal rod domain of talin (talin-R) interactswith talin-H and allosterically restrains talin in aclosed conformation. We further demonstrate thattalin-R specifically masks a region in talin-PTB whereintegrin b-MP-CT binds and competes with it forbinding to talin-PTB. The inhibitory interaction isdisrupted by a constitutively activating mutation(M319A) or by phosphatidylinositol 4,5-bisphos-phate, a known talin activator. These data definea distinct autoinhibition mechanism for talin andsuggest how it controls integrin activation and celladhesion.

INTRODUCTION

Talin is a high molecular weight protein containing an N-terminal

head (1–433, talin-H, 50 kDa) and a C-terminal rod domain (434–

2541, talin-R, 220 kDa) (Figure 1A) (Rees et al., 1990). Talin-H is

globular, containing a FERM domain composed of three lobes,

F1, F2, and F3 or phosphotyrosine-binding (PTB) domain (Gar-

cıa-Alvarez et al., 2003); talin-R is highly elongated, containing

a series of helical bundles separated by linkers (McLachlan

et al., 1994; Papagrigoriou et al., 2004; Fillingham et al., 2005;

Gingras et al., 2008). Discovered in high concentrations at re-

gions of cell-substratum contact (Burridge and Connell, 1983),

talin has long been known to be a physical linker between integ-

rins and the actin cytoskeleton, and a regulator of a variety of

cellular processes such as cell spreading, migration, and prolif-

124 Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc.

eration (Turner and Burridge, 1991; Calderwood et al., 2000). Re-

cent advances have revealed an additional and especially impor-

tant role for talin: by binding to integrin b CTs, talin induces high-

affinity ligand binding to integrins, integrin activation (Tadokoro

et al., 2003; Wegener et al., 2007), a major step in cell adhesion,

migration, and numerous physiological and pathophysiological

responses (for review, see Hynes, 2002). A surge of genetic,

cell biological, and biochemical experiments has now estab-

lished that talin F3, the PTB domain of talin-H, plays a dominant

role in inducing the integrin activation (Calderwood et al., 2002;

Tadokoro et al., 2003; Ma et al., 2006; Wegener et al., 2007).

Structural analyses have shown that talin-PTB binds to both

membrane-distal and membrane-proximal regions of integrin

b CTs (Vinogradova et al., 2002, 2004; Garcıa-Alvarez et al.,

2003; Ulmer et al., 2003; Wegener et al., 2007), and the latter

event causes integrin activation by unclasping the integrin

a/b CT complex (Vinogradova et al., 2002, 2004; Kim et al.,

2003; Wegener et al., 2007).

With rapidly accumulating in vitro (see review, Calderwood,

2004) and in vivo (Nieswandt et al., 2007; Petrich et al., 2007)

data demonstrating the central role of talin in integrin activation,

a fundamental question still remains: how is talin activity regu-

lated? Previous functional analyses have suggested that talin

activity may be autoinhibited for binding to integrins (Martel

et al., 2001; Yan et al., 2001). However, this mechanism remains

largely speculative, and the structural basis for such auto-

inhibition and how it impacts integrin activation have not been

established.

Here, we use structural, biochemical, and mutational analyses

to pinpoint the molecular details of the talin regulation. Intact

talin, but not its talin-H fragment, has low intrinsic integrin-

activating activity, directly supporting the notion that talin adopts

a default inactive conformation. By performing extensive NMR-

based structural analyses, we show that a middle segment of

talin-R specifically interacts with talin-FERM and competes

with integrin b CT for binding to talin-PTB. Mutations of key res-

idues in talin-PTB involved in binding to integrin b CT revealed

that talin-R specifically masks the integrin membrane-proximal

b CT-binding site on talin-PTB that is key for controlling the

cytoplasmic unclasping and activation of integrins. A structure-

based mutation on talin-PTB, which does not affect talin-H/in-

tegrin interaction but disrupts the talin-H/talin-R interaction,

Molecular Cell

Structural Basis of Talin Autoinhibition

leads to constitutive activation of full-length talin. Phosphatidyli-

nositol 4,5-bisphosphate (PIP2), a known talin activator (Martel

et al., 2001), disrupts the inhibitory talin-PTB/talin-R interaction.

These results define a structural mechanism of the talin autoinhi-

bition and suggest how it may serve as a specific cellular brake to

control integrin activation.

RESULTS

Talin Adopts a Default Low-Activity Statefor Regulating Integrin ActivationIt is well-established that talin-H can activate integrins (Calder-

wood et al., 1999, 2002; Tadokoro et al., 2003; Kim et al.,

2003; Ma et al., 2006; Bouaouina et al., 2007), and it is presumed

that this activity is blunted in full-length talin. To directly compare

the integrin-activating activity of these two talin forms, we trans-

fected vectors expressing full-length talin or talin-H, all as EGFP

constructs, into a CHO cell line stably expressing integrin aIIbb3

(aIIbb3-CHO). The activation state of the aIIbb3 was then mea-

sured by the binding of an activation-specific mAb to aIIbb3

antibody (PAC1) by FACS. To eliminate the influence of varied

Figure 1. Effects of Full-Length Talin and Talin-H on Integrin

Activation

(A) Domain organization of full-length talin. The three lobes of talin-H FERM

domain, F1, F2, F3, or PTB and the rod domain have been labeled.

(B) Comparison of the activation of integrin aIIbb3 by full-length talin and talin-H.

CHO cells stably expressing the integrin aIIbb3 were transfected with cDNAs for

full-length talin or talin-H, each as EGFP constructs. The EGFP-positive cells

were gated and used to monitor PAC-1 binding to determine the extent of aIIbb3

activation as previously described (Ma et al., 2008). The data are means ± SD

from three independent experiments. **p < 0.01.

expression of talin-H and full-length talin, only EGFP-positive

cells with similar expression levels were gated. The data in

Figure 1B are from three independent experiments and show

that, while talin-H induced substantial activation of aIIbb3 activa-

tion, full-length talin was significantly weaker as an activator.

Thus, our data provide direct functional evidence for the suppo-

sition that integrin activation by full-length talin is dampened

and is enhanced by events that change exposure of its head

region.

A Middle Segment of Talin-R, 1654–2344, Interactswith Talin-PTB in Talin-FERMSince talin-PTB (F3) in talin-H is solely responsible for binding to

integrin b CTs during integrin activation (Calderwood et al., 2002;

Garcıa-Alvarez et al., 2003; Tadokoro et al., 2003; Wegener et al.,

2007), we reasoned that talin-R might mask the integrin b CT-

binding site. To test this hypothesis, we used NMR-based 2D1H-15N HSQC (heteronuclear single quantum correlation) exper-

iments to examine the interaction between 15N-labeled talin-PTB

and unlabeled talin-R fragments. 2D 1H-15N HSQC is extremely

sensitive for probing protein-target interactions with a wide

range of affinities (Bonvin et al., 2005; Vaynberg and Qin, 2006;

Takeuchi and Wagner, 2006). The HSQC spectrum of an 15N-la-

beled protein contains many peaks; each peak correlates to

a proton attached to 15N within a particular residue in the protein.

Some peaks may be shifted or broadened in the HSQC spectrum

if the protein is bound to a target, an excellent indication of the

binding interface. The peak broadening or disappearance can

be due to the size increase or intermediate rate exchange of

the protein complex at the NMR timescale. If any of the unlabeled

talin-R fragments bind to 15N-labeled talin-PTB, some or all1H/15N amide signals of talin-PTB should be perturbed and

broadened, which in turn provides information on the intramolec-

ular interaction between talin-PTB and talin-R. Based on the

available helical bundle structure of talin-R (Papagrigoriou

et al., 2004; Fillingham et al., 2005; Gingras et al., 2008) and

a secondary structure prediction program (Bryson et al., 2005),

we dissected talin-R into nine consecutive fragments (R1,

434–947; R2, 944–1483; R3, 1482–1653; R4, 1654–1848; R5,

1841–1983; R6, 1984–2102; R7, 2103–2229; R8, 2225–2344;

and R9, 2338–2541), where the division regions were predicted

to be random coil or loop structures so the structural integrities

of these fragments should be preserved. A series of HSQC spec-

tra were collected for 15N-labeled talin F2F3 domain, which con-

tains talin-PTB (F3), in the absence and presence of individual

unlabeled talin-R fragments. Starting from the N terminus of

talin-R, we found that R1, R2, and R3 had little effect on the

HSQC spectrum of talin-F2F3 (see Figures S1A–S1C, available

online), whereas R4 (�21 kDa) caused significant line broadening

of talin-F2F3 (�22 kDa) (Figure S1D), suggesting that R4 binds to

talin-F2F3. From the C terminus, R9 did not bind (Figure S1E),

but R8 caused significant line broadening of talin-F3F3, suggest-

ing that it also interacts with talin-F2F3 (Figure S1F). These initial

mapping data indicated that talin-R does interact with talin-F2F3

and the binding site involves multiple regions, R4 and R8, but not

the N-terminal R1-R3 and C-terminal R9. Based on these initial

data, we then prepared another larger expression construct

encompassing R4 and R8, i.e., 1654–2344, with a total molecular

Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc. 125

Molecular Cell

Structural Basis of Talin Autoinhibition

weight of �76 kDa (termed talin-RM). Talin-RM was well-folded,

as assessed by its chemical shift dispersion pattern (Figure S2).

As predicted, talin-RM also bound to talin F2F3, as indicated by

the substantial line broadening and disappearance of talin F2F3

signals in HSQC (MW�100 kDa) (data not shown). By employing

a transverse relaxation optimized spectroscopy (TROSY) tech-

nique into HSQC, which is tailored for detecting NMR signals

of large proteins and protein complexes (Pervushin et al.,

1997), we were able to recover the majority of signals, some of

which were significantly shifted due to binding (Figure 2A).

To understand the nature of this interaction, we performed

backbone signal assignments of talin F2F3 using triple reso-

nance NMR, including HNCACB, CBCACONH, HNCA, HNCO,

HC(CO)NH, and C(CO)NH (Bax and Grzesiek, 1993). Chemical

shift mapping revealed that only NMR signals of F3 (PTB), but

not F2 in talin F2F3, were either significantly shifted or broadened

(Figure 2B), thus supporting our hypothesis that talin-PTB binds

to talin-RM. To improve the spectral quality and simplify the

spectral analysis, we made 15N/2H-labeled talin-PTB and per-

formed its TROSY-HSQC in complex with the unlabeled talin-

RM (total complex is �90 kDa). Both deuteration and TROSY

are known to dramatically reduce the line broadening of the pro-

teins, which led to an excellent and well-resolved spectrum of

talin-PTB bound to unlabeled talin-RM (Figure S3A). As expected,

talin-RM caused significant chemical shift perturbation for talin-

PTB (Figure S3A). Surface plasmon resonance (SPR) experi-

ments revealed that the dissociation constant (KD) between

talin-PTB and talin-RM is 577 nM (Table S1 and Figure S3B).

Talin-RM and Integrin Membrane-Proximal b3 CTCompete for an Overlapping Binding Site on Talin-PTBTo precisely map the talin-RM-binding site on talin-PTB, we per-

formed a series of NMR titration experiments. TROSY-HSQC

spectra were collected for 15N/2H-labeled talin-PTB in the

absence and presence of increasing concentrations of talin-RM

to obtain molar ratios of 1:0.0, 1:0.5, 1:1.0, and 1:2. This exper-

imental design allowed us to trace the significantly perturbed

signals (Figure S3A). Figure 3A shows the detailed chemical shift

perturbation profile. Remarkably, the perturbation pattern was

similar to that previously reported for integrin b3 CT or integrin

membrane-proximal b3 CT segment fused to PIPKIg peptide

(Wegener et al., 2007) (Figure 3A), suggesting that the talin-RM-

binding site on talin-PTB overlaps with that for integrin b3 CT.

Interestingly, chemical shift mapping revealed that talin-R4

(1654–1848) also induced a very similar perturbation profile

(Figure S4A versus Figure S4B), albeit with slightly reduced

chemical shift changes and lower affinity (KD �3.6 mM, Table

S1) than talin-RM (KD �0.58 mM, Table S1). On the other hand,

a larger fragment containing C-terminal talin-RM (R6-R8, 1984–

2344) induced different and very narrow-range chemical shift

changes peaking around 367–375 (Figure S4C) with much lower

affinity (KD �78.0 mM, Table S1) than talin-RM. Combining these

two fragments in a single construct (talin-RM) yields higher affin-

ity (0.58 mM, see Table S1). These data suggest that R4 and

R6–R8 bind to different regions in talin-PTB and that R4 plays

a more important role in binding to talin-PTB (see more data

below). Figure 3B highlights significantly shifted residues in

talin-PTB upon binding to talin-RM and integrin b3 CT chimera,

126 Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc.

respectively, thus providing a more direct view of how the two

binding sites might overlap.

To more precisely evaluate how the integrin b3 CT-binding

sites on talin-PTB may be involved in binding to talin-RM, we in-

troduced a series of structure-based talin-PTB point mutations,

L325R, W359A, S365D, S379R, and Q381V, each of which has

been previously shown to impair the talin-mediated integrin

activation without affecting the structural integrity of talin-PTB

(Wegener et al., 2007). L325R, S365D, S379R, and Q381V dis-

rupt the integrin b membrane-proximal CT binding to talin-

PTB, whereas W359A abolishes the integrin CT binding to

talin-PTB by removing the bulky interaction of the membrane-

distal CT with talin-PTB (Wegener et al., 2007). We also made

the M319A mutant. M319 is a surface-exposed hydrophobic res-

idue that is not involved in interacting with either integrin b3

membrane-proximal region (Wegener et al., 2007) or mem-

brane-distal region (Garcıa-Alvarez et al., 2003), and thus its

mutation to Ala had little effect on the interaction with integrin

b3 CT chimera (Figure S5). However, M319 is most significantly

perturbed by talin-RM (Figure 3A), suggesting that it may play

a crucial role in interacting with talin-RM. Table S1 summarizes

the KD values of talin-RM binding to WT talin-PTB, M319A,

L325R, W359A, S365D, S379R, and Q381V by SPR. Compared

Figure 2. The Binding of Talin-RM to Talin-F2F3

(A) 2D TROSY 1H-15N HSQC of talin-F2F3 in the absence (black) and presence

of unlabeled talin-RM (red). Well-resolved peaks, which are either significantly

broadened or shifted, are labeled in free-form talin-F2F3.

(B) Chemical shift mapping of the binding to talin-F2F3. Only the resonances

in F3 (306–405) are significantly perturbed. Residues whose signals were

diminished due to severe line broadening are indicated by gray bars.

Molecular Cell

Structural Basis of Talin Autoinhibition

to wild-type talin-PTB, S365D, S379R, and Q381V exhibited

markedly reduced binding to talin-RM, whereas L325R had

very small effect. As expected, while M319A still binds to integrin

b3 CT as WT talin-PTB (Figure S5A versus Figure S5B), it had

dramatically weakened interaction (�140-fold) with talin-RM

(Table S1).

The effects of S365D, S379R, and Q381V recapitulate those

for binding to the integrin membrane-proximal b3 CT and indi-

cate that this site significantly overlaps with that for talin-RM in

the talin-PTB domain. To confirm this conclusion, we prepared

large quantities of two representative mutants in 15N/2H-labeled

form, S365D (reduced binding to talin-RM by �2.9 3 103-fold)

and Q381V (reduced binding to talin-RM by �540-fold) (Table

S1) and examined their chemical shift perturbation in the

Figure 3. Mapping of the Talin-RM-Binding Site on Talin-PTB

(A) 1H/15N chemical shift changes of talin-PTB upon binding to talin-RM as

a function of residue number. As a comparison, the chemical shift changes

of talin-PTB upon binding to b3 CT chimera are also reproduced using the

chemical shift table deposited in the BioMagResBank (accession number

7150) by Wegener et al. (2007). The membrane-distal residues involved in

binding to integrin b3 CT chimera are colored in red in the upper panel. Note

that W359 is changed dramatically by b3 CT chimera (the change was so

big, due to its bulky interaction with talin-PTB [Wegener et al., 2007, that a

broken line was used to conserve space).

(B) Significantly perturbed residues on talin-PTB by b3-CT chimera (left) and

talin-RM (right) are highlighted using the structure of talin-PTB. The order of

dark blue, blue, and light blue indicates the extent of the chemical shift

changes, with the dark blue having the most significant changes. The changes

have some similarity indicating potential overlapping binding sites for b3 CT

and talin-RM, but there are significant differences indicating that the binding

sites are not identical.

absence and presence of unlabeled talin-RM. Consistent with

the SPR data, the extent of the chemical shift changes was

decreased for Q381V and much more decreased for S365D as

compared to WT talin-PTB (Figure 4).

Interestingly, W359A, which completely abolished the talin-

PTB binding to integrin b3 CT by disrupting the bulky hydropho-

bic interaction between talin-PTB and membrane-distal b3 CT

(Garcıa-Alvarez et al., 2003; Wegener et al., 2007), had little ef-

fect on the KD of the talin-PTB/talin-RM interaction (Table S1).

This observation, together with the above described effects of

other mutants, suggests that the binding sites on talin-PTB for

talin-RM and integrin b3 CT are overlapping but not identical.

To further investigate this possibility, we performed HSQC-

based competition experiments. As shown in Figures S6A and

S6B, while the majority of signals disappear from 15N-labeled

talin-PTB upon binding to the large talin-RM (total MW �90 kDa,

KD �577nM, see Table S1), these signals return upon addition

of equal molar b3 CT chimera (b3 membrane-proximal CT fused

to PIPKIg peptide, MW �3.5 kDa, KD �140 nM, see Wegener

et al. [2007]), yielding a spectrum identical to that for 15N-labeled

talin-PTB bound to the unlabeled b3 CT chimera (Figure S6C).

These data demonstrate that the small b3 CT chimera peptide

competed with large talin-RM for binding to talin-PTB. In contrast,

excess PIPKIg peptide, which mimics the b3 membrane-distal

CT binding and binds tightly to talin-PTB (KD �270 nM) (de Per-

eda et al., 2005), did not recover the broadened signals at all

(Figure S6D), indicating that PIPKIg and talin-RM do not have

overlapping binding sites on talin-PTB. Since PIPKIg mimics

the b3 membrane-distal CT for binding to talin-PTB (de Pereda

et al., 2005; Kong et al., 2006; Wegener et al., 2007), our NMR

data are in agreement with our SPR data on W359A mutant (Table

S1) indicating that while the integrin membrane-proximal CT-

binding site is significantly masked by talin-RM, the b3 mem-

brane-distal CT-binding site for talin-PTB is not. Consistently,

a F730A mutant in the chimera peptide, which has dramatically

reduced membrane-proximal b3 CT binding to talin-PTB (Wege-

ner et al., 2007), did not compete effectively with talin-RM

(Figure S7A versus Figure S7B). Note that the b3 CT also binds

to talin-RM (Tremuth et al., 2004). However, such binding does

not appear to interfere with the talin-PTB/talin-RM interaction,

since b3 CT did not affect the talin-RM interaction with talin-

PTB W359A (Figure S8). Note that W359A has the same affinity

for talin-RM as WT talin-PTB (Table S1) but does not bind to b3

CT (Wegener et al., 2007).

NMR-based competition experiments also revealed that talin-

R4 (Figure S9A), but not talin-R6-R8 (Figure S9B), competed with

b3 CT chimeria for binding to talin-PTB. Since talin-R4 induced

very similar chemical shift perturbations as talin-RM when bind-

ing to talin-PTB (Figure S4), this finding suggests that talin-R4

plays a major role in masking the membrane-proximal CT-bind-

ing site on talin-PTB. It also further supports our prior chemical

shift mapping and affinity-based results indicating that talin-

R4 and talin-R6-R8 bind to different regions of talin-PTB.

Figure 5A summarizes the surface depiction of the talin-PTB

structure in which key residues directly involved in binding to

talin-RM are highlighted based on the chemical shift mapping,

mutagenesis, and competition data. The binding surface is com-

pared to that for the talin-PTB/integrin b CT complex (Figure 5B)

Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc. 127

Molecular Cell

Structural Basis of Talin Autoinhibition

(Wegener et al., 2007) and shows that the membrane-proximal

b3 CT-binding site significantly overlaps with that for talin-RM,

thus providing a view of how talin-R may sterically suppress

the b CT binding to talin-PTB in intact talin.

To further evaluate the significance of the talin autoinhibition,

we examined the activity of talin M319A in integrin activation.

Since this mutant still maintains the integrin binding (Figure S5)

but has dramatically reduced the talin-PTB/talin-RM affinity (Ta-

ble S1), we speculated that full-length talin M319A would acti-

vate the integrin. As shown in Figure 5C, M319A indeed dramat-

ically enhanced talin-induced aIIbb3 activation as compared to

WT talin. This enhancement was abolished by RGDS peptide,

an inhibitor of ligand binding to b3 integrins (data not shown), in-

dicating specificity. Thus, this observation offers strong support

for our hypothesis. Note that the M319A-induced integrin activa-

tion is slightly more potent than talin-H. While the precise mech-

anism for this higher potency remains to be determined, one can

envision two possibilities: (1) full-length talin M319A has higher

affinity to integrin than talin-H alone, thus leading to the more po-

tent integrin activation. In addition to talin-H, isolated talin-R also

binds to integrin b CT (Xing et al., 2001; Yan et al., 2001; Tremuth

et al., 2004) at the membrane-distal region (Tremuth et al., 2004),

but not the membrane-proximal site (E.G. and J.Q., unpublished

data). Thus, the constitutively open conformation of M319A may

have higher affinity for integrin than talin-H alone due to both ta-

lin-H (M319A)/integrin and talin-R/integrin interactions. (2) Mem-

brane anchoring of talin is important for integrin activation (Vi-

nogradova et al., 2004; Wegener et al., 2007). It is possible that

Figure 4. Chemical Shift Perturbation Pro-

files for WT Talin-PTB and Its Mutants

Q381V and S365D by Talin-RM

Note that the chemical shifts are attenuated with

the Q381V mutant but are still similar to WT,

whereas they are dramatically reduced with the

S365D mutant.

the open conformation of M319A has

stronger capacity to anchor to the mem-

brane than talin-H, resulting in more

potent integrin activation.

Conformational Activation by PIP2Given the above findings, an obvious

question is how the closed conformation

of talin is opened. A well-known talin acti-

vator is PIP2, which promotes strong talin

binding to integrin b CT (Martel et al.,

2001), resulting in the formation of a ter-

nary PIP2/talin/integrin complex in living

cells for mediating integrin activation and

clustering (Cluzel et al., 2005). Our SPR

experiment revealed that talin-H, but not

talin-RM, can potently bind to biotinylated

PIP2 with high affinity (KD �89.2 ±

1.25 nM, Figure S10A). The biotinylated

PIP2 was mounted on a biotin sensor

chip, and such positioned PIP2 should mimic PIP2 anchored to

the membrane. Since talin-H FERM domain and multiple FERM

domains bind to PIP2 involving PTB/PH subdomains (Hamada

et al., 2000; Bompard et al., 2003; Cai et al., 2008), we considered

if talin-PTB is involved in binding PIP2. Our HSQC-based map-

ping experiment revealed that PIP2 can indeed interact with ta-

lin-PTB with the most significant perturbation around 370–378

(Figure S10B), which overlaps with the talin-RM-binding site

(Figure 5D). SPR experiments demonstrated that PIP2 can dis-

rupt the talin-RM/talin-PTB interaction in a concentration-

dependent manner (Figure 6A). Such competition was confirmed

by HSQC experiments in which soluble C4-PIP2 was able to

compete with talin-RM for binding to talin-PTB (Figure 6B). Since

PIP2/talin-H interaction has been shown not to interfere with the

talin-H/integrin interaction (see Figure 6 in Martel et al. [2001]) and

instead it induces conformational change of talin for high-affinity

integrin binding (Martel et al., 2001), our findings provide a mech-

anism by which PIP2 binds to talin-PTB and sterically displaces

the inhibitory talin-R to expose the integrin-binding site.

DISCUSSION

A combination of NMR spectroscopy, mutagenesis, and bio-

chemical experiments have revealed how talin-FERM via its

PTB subdomain interacts with talin-R to restrain talin in an inac-

tive/autoinhibited conformation. Autoinhibition is a widespread

phenomenon in controlling protein functions and occurs in mul-

tiple FERM-containing proteins (Pearson et al., 2000; Li et al.,

128 Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc.

Molecular Cell

Structural Basis of Talin Autoinhibition

2007; Lietha et al., 2007). However, only the talin FERM domain

can directly mediate integrin activation (Tadokoro et al., 2003) by

binding to the integrin membrane-proximal b CT (Vinogradova

et al., 2002; Wegener et al., 2007). The masking of the integrin

membrane-proximal b CT-binding site in talin-FERM by talin-R

is quite unique as compared to other autoinhibitory FERM-

containing proteins, which utilize FERM domains to autoinhibit

the functions of other parts of proteins (Pearson et al., 2000; Li

et al., 2007; Lietha et al., 2007). For example, focal adhesion ki-

nase (FAK) utilizes its N-terminal FERM F2 domain to mask the

C-terminal kinase active site (Lietha et al., 2007), whereas moe-

sin uses its N-terminal FERM F2 and F3 subdomains to mask its

C-terminal actin-binding site. Thus, we have unraveled a distinct

autoinhibition mechanism for talin in which other parts of the

molecule, talin-R, control the function of the FERM domain in

integrin activation. Our cell-based experiments provide direct

functional evidence to support this mechanism; full-length talin

was a poor activator of integrin aIIbb3, whereas talin-H was sub-

stantially more potent (Figure 1). These data are also consistent

with the report of Han et al. (2006) showing that talin must be ac-

Figure 5. Talin Autoinhibition and Its Effect

on Integrin Activation

(A) Surface representation of talin-PTB domain

with the talin-RM-binding site highlighted. Resi-

dues whose mutations impair talin-RM binding

are colored in green, whereas other potential bind-

ing residues are colored in cyan based on their sig-

nificant chemical shift perturbation in Figure 3 and

competition data in Figures S6–S9.

(B) The same view as (A) but with the integrin b3

CT-binding site highlighted. The integrin-binding

site is based on Garcıa-Alvarez et al. (2003) and

Wegener et al. (2007). The integrin membrane-

proximal b3 CT-binding residues are colored in

green, and the membrane-distal b3 CT-binding

residues are colored in red. Note the significant

overlap of the integrin b3 membrane-proximal

binding site in green as compared to that of talin-

RM in (A), also in green.

(C) Comparison of the activity of integrin aIIbb3 by

full-length talin, full-length talin M319A, and talin-

H activation. M319A is more potent than talin-H,

probably due to its higher affinity for integrin b3

CT than talin-H arising from its two sites of interac-

tion, talin-H/integrin (unmasked) and talin-R/integ-

rin. **p < 0.01; *p < 0.05.

(D) The same view of (A) but with potential PIP2-

binding site highlighted based on the chemical

shift mapping of significantly perturbed residues

(green, most significantly shifted; cyan, next signif-

icantly shifted).

tivated to display its integrin-activating

activity. Furthermore, talin M319A muta-

tion, which disrupts the talin-PTB/talin-R

interaction, but not talin-PTB/integrin in-

teraction, constitutively activated aIIbb3

(Figure 5C). These data provide strong

functional evidence for talin autoinhibition

in regulating integrin activation. The auto-

inhibitory domain was mapped to the middle region (1654–2344)

of talin-R, and its affinity for talin-PTB was found to be within the

submicromolar range as assessed by SPR using purified com-

ponents (Table S1). We note that the intramolecular interaction

involving the two domains in intact talin is expected to be even

stronger by placing the binding surfaces within the same mole-

cule and may provide the tight control of talin activity needed

to prevent spontaneous integrin activation. Interestingly, despite

its inhibitory conformation that prevents the talin-PTB/integrin

membrane-proximal b CT contact, full-length talin can still bind

weakly to integrin b CT (the affinity is about 6-fold weaker than

that of the talin-H/integrin interaction, see Yan et al. [2001]), sug-

gesting that the integrin-binding site on talin is not completely

masked. Our data are consistent with this observation in that

the integrin membrane-distal CT-binding site on talin-PTB

does not appear to be significantly masked by talin-R. The bind-

ing of isolated talin-R to the membrane-distal region of integrin

b CT (Xing et al., 2001; Yan et al., 2001; Tremuth et al., 2004)

may contribute to the full-length talin/integrin interaction. Based

on our observations, we propose a model for how talin controls

Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc. 129

Molecular Cell

Structural Basis of Talin Autoinhibition

integrin activation (Figure 7). In the closed state, the integrin

membrane-proximal b CT-binding site on talin-H is masked by

talin-R, although talin can still weakly associate with integrin

b CT via its unmasked sites. Upon activation by cellular stimuli,

talin undergoes a conformational change so that talin-PTB can

access the integrin membrane-proximal b CT, which leads to

the integrin a/b CT unclasping and inside-out activation. Since

talin also binds to actin via the C-terminal end of talin-R (outside

talin-RM) (Gingras et al., 2008), the enhanced talin/integrin inter-

action, consequent to the talin conformational change, may alter

integrin-actin linkage. In this manner, cells can undergo dynamic

cytoskeleton remodeling, in coordination with integrin activation

and ligation. A dynamic equilibrium exists between monomeric

and dimeric talin, which only shifts to the dimeric state at

Figure 6. PIP2 Disrupts the Inhibitory Talin-PTB/Talin-RM

Interaction

(A) SPR showing that C8-PIP2 (Echelon Biosciences, Inc.) suppresses the ta-

lin-PTB interaction with talin-RM in a concentration-dependent manner. Talin-

RM was immobilized on the activated chip. Talin-PTB mixed with increasing

amount of PIP2 was each injected at a flow rate of 20 ml/min.

(B) Overlay of HSQC spectra of 15N-labeled talin-PTB showing that C4-PIP2

(soluble at a high concentration in 150 mM NaCl, 50 mM phosphate buffer

[pH 7.0]) recovers many signals of talin-PTB (red), which were otherwise

broadened/disappeared in the presence of talin-RM (black) (talin-PTB:talin-

RM:PIP2 = 0.2 mM:0.4 mM:2.0 mM).

130 Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc.

high talin concentration (>3 mM) (Molony et al., 1987), and the

dimeric state may strengthen the integrin-actin linkage. Al-

though the exact dimerization topology for full-length talin is

not clear, recent crystallographic studies of the C-terminal ac-

tin-binding module (2300–2541) revealed a dimeric structure

and suggested that the full-length talin dimer may exist in three

possible conformations (Gingras et al., 2008): parallel, tail-

to-tail V-shaped, or tail-to-tail antiparallel fashion. Earlier EM

studies (Goldmann et al., 1994) suggested that the two globular

heads (talin-Hs) are on two opposite ends of the talin dimer and

suggested a head-to-tail antiparallel dimer, which raises a pos-

sibility that the middle talin-RM in one subunit interacts with

talin-H in the other. However, such head-to-tail antiparallel is

incompatible with the crystallographic studies (Gingras et al.,

2008), and thus intermolecular autoinhibition is less likely to

occur in cells.

What is the mechanism to trigger the conformational change

of talin to expose its integrin membrane-proximal b CT site

in vivo? One mechanism involves PIP2, which promotes strong

talin binding to the integrin b1 CT (Martel et al., 2001). Interest-

ingly, the PIP2-producing enzyme, PIPKIg, is also recruited to

integrin adhesion sites by talin (Ling et al., 2002; Di Paolo

et al., 2002), providing a mechanism for efficient PIP2/talin in-

teraction. Our NMR and biochemical data have indicated that

while the PIPKIg binding to talin does not affect the autoinhibi-

tory conformation of talin (Figure S6D), the PIPKIg product,

PIP2, does (Figure 6). Thus, upon agonist stimulation, talin

may recruit PIPKIg to locally enrich PIP2, which in turn induces

the conformational change of talin to expose its integrin mem-

brane-proximal b CT site, promoting high-affinity talin binding

to and activation of integrins. This model is supported by sev-

eral independent in vivo observations: (1) integrin aIIbb3 activa-

tion is associated with the increased PIPKIg activity and PIP2

levels in platelets (Hinchliffe et al., 1996). (2) PIP2 level is asso-

ciated with talin on activated platelets (in which aIIbb3 is acti-

vated), but not on resting platelets (Heraud et al., 1998); and

(3) a ternary complex involving PIP2, talin, and integrin avb3

was formed in the living cells to mediate avb3 activation and

clustering (Cluzel et al., 2005). Another emerging mechanism

for the talin activation involves RIAM (Han et al., 2006), which

forms a supramolecular complex with talin and Rap1 to activate

integrins. It remains to be determined whether RIAM changes

the conformation of talin in this supermolecular complex to re-

lieve its autoinhibition. Finally, calpain cleaves talin into a talin-

H/talin-R mixture, thereby releasing talin-H for enhanced bind-

ing to integrin (Yan et al., 2001). However, activation of integrin

aLb2 by ionomycin did not involve calpain-mediated talin cleav-

age (Dreolini and Takei, 2007). RIAM-mediated integrin activa-

tion also does not involve calpain-dependent talin cleavage

(Han et al., 2006). Most data implicate calpain in integrin out-

side-in signaling (Schoenwaelder et al., 1997; Hayashi et al.,

1999; Franco et al., 2004).

In summary, we have demonstrated the structural basis of the

autoinhibition for talin in regulating integrin activation. Our find-

ings, together with previous structural data (Vinogradova et al.,

2002; Garcıa-Alvarez et al., 2003; Wegener et al., 2007), now

provide a view of how a series of conformation changes occur

for talin-mediated integrin activation (Figure 7).

Molecular Cell

Structural Basis of Talin Autoinhibition

EXPERIMENTAL PROCEDURES

Sample Preparation for NMR Analyses

A pET15b vector containing a thrombin-cleavable N-terminal His tag

GSS(H)6SSGLVPRGSHM was used to subclone the talin-H fragments talin-

F3/PTB (306–405) and talin-F2F3 (206–405). A variant of pET15b, pET30a,

was used to clone larger talin-R fragments, talin-R 434–947 (R1), 944–1483

(R2), 1483–1653 (R3), 1654–1848 (R4), 1841–1983 (R5), 1984–2102 (R6),

2103–2229 (R7), 2225–2344 (R8), 2338–2541 (R9), and talin-RM (1654–2344)

and a talin R6-R8 construct. The fragments were expressed in E. coli BL21

(DE3), and the cells were harvested and lysed using 10 mg/ml lysozyme. All

fragments were purified on nickel columns with 250 mM imidazole in the elu-

tion buffer (50 mM phosphate buffer, 100 mM NaCl, 1 mM DTT [pH 7.0]). The

eluted proteins were dialyzed against elution buffer, and His tags were cleaved

using thrombin. The proteins were then further purified by size exclusion chro-

matography on Superdex 200 (Amersham Bioscience). Talin-H was prepared

as described (Vinogradova et al., 2002). To prepare 15N and/or 13C-labeled

protein, the cells were grown in M9 minimal media containing 15NH4Cl and/

or 13C-glucose, and all other conditions were the same as for the unlabeled

proteins. To prepare 15N/90%2H-labeled talin-PTB, cells were grown in M9

minimal media containing 15NH4Cl and 90% 2H2O. Point mutants of talin-

PTB (M319A, L325R, W359A, S365D, S379R, and Q381V) were made using

QuikChange Site-Directed Mutagenesis (Stratagene, Inc) and prepared in

the same way as WT proteins. A point mutation (C336S) was also introduced

in talin-PTB as reported (de Pereda et al., 2005) to improve protein stability.

The b3 CT chimera peptide TIHDRKEFAKFEEERARAKWVYSPLHYSAR (the

sequence in bold is PIPKIg tail), b3 CT chimera mutant (F730A), and PIPKIg

(SWVYSPLHYSAR) were synthesized by our Biotechnology Core. For impro-

ved solubility, the KLLI sequence was not included at the N terminus of the

b3 CT, since it is not involved in interacting with talin-PTB (Wegener et al.,

2007). The b3 CT chimera binds tightly to talin-PTB (Kd �140 nM) in slow ex-

change, which mimicks the b3 CT that has weaker binding to talin-PTB in

intermediate exchange (Wegener et al., 2007).

NMR Spectroscopy

All heteronuclear NMR experiments were performed as described by Bax and

Grzesiek (1993). All triple resonance NMR experiments for 1 mM talin-F2F3

were performed on a Varian 600 MHz instrument equipped with a triple reso-

nance probe and shielded z-gradient unit. These experiments were performed

at 25�C (pH 7.0) in 150 mM NaCl, 50 mM phosphate buffer, and 1 mM DTT. All

HSQC experiments were performed on Bruker Avance 600 or 900 MHz spec-

trometers using the cryogenic triple resonance probes. 15N-labeled and/or15N/90%2H-labeled talin-PTB or talin-F2F3 (0.2 mM) were used with and with-

out the talin-R fragments at 1:2 ratio in all HSQC experiments. For chemical

Figure 7. Model for the Talin Activation in

Inducing the Integrin Activation

In the closed state, the integrin membrane-proxi-

mal b CT-binding site (blue bar) on talin-H is

masked by talin-R, although talin can still weakly

bind to integrin b CT via unmasked sites. Upon ac-

tivation by some cellular factor such as PIP2 or

RIAM, talin undergoes conformational change so

that talin-PTB can access to the integrin mem-

brane-proximal b CT and induces integrin a/b CT

unclasping and integrin activation. The ternary

complex involving PIP2/talin/integrin has been indi-

cated by Martel et al. (2001) and Cluzel et al. (2005).

shift mapping, TROSY-based HSQC (Pervushin

et al., 1997) for talin-PTB or talin-F2F3 in complex

with talin-RM was performed on a Bruker 900 MHz

spectrometer. Weighted chemical shift changes

were calculated using the equation Ddobs[HN,N] =

([DdHNWHN]2 + [DdNWN]2)1/2, where WHN = 1 and

WN = 0.154 are weighting factors based on the gyromagnetic ratios of 1H

and 15N. All the spectra were processed with nmrPipe (Delaglio et al., 1995)

and visualized with PIPP (Garrett et al., 1991).

Surface Plasmon Resonance Measurements

SPR was performed using a BIA Core 3000 instrument (Amersham Pharmacia

Biotech). CM5 sensor chips were activated using the amine coupling kit from

Amersham Pharmacia Biotech. Talin-RM was immobilized to the activated sur-

face, and talin-PTB and its mutants were injected at 0.1–5 mM and flow rates of

20 ml/min. For PIP2/talin-H interaction, biotinylated C6-PIP2 (C-45B6a, Echelon

Biosciences, Inc.) was immobilized on a biotin chip and talin-H was injected at

20 ml/min. Experiments were performed at 37�C in 10 mM HEPES (pH 7.4).

Binding affinities were calculated by using the BIAcore 3000 evaluation soft-

ware (Biacore AB, Uppsala, Sweden).

Plasmids and Transfections

Plasmid DNA encoding full-length mouse talin was kindly provided by Richard

Hynes, Massachusetts Institute of Technology, and it was cloned into pEGFP-

N1 vector (Clontech Lab, Inc) encoding a red-shifted variant of WT GFP using

EcoRI and AgeI restriction sites. The final DNA had a C-terminal GFP fusion

and was confirmed by sequencing. Full-length talin M319A DNA mutant was

prepared employing the QuickChange Site-Directed Mutagenesis Kit (Strata-

gene) using the cloned GFP-fusion talin DNA as a template. The resulting

mutant DNA was confirmed by sequencing.

Integrin Activation

The effects of full-length talin, full-length talin M319A, and talin-H on integrin

activation were analyzed using aIIbb3-CHO and an activation-specific mAb

(PAC1) as described previously (Ma et al., 2008). Briefly, aIIbb3-CHO cells

were transfected with EGFP-talin, EGFP-talin-H, or EGFP vector alone; 24 hr

after transfection, the cells were harvested and stained with PAC1 followed

by Alexa Fluor 633 goat anti-mouse IgM conjugate. After washing, the cells

were fixed and analyzed on a FACSCalibur instrument (BD Scientific, Franklin

Lakes, NJ). PAC1 binding was analyzed only on a gated subset of cells positive

for EGFP expression. The mean fluorescence intensities of PAC1 bound to the

EGFP-talin or EGFP-talin-H-expressing cells were compared to that of PAC1

bound to the control EGFP expressing cells. Three independent experiments

were performed, and the Student’s t test was used for statistical analyses.

ACCESSION NUMBERS

The Biological Magnetic Resonance Data Bank accession number for the

chemical shift assignments reported in this paper is 15792.

Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc. 131

Molecular Cell

Structural Basis of Talin Autoinhibition

SUPPLEMENTAL DATA

The Supplemental Data include one table and ten figures and can be found

with this article online at http://www.molecule.org/cgi/content/full/31/1/124/

DC1/.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health (NIH) grants awarded

to J.Q. (HL5875) and to J.Q. and E.F.P. (P01HL073311). We thank Xi-An Mao,

Sujay Ithychanda, Jun Yang, Keyang Ding, Jianmin Liu, and Julia Vaynberg for

assistance and useful discussions.

Received: January 25, 2008

Revised: May 15, 2008

Accepted: June 24, 2008

Published: July 10, 2008

REFERENCES

Bax, A., and Grzesiek, S. (1993). Methodological advances in protein NMR.

Acc. Chem. Res. 26, 131–138.

Bompard, G., Martin, M., Roy, C., Vignon, F., and Freiss, G. (2003). Membrane

targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via

binding to phosphatidylinositol 4,5-biphosphate. J. Cell Sci. 116, 2519–2530.

Bonvin, A.M., Boelens, R., and Kaptein, R. (2005). NMR analysis of protein

interactions. Curr. Opin. Chem. Biol. 9, 501–508.

Bouaouina, M., Lad, Y., and Calderwood, D.A. (2007). The N-terminal domains

of talin cooperate with the phosphotyrosine binding-like domain to activate

beta1 and beta3 integrins. J. Biol. Chem. 283, 6118–6125.

Bryson, K., McGuffin, L.J., Marsden, R.L., Ward, J.J., Sodhi, J.S., and Jones,

D.T. (2005). Protein structure prediction servers at University College London.

Nucleic Acids Res. 33, W36–W38.

Burridge, K., and Connell, L. (1983). Talin: a cytoskeletal component concen-

trated in adhesion plaques and other sites of actin-membrane interaction. Cell

Motil. 3, 405–417.

Cai, X., Lietha, D., Ceccarelli, D.F., Karginov, A.V., Rajfur, Z., Jacobson, K.,

Hahn, K.M., Eck, M.J., and Schaller, M.D. (2008). Spatial and temporal regula-

tion of focal adhesion kinase activity in living cells. Mol. Cell. Biol. 28, 201–214.

Calderwood, D.A. (2004). Talin controls integrin activation. Biochem. Soc.

Trans. 32, 434–437.

Calderwood, D.A., Zent, R., Grant, R., Rees, D.J., Hynes, R.O., and Ginsberg,

M.H. (1999). The Talin head domain binds to integrin beta subunit cytoplasmic

tails and regulates integrin activation. J. Biol. Chem. 274, 28071–28074.

Calderwood, D.A., Shattil, S.J., and Ginsberg, M.H. (2000). Integrins and actin

filaments: reciprocal regulation of cell adhesion and signaling. J. Biol. Chem.

275, 22607–22610.

Calderwood, D.A., Yan, B., de Pereda, J.M., Alvarez, B.G., Fujioka, Y., Lid-

dington, R.C., and Ginsberg, M.H. (2002). The phosphotyrosine binding-like

domain of talin activates integrins. J. Biol. Chem. 277, 21749–21758.

Cluzel, C., Saltel, F., Lussi, J., Paulhe, F., Imhof, B.A., and Wehrle-Haller, B.

(2005). The mechanisms and dynamics of {alpha}v{beta}3 integrin clustering

in living cells. J. Cell Biol. 171, 383–392.

Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. (1995).

NMRPipe: a multidimensional spectral processing system based on UNIX

pipes. J. Biomol. NMR 6, 277–293.

de Pereda, J.M., Wegener, K.L., Santelli, E., Bate, N., Ginsberg, M.H., Critch-

ley, D.R., Campbell, I.D., and Liddington, R.C. (2005). Structural basis for

phosphatidylinositol phosphate kinase type Igamma binding to talin at focal

adhesions. J. Biol. Chem. 280, 8381–8386.

Di Paolo, G., Pellegrini, L., Letinic, K., Cestra, G., Zoncu, R., Voronov, S.,

Chang, S., Guo, J., Wenk, M.R., and De Camilli, P. (2002). Recruitment and

regulation of phosphatidylinositol phosphate kinase type 1 gamma by the

FERM domain of talin. Nature 420, 85–89.

132 Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc.

Dreolini, L., and Takei, F. (2007). Activation of LFA-1 by ionomycin is indepen-

dent of calpain-mediated talin cleavage. Biochem. Biophys. Res. Commun.

356, 207–212.

Fillingham, I., Gingras, A.R., Papagrigoriou, E., Patel, B., Emsley, J., Critchley,

D.R., Roberts, G.C., and Barsukov, I.L. (2005). A vinculin binding domain

from the talin rod unfolds to form a complex with the vinculin head. Structure

13, 65–74.

Franco, S.J., Rodgers, M.A., Perrin, B.J., Han, J., Bennin, D.A., Critchley, D.R.,

and Huttenlocher, A. (2004). Calpain-mediated proteolysis of talin regulates

adhesion dynamics. Nat. Cell Biol. 6, 977–983.

Garcıa-Alvarez, B., de Pereda, J.M., Calderwood, D.A., Ulmer, T.S., Critchley,

D., Campbell, I.D., Ginsberg, M.H., and Liddington, R.C. (2003). Structural de-

terminants of integrin recognition by talin. Mol. Cell 11, 49–58.

Garrett, D.S., Powers, R., Gronenborn, A.M., and Clore, G.M. (1991). A com-

mon sense approach to peak picking in two- three- and four-dimensional

spectra using automatic computer analysis of contour diagrams. J. Magn. Re-

son. 95, 214–220.

Gingras, A.R., Bate, N., Goult, B.T., Hazelwood, L., Canestrelli, I., Grossmann,

J.G., Liu, H., Putz, N.S., Roberts, G.C., Volkmann, N., et al. (2008). The struc-

ture of the C-terminal actin-binding domain of talin. EMBO J. 27, 458–469.

Goldmann, W.H., Bremer, A., Haner, M., Aebi, U., and Isenberg, G. (1994).

Native talin is a dumbbell-shaped homodimer when it interacts with actin.

J. Struct. Biol. 112, 3–10.

Hamada, K., Shimizu, T., Matsui, T., Tsukita, S., and Hakoshima, T. (2000).

Structural basis of the membrane-targeting and unmasking mechanisms of

the radixin FERM domain. EMBO J. 19, 4449–4462.

Han, J., Lim, C.J., Watanabe, N., Soriani, A., Ratnikov, B., Calderwood, D.A.,

Puzon-McLaughlin, W., Lafuente, E.M., Boussiotis, V.A., Shattil, S.J., and

Ginsberg, M.H. (2006). Reconstructing and deconstructing agonist-induced

activation of integrin alphaIIbbeta3. Curr. Biol. 16, 1796–1806.

Hayashi, M., Suzuki, H., Kawashima, S., Saido, T.C., and Inomata, M. (1999).

The behavior of calpain-generated N- and C-terminal fragments of talin in in-

tegrin-mediated signaling pathways. Arch. Biochem. Biophys. 371, 133–141.

Heraud, J.M., Racaud-Sultan, C., Gironcel, D., Albiges-Rizo, C., Giacomini, T.,

Roques, S., Martel, V., Breton-Douillon, M., Perret, B., and Chap, H. (1998).

Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 40,50-bi-

sphosphate are both required for ADP-dependent platelet spreading. J. Biol.

Chem. 273, 17817–17823.

Hinchliffe, K.A., Irvine, R.F., and Divecha, N. (1996). Aggregation-dependent,

integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels

in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to

the cytoskeleton. EMBO J. 15, 6516–6524.

Hynes, R.O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell

110, 673–687.

Kim, M., Carman, C.V., and Springer, T.A. (2003). Bidirectional transmembrane

signaling by cytoplasmic domain separation in integrins. Science 301, 1720–

1725.

Kong, X., Wang, X., Misra, S., and Qin, J. (2006). Structural basis for the phos-

phorylation-regulated focal adhesion targeting of type Ig phosphatidylinositol

phosphate kinase (PIPKIg) by talin. J. Mol. Biol. 359, 47–54.

Li, Q., Nance, M.R., Kulikauskas, R., Nyberg, K., Fehon, R., Karplus, P.A.,

Bretscher, A., and Tesmer, J.J. (2007). Self-masking in an intact ERM-merlin

protein: an active role for the central a-helical domain. J. Mol. Biol. 365,

1446–1459.

Lietha, D., Cai, X., Ceccarelli, D.F., Li, Y., Schaller, M.D., and Eck, M.J. (2007).

Structural basis for the autoinhibition of focal adhesion kinase. Cell 129, 1177–

1187.

Ling, K., Doughman, R.L., Firestone, A.J., Bunce, M.W., and Anderson, R.A.

(2002). Type I gamma phosphatidylinositol phosphate kinase targets and reg-

ulates focal adhesions. Nature 420, 89–93.

Ma, Y.Q., Yang, J., Pesho, M.M., Vinogradova, O., Qin, J., and Plow, E.F.

(2006). Regulation of integrin alpha(IIb)beta(3) activation by distinct regions

of its cytoplasmic tails. Biochemistry 45, 6656–6662.

Molecular Cell

Structural Basis of Talin Autoinhibition

Ma, Y.Q., Qin, J., Wu, C., and Plow, E.F. (2008). Kindlin-2 (Mig-2): a co-activa-

tor of beta3 integrins. J. Cell Biol. 181, 439–446.

Martel, V., Racaud-Sultan, C., Dupe, S., Marie, C., Paulhe, F., Galmiche, A.,

Block, M.R., and Albiges-Rizo, C. (2001). Conformation, localization, and in-

tegrin binding of talin depend on its interaction with phosphoinositides. J.

Biol. Chem. 276, 21217–21227.

McLachlan, A.D., Stewart, M., Hynes, R.O., and Rees, D.J. (1994). Analysis of

repeated motifs in the talin rod. J. Mol. Biol. 235, 1278–1290.

Molony, L., McCaslin, D., Abernethy, J., Paschal, B., and Burridge, K. (1987).

Properties of talin from chicken gizzard smooth. J. Biol. Chem. 262, 7790–

7795.

Nieswandt, B., Moser, M., Pleines, I., Varga-Szabo, D., Monkley, S., Critchley,

D., and Fassler, R. (2007). Loss of talin1 in platelets abrogates integrin activa-

tion, platelet aggregation, and thrombus formation in vitro and in vivo. J. Exp.

Med. 204, 3113–3118.

Papagrigoriou, E., Gingras, A.R., Barsukov, I.L., Bate, N., Fillingham, I.J., Patel,

B., Frank, R., Ziegler, W.H., Roberts, G.C., Critchley, D.R., and Emsley, J.

(2004). Activation of a vinculin-binding site in the talin rod involves rearrange-

ment of a five-helix bundle. EMBO J. 23, 2942–2951.

Pearson, M.A., Reczek, D., Bretscher, A., and Karplus, P.A. (2000). Structure

of the ERM protein moesin reveals the FERM domain fold masked by an ex-

tended actin binding tail domain. Cell 101, 259–270.

Pervushin, K., Riek, R., Wider, G., and Wuthrich, K. (1997). Attenuated T2 re-

laxation by mutual cancellation of dipole-dipole coupling and chemical shift

anisotropy indicates an avenue to NMR structures of very large biological mac-

romolecules in solution. Proc. Natl. Acad. Sci. USA 94, 12366–12371.

Petrich, B.G., Marchese, P., Ruggeri, Z.M., Spiess, S., Weichert, R.A., Ye, F.,

Tiedt, R., Skoda, R.C., Monkley, S.J., Critchley, D.R., and Ginsberg, M.H.

(2007). Talin is required for integrin-mediated platelet function in hemostasis

and thrombosis. J. Exp. Med. 204, 3103–3111.

Rees, D.J., Ades, S.E., Singer, S.J., and Hynes, R.O. (1990). Sequence and do-

main structure of talin. Nature 347, 685–689.

Schoenwaelder, S.M., Yuan, Y., Cooray, P., Salem, H.H., and Jackson, S.P.

(1997). Calpain cleavage of focal adhesion proteins regulates the cytoskeletal

attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the

cellular retraction of fibrin clots. J. Biol. Chem. 272, 1694–1702.

Tadokoro, S., Shattil, S.J., Eto, K., Tai, V., Liddington, R.C., de Pereda, J.M.,

Ginsberg, M.H., and Calderwood, D.A. (2003). Talin binding to integrin beta

tails: a final common step in integrin activation. Science 302, 103–106.

Takeuchi, K., and Wagner, G. (2006). NMR studies of protein interactions. Curr.

Opin. Struct. Biol. 16, 109–117.

Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S.,

Takeda, K., and Kieffer, N. (2004). A fluorescence cell biology approach to

map the second integrin-binding site of talin to a 130-amino acid sequence

within the rod domain. J. Biol. Chem. 279, 22258–22266.

Turner, C.E., and Burridge, K. (1991). Transmembrane molecular assemblies

in cell-extracellular matrix interactions. Curr. Opin. Cell Biol. 3, 849–853.

Ulmer, T.S., Calderwood, D.A., Ginsberg, M.H., and Campbell, I.D. (2003). Do-

main-specific interactions of talin with the membrane-proximal region of the

integrin beta3 subunit. Biochemistry 42, 8307–8312.

Vaynberg, J., and Qin, J. (2006). Weak protein-protein interactions as probed

by NMR spectroscopy. Trends Biotechnol. 24, 22–27.

Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and Qin,

J. (2002). A structural mechanism of integrin alpha(IIb)beta(3) ‘‘inside-out’’ ac-

tivation as regulated by its cytoplasmic face. Cell 110, 587–597.

Vinogradova, O., Vaynberg, J., Kong, X., Haas, T.A., Plow, E.F., and Qin, J.

(2004). Membrane-mediated structural transitions at the cytoplasmic face dur-

ing integrin activation. Proc. Natl. Acad. Sci. USA 101, 4094–4099.

Wegener, K.L., Partridge, A.W., Han, J., Pickford, A.R., Liddington, R.C., Gins-

berg, M.H., and Campbell, I.D. (2007). Structural basis of integrin activation by

talin. Cell 128, 171–182.

Xing, B., Jedsadayanmata, A., and Lam, S.C. (2001). Localization of an integrin

binding site to the C terminus of talin. J. Biol. Chem. 276, 44373–44378.

Yan, B., Calderwood, D.A., Yaspan, B., and Ginsberg, M.H. (2001). Calpain

cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain.

J. Biol. Chem. 276, 28164–28170.

Molecular Cell 31, 124–133, July 11, 2008 ª2008 Elsevier Inc. 133