The molecular basis of talin2’s high affinity toward β1-integrin

Talin interacts with β-integrin tails and actin to control integrin activation, thus regulating focal adhesion dynamics and cell migration. There are two talin genes, Tln1 and Tln2, which encode talin1 and talin2, and it is generally believed that talin2 functions redundantly with talin1. However, we show here that talin2 has a higher affinity to β1-integrin tails than talin1. Mutation of talin2 S339 to leucine, which can cause Fifth Finger Camptodactyly, a human genetic disease, completely disrupted its binding to β–integrin tails. Also, substitution of talin1 C336 with Ser enhanced the affinity of talin1, whereas substitution of talin2 S339 with Cys diminished that of talin2. Further computational modeling analysis shows that talin2 S339 formed a hydrogen bond with E353, which is critical for inducing key hydrogen bonds between talin2 N326 and β1-integrin R760, and between talin2 K327 and β1-integrin D759. Mutation at any of these residues significantly diminished the interaction of talin2 with β1- integrin tails. These hydrogen bonds were not observed in talin1/β1-integrin, but did exist in talin1C336S/β1-integrin complex. These results suggest that talin2 S339 forms a hydrogen bond with E353 to mediate its high affinity to β1-integrin.

Integrins are a family of transmembrane adhesion receptors that mediate cell-matrix and cell-cell adhesion 1 . Integrins are heterodimers, comprising α (alpha) and β (beta) subunits. Talin, a large focal adhesion protein, binds to the beta subunit, consequently activating integrin, which in turn regulates cell migration 2,3 , invasion 4,5 , growth 6,7 , differentiation 6,8 , and apoptosis 9 . As a result, integrin activation modulates a variety of physiological and pathological processes, such as development, immunity, inflammation, and tumor metastasis. Thus, the talin-integrin interaction is one of the most important protein-protein interactions.
Talin contains an amino-terminal globular head domain and a carboxy-terminal rod domain 10 . The talin head domain contains a FERM (band four-point-one, ezrin, radixin, moesin homology) domain, which comprises three subdomains, F1, F2 and F3. The F2 domain is entirely α -helical with a short linked region, and the F3 domain is a sandwich of two orthogonal antiparallel β -sheets followed by an α -helix 11 . The FERM domain is responsible for the binding of talin to β -integrin tails 12 , type I phosphatidylinositol 4-phosphate 5-kinase γ (PIPKIγ ) 13,14 , and focal adhesion kinase (FAK) 15 . The rod domain has several vinculin-binding sites, and two actin-binding sites 16 .
The binding of talin to β integrin tails is essential for integrin activation 12,17 , which in turn regulates focal adhesion (FA) dynamics, a key step in cell migration 18,19 . Talin also mediates calpain-induced FA disassembly 19 . We and our collaborators have shown that talin phosphorylation by Cdk5 regulates FA dynamics, cell migration and invasion 20,21 . Talin interacts with PIPKIγ , which produces PIP 2 to regulate FA dynamics, cell migration and invasion 13,14,22-28 . There are two talin genes, Tln1 and Tln2, encoding talin1 and talin2, respectively. Talin1 has been well studied, while the biological function of talin2 is less clear. It was presumed that talin2 functions redundantly with talin1. However, recent evidence indicates that talin2 is functionally different from talin1. Previous study shows that talin2 regulates focal adhesion assembly and focal adhesion kinase (FAK) signaling in the absence of talin1 29 . Talin2 is usually localized at large FAs and fibrillar adhesions, whereas talin1 is usually found at smaller FAs in the peripheral region 30,31 . Trastuzumab, a HER2-targeting antibody drug for cancer therapy, inhibits cell migration and invasion, most likely through down-regulating talin2 32 . Recently, we reported that talin2 binds to β integrin tails more strongly than talin1, and a strong interaction of talin2 with β integrins is required for the generation of traction force, which in turn drives invadopodium formation and cell invasion 33 . Nevertheless, the underlying molecular basis for the difference between talin1 and talin2 remains to be elucidated.
In the present study, we demonstrated that talin2 had a higher affinity to β 1-integrin tails than talin1, talin2 Ser339 is largely responsible for this affinity difference, and mutation at Ser339 reduced its binding to β 1-integrin tails. Coincidently, a new study shows that Fifth Finger Camptodactyly, a human genetic disease, is caused by a Leucine mutation at talin2 Ser339 34 . We defined the molecular basis of talin2 binding to β 1-integrin using computational modeling methods, performed experiments to verify the computational model, and examined the role of talin2 S339 in focal adhesion assembly.

Results
Talin2 has a higher affinity to β1-integrin tails than talin1. Recently, we demonstrated that talin2 had a stronger binding to β -integrin tails than talin1 33 . There are several residues that are different between the integrin-binding sequences of talin1 and talin2 (Fig. 1A), but Cys336 of talin1 and the corresponding Ser339 on talin2 are largely responsible for their binding difference. Substitution of Cys336 with Ser enhanced talin1's binding to β 1A-integrin tails, whereas substitution of Ser339 with Cys or Leu significantly attenuated talin2 binding, suggesting the critical role of Ser339 in mediating talin2 binding to β -integrins.
To study the interaction of talin and mutants with β 1-integrin tails, we purified His tagged talin1 1-446 WT , talin1 1-446 C336S , talin2 1-449 WT , and talin2 1-449 S339C overexpressed in E. coli. To examine the quality of the proteins, purified proteins were analyzed by gel filtration chromatography combined with static light scattering detector. As shown in Supplementary Fig. S1 and Table S1, all proteins were mainly monomeric in solution (judged by static light scattering), with molecular weights approximately 59 kDa, which is consistent with theoretical molecular weights (~53 kDa). Also, the eluted protein peak was rather symmetric in the case of all the protein forms, suggesting that these proteins are soluble and folded.
To determine the affinity of talin1 to β 1-integrin tails, different concentrations of His-tagged-talin1 1-446 were incubated with glutathione agarose beads that were preloaded with GST-β 1A-integrin tails. Unbound protein was washed away and bound protein was separated by SDS-PAGE, stained with Coomassie blue, and quantitated with standards run on the same gel. The β 1A-integrin tails bound to talin1 1-446 moderately, but GST did not (Fig. 1B). The dissociation constant (K d ) between talin1 1-446 and β 1A-integrin tails is approx. 0.88 ± 0.07 μ M (n = 5) (Fig. 1C,D). We employed the same method to determine the K d between talin2 1-449 and β 1 A integrin tails. The K d between talin2 1-449 and β 1A-integrin tails is approx. 0.35 ± 0.09 μ M (n = 5) (Fig. 1B-1D). Substitution of talin1 C336 with Ser caused a slight increase in its affinity, whereas substitution of talin2 S339 with Cys diminished its affinity (Fig. 1C,D). Surprisingly, substitution of talin2 S339 with Leu completely abolished its interaction with β 1 A integrin tails (Fig. 1E). These results indicate that S339 is critical for talin2's high affinity for β integrins.
Talin2 forms closer contacts and more hydrogen bonds with β1 integrin tails than talin1. First, we compared the energy minimization and molecular dynamics (MD) in talin2 WT /integrin, talin1 WT /integrin, and talin1 C336S /integrin complexes. Depicted in Fig. 2 are the structural dynamics tracked as positional root-mean square deviation (RMSD) of Cα atoms along the 40 ns MD simulations in the three talin/integrin complex structures. After a time period of ~10 ns, all the RMSD curves become flat, indicating the complex structure in the three MD systems are relaxed and equilibrated.
The sequences of mouse talin2 (mTalin2), human talin2 (hTalin2), human talin1 (hTalin1) and human β 1D-integrin tail are shown in Fig. 3A. To understand the structural basis of talin1 and talin2 binding to β 1-integrin tail, we performed molecular modeling and MD simulations to explore the structural differences in talin1 and talin2. Briefly, the talin1/integrin tail complex structure is constructed based on talin2/integrin complex structure 11 with an homology modeling method implemented by the MODELLER module of Discovery Studio 2.5 as described previously 35,36 . As shown in Fig. 3B1, the β 1-integrin tail mostly contacts with these two β -sheets of F3 domain. Based on the energy minimized complex structures from 40 ns MD simulation, we found that the β 1-integrin tail binds to talin1 WT with a very different orientation comparing with its binding to talin2 WT (Fig. 3B2), while the single mutation C336S of talin1 switches the β 1-integrin in Talin1 WT /integrin complex to a similar orientation seen in talin2 WT /integrin complex (Fig. 3B3,B4). Furthermore, the average structures of the talin2 WT /integrin, talin1 WT /integrin, and talin1 C336S /integrin complexes of 40 ns simulation also confirm the systematic difference between talin2 WT /integrin and talin1 WT /integrin and the systematic similarity between tal-in2 WT /integrin and talin1 C336S /integrin ( Supplementary Fig. S2). Interestingly, the β -sheet 1, especially the loop region between β strand 1 (β 1) and β strand 2 (β 2) in β -sheet 1 adopts significantly different conformations in talin2 WT /integrin and talin1 WT /integrin complex (Fig. 3B5), while the C336S mutant of talin1 eliminates the difference (Fig. 3B6,3B7). This is consistent with experimental results that talin2 has a higher affinity to β 1-integrin tails than talin1, and that the substitution of talin1 C336 with Ser enhances the affinity of talin1 (Fig. 1).

Role of S339 in talin/integrin interaction.
As the β 1-β 2 loop of talin contacts directly with β 1-integrin, conformation of β -sheet 1 should be one of the decisive factors for the orientation of β 1-integrin binding with talin. However, β -sheet 1 of talin1 and talin2 are fully conserved except in two residues, D338 and S339 in tal-in2 WT corresponding to E335 and C336 in talin1 WT , respectively (Fig. 3A). As the side chains of aspartic acid and glutamic acid have similar chemical property, S339 of talin2 WT and C336 of talin1 WT could be considered as the major difference in β -sheet 1 of the two isoforms. As a result, we expect that the C336S mutation of talin1 WT would adapt the conformation of β -sheet 1 similar to that in talin2 WT , thus talin1 C336S and talin2 WT would bind to β 1-integrin with similar binding orientation. Fig. 4A depicts the tracked distances and typical snapshots of the hydrogen bond between S336/339 and E350/353 in talin/integrin complex from the MD trajectories (residue number with slash indicates residue in talin1/talin2, respectively). The average distance of this hydrogen bond (in the last 10 ns of simulation) is 1.75 ± 0.18 Å (frequency of hydrogen bond occurrence: 99.3%, with 2.5 Å H-O distance threshold) and 1.78 ± 0.24 Å (frequency: 98.2%) in talin1 C336S /integrin and talin2 WT /integrin complex respectively, while a much larger and more fluctuating average distance of 4.39 ± 1.92 Å (frequency: 27.0%)   between terminus hydrogen of C336 and oxygen of carboxyl group of E350 is observed in talin1 WT /integrin complex. Thus, the hydrogen bond between S339 and E353 in talin2 WT does not exist in talin1 WT due to the weak hydrogen bond capacity of corresponding residue C336 in talin1 WT comparing with S339 in talin2 WT (Fig. 4D,E). Therefore, C336S mutation of Talin1 is expected to restore this hydrogen bond (Fig. 4F). In addition, as residues S336/339 and E350/353 could form hydrogen bond between the β 3 and β 4 strand of β -sheet 1, loss of this strong interaction in talin1 WT /integrin complex would influence the steric alignment of β strands in β -sheet 1, which is also observed in the typical snapshots from MD simulation (Fig. 3B5-7).
The hydrogen bonds between talin and β1-integrin contribute to talin's affinity to β1-integrins. Since C336 in talin1 WT alters the conformation of β -sheet 1, the orientation of β 1-integrin tail binding to talin1 WT is significantly shifted from its binding status in talin2 WT /integrin complex. Therefore, the interaction pairs involved in the binding interface of talin1 WT /integrin and talin2 WT /integrin complex are much different. Besides, vibration of β 1-integrin tail in these complex structures is also observed during 40 ns simulations, which implies that the static comparison of interaction pairs would be biased. We calculated the statistical hydrogen bond profile of the binding interface to reveal the difference among the three binding complexes. Figure 5 depicts is the heat map that shows the change in the hydrogen bond profile of talin1 WT /integrin and talin1 C336S /integrin compared with talin2 WT /integrin. More hydrogen bonds are formed by Talin2 WT / integrin complex than talin1 WT /integrin complex, supporting the statement that β 1-integrin binds to talin2 WT with higher affinity than binding to talin1 WT (Fig. 5A). Moreover, substitution of talin1 C336 with Ser promotes hydrogen bond formation between talin1 and β 1-integrin (Fig. 5B). This is consistent with the experiment result that substitution of talin1 C336 with Ser enhances the affinity of talin1 to β 1-integrin tails. Based on the MD simulation, two hydrogen bonds between β 1-β 2 loop of talin and β 1-integrin tails, i.e., hydrogen bonds between Tln-K324/327 and Int-D759, and between Tln-N323/326 and Int-R760 (Tln refers to talin, Int for β 1-integrin tails), are observed in typical snapshots of talin1 C336S /integrin and talin2 WT /integrin complexes (Fig. 4G,I), but are missing in talin1 WT /integrin complex (Fig. 4H),. According to the tracked distance of this hydrogen bond depicted in Fig. 4B, the average distance (in the last 10 ns of simulation) of hydrogen bond between Tln-K324/327 and Int-D759 is 2.02 ± 0.52 Å (frequency of hydrogen bond occurrence: 95.1%, with 2.5 Å H-O distance threshold) and 2.00 ± 0.53 Å (frequency: 90.6%) in talin1 C336S /integrin and talin2 WT /integrin complex, respectively, while this distance increases to 5.07 ± 1.07 Å (frequency: 0.1%) in talin1 WT /integrin complex. Similarly, the average distance (in the last 10 ns of simulation) of the hydrogen bond between Tln-N323/326 and Int-R760 is 2.39 ± 0.46 Å (frequency: 76.2%) and 2.51 ± 0.79 Å (frequency: 63.2%) in talin1 C336S /integrin and talin2 WT /integrin complex, respectively, while this distance increases to 7.40 ± 1.47 Å (frequency: 0.0%) in talin1 WT /integrin complex (Fig. 4C). Although there are many other differences among the three complexes, status of these two hydrogen bonds could be considered as key indicators of talin-integrin binding state. It should be noted that both Tln-K324/327 and Tln-N323/326 are located in the β 1-β 2 loop (Fig. 4G), so the conformation of the β 1-β 2 loop is expected to influence the interaction between talin and intergrin. In conclusion, residue C336 of talin1 WT disturbs the conformation of β -sheet 1 as well as β 1-β 2 loop, thus eliminating both hydrogen bonds between Tln-K324 and Int-D759, and Tln-N323 and Int-R760, which results in an off-switch in the orientation of β 1-integrin binding and a decrease in the affinity of talin1 WT to β 1-integrin tails. On the contrary, talin1 C336S adapts a conformation of the β 1-β 2 loop seen in talin2/integrin complex, thus restoring the hydrogen bonds between Tln-K324 and Int-D759, and Tln-N323 and Int-R760, which is expected to enhance the affinity of talin1 C336S to β 1-integrin tails.
The critical role of the hydrogen bonds between Tln-S339 and Tln-E353, Tln-K327 and Int-D759, and Tln-N326 and Int-R760 for talin2's high affinity to β1-integrin tails. Next, we set out to verify our computational model. The phosphotyrosine binding (PTB)-like domain is responsible for talin1 binding to β 1-integrin tails. To prove whether this is true for talin2, R361, W362, and S365 within the talin2 PTB-like domain were substituted with Alanine, and the talin2 mutants and the WT were transiently transfected into CHO-K1 cells. The binding of these proteins to β 1-integrin tails was determined using GST pulldown assays. As shown in Fig. 6A, mutation at any of these residues caused dramatic reduction in talin2 binding to β 1-integrin tails, indicating that talin2 and talin1 employ the same motif to bind β 1-integrin tails.
To confirm the critical role of S339 in modulating talin2 binding to β 1-integrin tails, S339 and the adjacent residues D338 and V340 were mutated to Ala. These mutants and the WT were transfected to CHO-K1 cells and their binding to β 1 A tails was examined. Substitution of either S339 or V340 with Ala significantly diminished talin2 binding to β 1-integrin tails; Substitution of D338 with Ala also slightly decreased talin2 binding (Fig. 6B).
Based on our computational model, talin2 S339 and E353 form a hydrogen bond between the β 3 and β 4 strand of β -sheet 1, which promotes talin2 binding to β 1-integrin tails. To verify the role of E353 in mediating the talin2 β 1-integrin interaction, talin2 E353 was substituted with Gly and Lys, respectively, and the interaction of these mutants with β 1-integrin tails was determined. Substitution of talin2 E353 with either Gly or Lys significantly diminished the binding of talin2 to β 1-integrin tails (Fig. 6C), suggesting that the hydrogen bond between S339 and E353 is required for the high affinity of talin2 to β 1-integrin tails.
To know whether the hydrogen bond between talin2 K327 and β 1-integrin D759 is critical for the talin2-integrin interaction, talin2 K327 was mutated to Glu and Ala, respectively, and the binding of these mutants to β 1-integrin tails was examined. Substitution of K327 with Glu abolished the interaction of talin2 with β 1-integrin tails (Fig. 6C), and substitution of K327 with Ala also reduced the binding of talin2 (Fig. 6D). In The difference of average inter-molecular hydrogen bond number between talin2 WT / integrin and talin1 WT /integrin. As the difference is dependent on the threshold of hydrogen bond definiation, the profile of various hydrogen bond distances and angle cutoffs is shown. Here the hydrogen bond distance is defined as the distance between hydrogen bond donor and acceptor, while the hydrogen bond angle is defined as the angle between donor, hydrogen, and acceptor. For a typical hydrogen bond distance cutoff of 3.0 Å and angle cutoff of 120°, talin2 WT /integrin complex has about 1 more inter-molecular hydrogen bond than talin1 WT / integrin complex. (B) The difference of the average inter-molecular hydrogen bond number between talin2 WT / integrin and talin1 C336S /integrin. It could be observed that these two complexes form an inter-molecular hydrogen bond in a similar level.
Scientific RepoRts | 7:41989 | DOI: 10.1038/srep41989 addition, substitution of N326 with Ala significantly diminished the interaction of talin2 with β 1-integrin tails (Fig. 6D), which also supports the predicted hydrogen bond between N326 of talin2 and R760 of β 1-integrin. Taken together, the hydrogen bonds between talin2 S339 and E353 and between talin2 K327 and β 1-integrin D759 are critical for talin2 to maintain its high affinity to β 1-integrins. Talin2 is a focal adhesion protein. To know whether talin2 S339 is critical for focal adhesion formation, talin2-null human osteosarcoma cells U2 OS were transfected with EGFP-talin2 WT and -talin2 S339C , respectively. Cells were plated on glass-bottom dishes that were pre-coated with fibronectin (5 μ g/ml), fixed, and stained with an anti-phospho-FAK antibody. The images of EGFP and phospho-FAK were recorded with a TIRF microscope. EGFP-talin2 WT was co-localized with phospho-FAK, whereas EGFP-talin2 S339C was deficient in focal adhesion formation and had a diminished co-localization with phospho-FAK (Fig. 7A,B), suggesting that a strong interaction of talin2 with β 1-integrins is essential for efficient focal adhesion assembly.

Discussion
We found that talin1 and talin2 interacted with β 1A-integrin tails with K d of 0.88 and 0.35 μ M, respectively. Substitution of S339 with Leucine completely disrupted talin2 binding to β 1-integrins, and mutation of S339 to Cys also diminished its binding, indicating that S339 is critical for talin2 binding to β 1-integrins. Our findings suggest that the incapacity of S339L mutant binding to β -integrins could be the pathological cause of fifth finger camptodactyly.
Substitution of talin1 C336 with Ser caused only a small increase in talin1's affinity (Fig. 1), but C336S mutant bound to β integrin tails more strongly than talin1 WT when talin1 and C336S were expressed in mammalian cells 33 . Similar result was observed as comparing the binding of talin2 WT and talin2 S339C . These results suggest that talin and mutants from mammalian cells may have post-translational modifications that influence their binding to β -integrin tails. However, the possibility that proteins from cell lysates have superior folding compared to those from bacteria cannot be ruled out.
Previous study reported that the F3 domains of talin1 and talin2 bound to β 1 A tails with K d of 491 and 652 μ M, respectively, as measured by NMR 11 . Our findings are different from the results in this report. As we discussed previously, the difference could be caused by using different fragments of the talin head domains and different binding buffers and blocking reagents in the assays 33 . The folding of talin head domains purified from bacteria could also cause this difference. Although our gel filtration chromatography analysis indicates that these proteins are folded ( Supplementary Fig. S1), this method does not prove that the proteins are folded and post-translationally modified exactly as in their natural host. Our unpublished data show that talin head domains induced by IPTG at 37 °C had higher β 1 A integrin binding capacities than those induced at 19 °C, probably because the expression of chaperones that control protein folding are inhibited at a low temperature 37,38 . We used talin head domains that were induced by IPTG at 37 °C in our affinity assays. We do not have evidence that the proteins studied here would have been folded better than in other studies. However, our affinity assays with purified recombinant proteins from E. coli are consistent with the pulldown assay results conducted using mammalian cell lysates. Talin head domain purified from CHO cells will be useful to further clarify this difference.
Our computational modeling results show that talin2 S339 forms hydrogen bonds with E353, which is critical for key hydrogen bonds between talin2 K327 and β 1-integrin D759, and between talin2 N326 and β 1-integrin R760 (Fig. 4D,G). These hydrogen bonds were not observed in the talin1/integrin complex (Fig. 4E,H). Mutation at any of these residues significantly diminished the binding of talin2 with β 1-integrin tails (Fig. 6). However, substitution of talin1 C336 with Ser promoted the formation of these hydrogen bonds (Fig. 4F,I), accompanying an increase in its affinity toward β 1-integrin tails. Interestingly, mutation of talin2 S339 to Leu caused a significant reduction in its binding to β 1-integrin tails (unpublished data) and Fifth Finger Camptodactyly, suggesting that the reduction in the talin2-integrin interaction could be the cause for the genetic disease. These results suggest that the hydrogen bonds between talin2 S339 and E353 play a critical role in maintaining talin2's high affinity to β 1-integrin. Because these residues are located outside of the well-characterized β 1-integrin-binding motif, it is possible to design a small molecule to diminish talin2 binding to β 1-integrins without affecting talin1. Besides mediating β -integrin interaction, talin2 K327 may also be involved in other functions of talin2. Previous studies show talin1 K324, which is aligned with K327 on talin2, to be a key residue for talin1 binding to PIP2 and is essential for integrin clustering 39 . This residue also mediates the head-and-rod auto-inhibition of talin1 [40][41][42] . Studies have also shown that PIP2 binds to talin1 and disrupts the head-and-rod interaction, thus causing talin1 activation 40,43 . It is likely that talin2 K327 is a key residue for the talin2-PIP2 interaction and is also involved in the head-and-rod auto-inhibition of talin2. However, it is unknown whether binding to PIP2 disrupts or enhances talin2-β 1-integrin interaction.
Talin2 N236 is another key residue that mediates talin2-β 1-integrin interaction. In fact, substitution of talin2 N326 with Ala had more significant effect on the binding of talin2 to β 1-integrin tails than that of K327 with Ala (Fig. 6D). Although substitution of talin2 K327 with Glu disrupted the interaction of talin2 with β 1-integrin tails (Fig. 6C), this could be caused by the perturbation of the negative charge of the Lys residue on talin2 conformation. Since talin2 K327 may involve in PIP2 interaction, the hydrogen bond between Tln-N326 and Int-R760 could play a key role in the talin2-β 1 integrin interaction, especially in the presence of PIP2.

Materials and Methods
Reagents. Anti-FAK[pY397] was from BD Biosciences. Anti-tubulin antibody was from Sigma. DyLight 549 conjugated goat anti-mouse IgG (H + L) was from Thermo Scientific. Fibronectin and recombinant human EGF were from Akron Biotech; Growth factor reduced Matrigel was from BD Bioscience. Pfu Ultra was from Agilent Technologies. Cold Fusion Cloning Kit was from System Biosciences (Palo Alto, CA). Anti-GFP monoclonal antibody and Safectine RU50 transfection kit were purchased from Syd Labs (Malden, MA). DNA primers were synthesized by Sigma-Aldria.

Protein interaction assays.
The binding of purified His-tagged proteins to GST-β 1-integrin tails was performed in lysis Buffer A containing 0.5 mg/ml bovine gelatin unless where specified. Bound proteins were separated using SDS-PAGE, stained with Coomassie blue. The gels were scanned with LI-COR Infrared Imager using 700 nm channel. Protein bands was quantified by analyzing inverted images using ImageJ as described previously 20,44 , and calibrated with standards run on the same gels. The binding curves and the K d were analyzed with SigmaPlot.

Molecular Dynamic Simulations.
For the three talin/integrin complex systems, i.e., talin2 WT /integrin, talin1 WT /integrin, and talin1 C336S /integrin, Amber 12 package (Case, D.A. et al., University of California, San Francisco, 2012) is used to perform energy minimization and MD simulations in TIP3P solvent environment in a way similar to that used in our previous studies 35,36,45 . Talin2/integrin complex structural model. X-ray crystal structure of Mus musculus talin2 and Homo sapiens β 1D-integrin complex (PDB entry as 3G9W with resolution of 2.16 Å) reveals the interaction between F2-F3 domains (residue #200-406) of talin2 and tail region (residue #750-789) of β 1D-integrin 11 . Sequence identity between Mus musculus talin2 (accession number as Q71LX4 in protein sequence database of UniProt 46 ) and Homo sapiens talin2 (accession number as Q9Y4G6) is 92.0%. However, sequence of F2-F3 domains of Mus musculus talin2 is fully conserved with corresponding domains of Homo sapiens talin2. Thus, the reported Mus musculus talin2 and Homo sapiens β 1D-integrin complex structure could be considered equivalent to Homo sapiens complex structure. For convenience, the talin/integrin complex model in this manuscript refers to the truncated complex structure, i.e., F2-F3 domains of talin complex with tail region of β 1D-integrin, unless otherwise noted.
Building the talin1/integrin complex structural model. Talin1/integrin model is constructed based on talin2/integrin structure with homology modelling method implemented by MODELLER module of Discovery Studio 2.5 (Accelrys Inc., San Diego, CA, 2009). According to the sequence alignment performed by using PROMALS3D server (http://prodata.swmed.edu/promals3d/) 47 , sequence identity between wild type Homo sapiens talin2 (talin2 WT ) and Homo sapiens talin1 (talin1 WT , accession number as Q9Y490) is 76.0%. Furthermore, the aligned sequences (Fig. 3A) indicate that the F2-F3 domains of talin2 are homologous to the corresponding F2-F3 domains of talin1 with a much higher sequence identity of 86.7%. Based on the information from the sequence alignment, the initial coordinates of atoms in the conserved regions of talin1 and whole tail region of β 1-integrin are directly transformed from the template structure of talin2/integrin complex, whereas the initial coordinates for the non-conserved residues of talin1 are mutated from these of the corresponding residues in the template. The model with best DOPE scores 48 is selected as the initial talin1/integrin complex structure (see below). Moreover, the in silico C336S mutant of talin1 (talin1 C336S ) and wild type β 1-integrin complex is obtained by using the mutagenesis function of PYMOL (http://www.pymol.org/) 49 based on the constructed talin1 WT / integrin complex structure.
Cell culture and transfection. CHO-K1 Chinese hamster ovary cells and U2 OS human bone osteosarcoma cells were from the American Type Culture Collection and were maintained in DMEM medium (Corning Inc.) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μ g/ml). CHO-K1 and U2 OS cells were transfected with Safectine RU50 according to the manufacturer's protocol.
FA staining. Endogenous talin2 in U2 OS cells was ablated using CRISPR techniques and EGFP-talin2 WT and -talin2 S339C were then stably re-expressed in talin2-null cells, respectively, as described previously 33 . The resulted cells were plated on glass-bottom dishes that were pre-coated with fibronectin (5 μ g/ml) and cultured for 4 h. The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized for 15 min with 0.5% Triton X-100, and then blocked with 5% BSA in PBS for 1 h. The cells were then incubated with anti-phospho-FAK[pY397] antibody, washed with PBS, and then incubated with Dylight550-labeled goat anti-mouse secondary antibody. After washing with PBS, the images of EGFP and phospho-FAK[pY397] were acquired with a Nikon Eclipse Ti TIRF microscope equipped with a 60 × , 1.45 NA objective, CoolSNAP HQ2 CCD camera (Roper Scientific). Focal adhesion area distribution was analyzed with Nis-Elements.