How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism
[['Talin physically links integrins to the contractile cytoskeleton [1 , 2] .', 'The talin head ( TH ) , residues 1–432 , has binding sites for integrin β-tails [3] , PIP kinase γ [4] , focal adhesion kinase ( FAK ) [5] , layilin [5] and actin [6] ( Figure 1B ) .', 'Binding of the talin head to the cytoplasmic tail of β-integrins can cause integrin activation [7] .', 'The 60 nm long talin rod ( TR ) , residues 433-2541 , is composed of bundles of amphiphatic α-helices [8 , 9] ( Figure 1 ) .', 'The talin rod contains up to eleven vinculin binding sites [10] ( Figures 1B and S1 ) , including five located within fragment H1–H12 , residues 486–889 , which is studied here ( Figure 1 ) .', 'All of these five binding sites are buried inside helix bundles ( native talin shows considerably lower affinity to vinculin compared to peptide fragments isolated from talin ) .', 'In addition to the VBSs , the talin rod has binding sites for actin [11] and for integrins [12] .', 'Upon cell adhesion , talin rapidly accumulates in focal contacts prior to vinculin recruitment [13] .', 'Only talin , but not vinculin or paxillin , is recruited to the clustered integrins , if β3 integrins are activated not by ligand binding but by manganese ( Mn2+ ) , i . e . , in cases where integrin activation occurs without the application of force and integrin is thus not part of a force-bearing protein network [14] .', 'Indeed , the recruitment of vinculin to cell adhesion sites has been shown to be force-dependent [15–19] .', 'Vinculin recruitment strengthens cell adhesions [20] , and reduces focal adhesion turnover [21] .', 'In focal adhesions , vinculin is in an activated state while it is autoinhibited in the surrounding cytoplasm [22] .', 'Important to the model proposed here is that vinculin activation can also be induced in solution by isolated talin peptides containing VBSs [23] .', 'As talin , vinculin is an alpha-helical protein sharing considerable structural similarity .', 'The x-ray structure of full-length vinculin [PDB: 1TR2] reveals three head domains , VH , VH2 , and VH3 , that are connected to the tail domains , VT2 and VT , via a flexible , proline-rich linker .', "Vinculin's talin binding site is located in the VH head domain , which is composed of two four-helix bundles sharing one helix [24] ( Figure 1A ) .", 'Since VH can bind to VT , thereby confining the molecule to an autoinhibitory closed conformation , vinculin activation is needed to increase its affinity to talin and actin [25 , 26] .', "While it was suggested that vinculin recruitment to focal adhesions is force-regulated [15–19] , no high resolution structural mechanism has been derived experimentally by how force can activate talin's buried vinculin binding sites .", "What is known , however , is that fragmentation of the talin rod into smaller isolated helix bundles can activate talin's VBS [27] , and that point mutations that destabilize helix-helix interactions can have a similar activating effect [27] .", "Finally , vinculin binding to talin's VBS can cause significant loss of secondary structure of the adjacent non-VB-helices [28 , 29] .", 'Alternatively , it has been hypothesized that binding of PIP2 lipids to the talin rod may also lead to the activation of VB sites as the presence of PIP2 slightly increased the binding of vinculin to talin [30] .', 'No mechanism has been suggested though how activation by PIP2 could directly explain force-upregulated vinculin recruitment to newly formed cell adhesion sites .', "To establish a potential mechanism of force-regulated activation of talin's VBSs , we used steered molecular dynamics ( SMD ) to study the force-induced conformational changes in the N-terminal talin rod fragment H1–H12 ( a model created by merging two experimentally determined overlapping structures covering residues 486–889 [28] ) .", 'This computational method is valuable since no experimental techniques are available to obtain high resolution information how the structure of proteins is changed when they are mechanically stretched .', 'Access to such information is essential to learn how force might activate proteins by switching their structure-function relation ( as reviewed in [31 , 32] ) .', 'Therefore , known equilibrium structures of the talin rod were solvated here computationally in a box filled with explicit water molecules .', 'Conducting the simulations in the presence of explicit water rather than in an unstructured dielectric medium is important , since the access of free water molecules to often buried force-bearing hydrogen bonds or salt bridges can regulate mechanical stability [31–34] .', 'After equilibration for 1 ns , constant force was applied to either the termini of the rod fragment , or alternatively to putative force-bearing interfaces which might stabilize this rod fragment in the intact talin molecule .', 'We then characterized the structural changes that lead to the sequential exposure of the vinculin binding sites .', 'A mechanism of how stretching of the talin rod might activate vinculin binding is proposed .'], ['When applying various constant forces locally to the terminal atoms of the H1–H12 bundle ( 100 pN , 200 pN , 300 pN , and 400 pN ) , the intial end-to-end distance of the talin rod H1–H12 of 3 . 2 nm increases rapidly with time due to progressive unfolding ( loss of secondary structure ) of the terminal helices ( Figure 2B ) .', 'The ease by which they unravel even at low forces indicates that relatively little energy is required to sequentially break the backbone hydrogen bonds , turn-by-turn , that stabilize their helical secondary structure [39 , 40] .', 'As shown in Figure 2A , a first short-lived plateau can be seen that has a lifetime of less than 1 ns if we pull at 100 pN ( Intermediate State I1 ) .', 'Just little activation is then needed to begin the unfolding of both of the terminal helices , H1 and H12 , which leads to an extension with respect to the resting length of altogether 15 nm , until a second plateau is reached ( Intermediate State I2 ) .', 'Notice though that both helices do not initially unfold completely .', 'Only the N-terminal half of H1 ( residues 499–503 ) is unraveled in state I2 , due to a bent in H1 at Ser502 .', 'The bending site seems to be defined by the side chain of residue Ser502 , which competes for the backbone hydrogen bond between the Ile499 and Met503 ( see Figure 3B for presentation of such an event ) .', 'Similarly for the H12 , which is a vinculin binding helix , its C-terminal residues 874–879 are unraveled while its N-terminal part of the helix remains buried in the helix bundle .', 'Further extension results from the sequential turn-by-turn unraveling of the N-terminal end of H1 ( D2; in all of 8 local force simulations; Figure 2 ) , and the H12 by ∼3 additional turns ( Intermediate State I3 ) which leads to a total extension of 28 nm while pausing in the third plateau in Figure 2A .', 'This intermediate I3 , which is seen to last for ∼15 ns at 200 pN , is characterized by a completely unraveled H1 , whereas residues 868–879 of H12 are unraveled while residues 849–867 of H12 are still in a helical conformation and in tight contact with the rest of the H9–H12 bundle .', 'At higher forces , we see a rapid transitioning into I4 ( multiple unraveled helices ) where both H2 ( ∼8 turns unraveled ) and H12 ( ∼6 turns unraveled ) are mostly unraveled .', 'I4 is characterized by an extension of 35–36 nm .', 'Once the major energy barrier to rupture I4 is passed ( as described below ) , the remaining bundle H2–H11 rapidly breaks down into the bundles H2–H5 , H6–H8 and H9–H12 ( T4 in Figure 2 ) .', 'Each of these three helix bundles unfolds independently from each other at later times as discussed below .', 'Note that the two vinculin binding helices , H11 and H12 , are already completely unraveled when T4 is reached , i . e . , they convert early on into an extended polypeptide chain held under tension .', 'To assess whether the just described intermediates are characteristic only for an isolated talin fragment where force is applied to the terminal atoms , or also for the case where force is distributed over the cross-section of the talin rod , we ran constant force simulations in which we distributed the force ( 200 pN , 250 pN , 300 pN , 400 pN ) along the Cα-atoms of the terminal helices H1 and H12 as shown in Figure', '3 . Additional constant velocity simulations were carried out under otherwise similar conditions , but by applying force distributed along the C-terminal H12 ( see Methods ) and harmonically constraining H1 ( Figure S2 , only stimulation with harmonical constrained helix ) .', 'Distributing the force over a putative interface had a major stabilizing impact on the resulting unfolding trajectories and the forces needed to activate the VBSs ( Figures 2 and 3 ) : The most notable difference is that the talin rod H1–H12 could withstand considerable higher forces before breaking apart ( Figure 3C ) , stably exceeding our simulation window of 20 ns in a prestretched intermediate state I1 ( Figure 3 ) if pulled with a constant force ( 200 pN ) .', 'Earlier , this force had resulted in the unraveling of the terminal helices within the first 5 ns ( Figure 2A ) if force was only applied to the terminal atoms .', 'Distributing the force over the terminal helices thus stabilized the entire H1–H12 bundle against force-induced breakage and terminal helix unraveling: unfolding of H1 was not detected as a first major unfolding event even at higher forces ( 7 simulations , 250–400 pN ) .', 'Plotting the sequence by which hydrogen bonds between the helix bundles broke ( Figure 4 ) reveals that straining the talin rod leads to the appearance of some new hydrogen bonds ( for example bond 8 between residues Asn559 and Glu738 ) while others break ( for example bond 5 between residues Ser658 and Pro725 ) .', 'When H1–H12 is stretched with a constant force of 250–400 pN distributed over the terminal helices , interhelical bundle contacts start to loosen up after a few ns leading to a slight opening of the interface between the bundles H9–H12 and H1–H8 ( I2 ) .', 'This passage leads to the breakdown of some hydrogen bonds in the interface between H1–H8 and H9–H12 , namely between residues Gln887-Ser752 , Gln886-Gln755 , His784-Glu733 , His788-Gln733 ( referred to as bonds 19 , 20 , 21 , 25 in Figure 4 ) .', 'Also the bonding between bundles H1–H5 and H6–H8 is weakened significantly by breakdown of hydrogen bonds between residues Asn559-Gln735 , Ser729-Asp548 , Gln715-Asn559 ( referred to as bonds 1 , 2 , 3 in Figure 4 ) .', 'Further pulling results in a gradual opening of the interface between the bundles and water starts to penetrate between the helices ( Figure 3E ) .', 'The stress applied leads finally to a bending of H9 around residue 770 due to breakage of the backbone hydrogen bond formed between residues Gly768 and Thr772 ( Figure 3B ) .', 'After this , H9 stays attached to H1–H8 on the N-terminal end during the short-lived intermediate state I3 seen in many but not in all the simulations ( 5 of 7 simulations 250–400 pN ) .', 'After passing the I3 intermediate , a rapid disintegration of the talin rod is seen ( T3− ) which then allows for the sequential unfolding of the now separated bundles .', 'Common to all simulations is the break-up of the talin rod , H1–H12 , into smaller helix bundles that have far smaller mechanical stabilities .', 'We observed that the first split occurred between H1–H8 and H9–H12 ( 6 times in 7 simulations where constant force is applied over terminal helices ) , after which H1–H8 splits into H1–H5 and H6–H8 ( Figures 2 , 3 , and 5 ) .', 'In the other case observed only once in seven simulations , H1–H5 separated first followed by the H9–H12 separation .', 'The break-up into these well-defined bundles as a major event is most clearly seen if the force is distributed over the terminal helices ( Figure 3 ) .', 'Once the force-bearing interfaces are broken apart , the bundles have no stabilizing effects upon each other any longer .', 'Consequently , the force is thus transmitted at these later times through the N- and C-terminal atoms of the resulting helix bundles .', 'We thus simulated separately the unfolding of the helix bundles H1–H9 ( crystallographically determined structure of talin rod fragment , PDB 1SJ8 ) , H2–H8 ( intermediate found in terminal atom pulls ) , and H9–H12 ( intermediate seen only in force-bearing interface simulations ) .', 'The starting end-to-end distances of the terminal atoms of these helix bundles prior to stretching were 3 . 6 nm ( H1–H9 ) , 7 . 9 nm ( H2–H8 ) and 1 . 5 nm ( H9–H12 ) .', 'Most noticeable when looking at all their unfolding pathways ( Figure 5 ) is the lack of well pronounced plateaus and thus clearly distinguishable intermediate states , especially in the case of H9–H12 .', 'If pulled apart at 300 pN , the H1–H12 bundle breaks into bundles H1–H8 and H9–H12 after 7 ns ( Figure 3 ) .', 'This indicates that different structural changes are happening in parallel at different positions .', "Furthermore , when simulating the mechanical stabilities of alternate talin rod fragments that were not truncated along a ‘natural' bundle-bundle interface , for example simulating H1–H9 instead of H2–H8 , it is interesting to note that H9 is the least stable helix in those cases ( Figure 5 ) since it belongs structurally to the H9–H12 bundle .", 'Unraveling of H9 is the first major unfolding event of H1–H9 even if the force is distributed over terminal helices , since H9 is easily detached from the H1–H8 bundle .', 'Activation of the VBS requires that the VB helices H4 , H6 , H9 , H11 , and H12 , are at least partially exposed to water .', 'As seen in Figure 3D , these five VB helices are buried in the H1–H12 bundle under equilibrium , and become sequentially exposed only after the fragmentation of the talin rod into smaller helix bundles has occurred .', 'The asterix defines the point where the strain-exposed solvent-buried surface area of a VB helix is equal to the solvent-buried area of the helix when complexed to the vinculin head .', 'The asterix thus marks the unique points in the unfolding trajectory in which each of the VB helices gets activated ( Figure 3D ) .', 'Breaking the talin rod apart thus defines the highest energy barrier that has to be overcome to initiate the exposure and activation of the VBSs , and the implications thereof will be discussed below .', 'To better understand the molecular mechanism behind the interactions that regulate helix bundle separation , we analyzed the hydrogen bonding pattern between the defined helix bundles as shown in Figure', '4 . The blue fluctuatuions represent bonds formed between H1–H5 and H6–H8 , while the green represents bonds formed between H6–H8 and H9–H12 .', 'While most hydrogen bonds fluctuate between formation and breakage even during the equilibration , only a few of the side-chain hydrogen bonds , like the bond 2 , are longer-lived .', 'This , together with the fact that there appears to be considerable statistical variability in bond breaking events between different simulations suggests that at least most of these side-chain hydrogen bonds are not force-bearing .', 'While it cannot be excluded that one or the other of these bonds slightly contributes to the mechanical stabilization , hydrophobic contacts between the helix bundles seem to play the dominant role in upregulating the mechanical stability of the N-terminal part of the talin rod .', 'A separate SMD analysis of the talin rod fragments H1–H5 was done recently by applying force to the polar side chains T498 , S501 and S502 close to the N-terminus of H1 and Q635 , Q646 , E650 , and Q653 close to the C-terminus of H5 , assuming that the force-bearing interactions were mediated by side chain hydrogen bond formation across the interfaces of adjacent α-helix bundles [41] .', 'When using an implicit water model in which the protein structure was solvated in a dielectric medium , they observed a rotation of VBS1 ( H4 ) under applied force and suggested this to be a potential activating mechanism .', 'They also observed that the H4 rotation was strongly reduced when repeating the simulation in the presence of explicit water molecules ( these computationally more elaborate conditions were used in our simulations as well ) .', 'We thus analyzed for how long the polar side chains of H5 are hydrogen-bonded across the interface formed between H1–H5 and H6–H8 .', 'Our simulations of H1–H12 reveal that water penetrates into the interface H1–H5 and H6–H8 , thus breaking these side-chain hydrogen bonds , even before we can see a major force-induced structural change within the H1–H5 bundle ( Figure S3 ) .', 'Among the residues that were previously used to model the contact interface between bundles H1–H5 and H6–H8 [41] , only residues Gln635 ( bonded to Ser714 ) and Gln646 ( bonded to Lys721 ) are hydrogen bonded across the interface , and those side-chain bonds show quite low stability during our simulations ( Figures 4 , bonds 6 and 14 , and S3 ) , suggesting that those polar residues are also not force-bearing during the activation process of the H4 helix .', 'To compare the mechanical properties of the talin1 and talin2 rods , H1–H12 , we generated a homology model of talin2 based on the talin1 structure ( Figure S4 ) .', 'Homology modeling is a reasonable approach since their sequences are highly similar ( 74% identical ) in this region .', 'Furthermore , a sequence analysis of the hydrogen bonding partners when comparing talin1 and talin2 from human , mouse , and chicken ( Figure 4 ) revealed that 29 of the 43 residues participating in hydrogen bonds between helix bundles are fully conserved , eleven of the residues ( Ser658 , Ser688 , Thr693 , Thr720 , Ser754 , Gln755 , Gln762 , Arg765 , Glu780 , His788 , Lys869 ) are similar and only three residues are non-conserved ( Arg692 , Ser752 , Gly766 ) ( Figure S1 ) .', 'The RMSD for backbone atoms after energy minimization and thermalization was 1 . 3 Å .', 'Also for talin 2 , the fragmentation of the rod H1–H12 constituted the major energy barrier as seen in constant force and constant velocity SMD simulations , in which the force was distributed over force-bearing interfaces .', 'The split of the rod occurred in identical positions as described above for talin1 , namely between the bundles H1–H5 , H6–H8 and H9–H12 .', 'Also the sequence of early events was similar: First , the H9–H12 bundles separated , followed by the rupture of the H1–H8 fragment .', 'Our simulations of early events further indicate that the major energy barrier of fragmenting the rod are not greatly different between the talin1 and talin2 rods , H1–H12 , at least within the stochastic variability that is intrinsic to single molecule studies ( Figure S5 ) .', 'Finally , it is important to note that the SMD simulations are carried out on time scales that differ significantly from those at which biological molecules are stressed .', 'Since the force needed to unfold a protein is logarithmically dependent on the pulling velocity , significantly smaller forces may be able to cause the here described structural rearrangements at physiological timescales .', 'Unfolding forces measured by SMD in nanosecond timescale are thus significantly higher compared to those measured using AFM on millisecond timescales , yet , SMD has correctly predicted in the past the relative mechanical stabilities of some protein domains and the position of key energy barriers [32–34] .'], ['Five VB helices have been identified in H1–H12 ( Figures 1 and S1; Table 1 ) [10] , namely H4 , H6 , H9 , H11 , and H12 .', 'Breaking the H1–H12 talin rod into smaller bundles is the prerequisite for the strain-dependent decrease in the buried surface area of these amphiphatic VB helices ( Figure 3 ) .', 'Starting with the first split where H1–H8 separates from the H9–H12 bundle ( Figure 3 ) , we observe a significant decrease in the buried surface areas of the three VB helices ( H6 , H9 , and H12 ) , while the buried surface area of the VB helix H4 is decreasing only at later time points when helix bundle H1–H5 is fragmented into smaller pieces .', 'Note that H6 , H9 and H12 are located at the interfaces of the H1–H5 , H6–H8 and H9–H12 bundles .', 'Since H11 is buried in the interior of the subbundle , there is only a slight drop in the buried area observed during the simulated time frame .', 'Is the increased solvent exposure sufficient to activate the VBSs ?', 'Considerable experimental evidence has demonstrated that bundle fragmentation by clipping off helices can indeed expose the VB sites that are otherwise buried in the full length talin rod under equilibrium conditions .', 'The isolated H1–H12 bundle shows a higher affinity to vinculin than does the full-length talin or talin rod domain [30] , and mutations stabilizing the H9–H12 bundle result in decreased vinculin binding [30] .', 'NMR studies have revealed that the helix H10 of the isolated H9–H12 bundle unfolds into a flexible random coil when the fragment complexes with VH [28] .', 'Taken together , these results support the model where the H9–H12 bundle is stabilized by the neighboring helices which bury the VB helices , H9 , H11 and H12 .', 'Similar trends are seen for the VB helix H4 .', 'Experiments show that the H1–H5 bundle does not bind to vinculin but removal of H5 from the bundle activates the otherwise buried VB helix H4 [30] .', 'Taking together all of these experimental and computational findings , we propose the following model of how tensile force acting on talin can activate its binding to vinculin: What drives the force-induced association of talin helices with vinculin once they have been activated by water exposure ?', 'Key to the VB helices are hydrophobic residues that are located on one face of these amphiphatic α-helices which share the consensus ( LxxAAxxVAxxVxxLIxxA ) , where x is a variable amino acid residue [10] .', "As shown for talin's isolated VB helices H4 , H11 and H12 ( the only ones for which sufficient data are available ) , association with the vinculin head is energetically favored since the vinculin head is thermodynamically stabilized by complexation with VB helices [23 , 26] , and has considerable structural homology to the respective equilibrium talin bundle structures [10 , 27 , 28] .", "At equilibrium , the buried surface areas of talin's VB helices complexed with talin versus the vinculin head , respectively , are 1537 Å2 vs . 1310 Å2 for H4 ( residues 607–631 ) , 1190 Å2 versus 1144 Å2 for H11 ( residues 821–842 ) , and 1706 Å2 versus 1291 Å2 ( residues 853–876 ) ( Table 1 ) .", 'Accordingly , the affinities of isolated VB helices with vinculin ( Kd = 3–33 nM ) [47] differ considerably from that of full length talin ( Kd = 8 . 9 μM ) [30] .', "It has thus been proposed earlier that once released from the hydrophibic core of the talin rod , the H4 ( VBS1 ) and potentially the other VB helices are available to induce ‘bundle conversion' thereby displacing the intramolecular interaction of the vinculin head to its tail [24 , 27 , 28 , 48] .", 'This suggestion was further supported by findings that vinculin can be activated by helical peptides that contain the VBSs , subsequently leading to an increased affinity of the vinculin tail to actin [22 , 23] .', 'Following these earlier suggestions derived for unstrained systems , we now propose that the complementary design between the talin and vinculin structures facilitates force-induced α-helix swapping: the VB helices find a thermodynamically more stable environment in talin under equilibrium conditions ( Table 1 ) , the biologically inactive state , but as soon as talin is sufficiently strained , the association with the unstrained vinculin head is energetically preferred once a collision has occurred and occurs upon a collision with the vinculin head .', 'Vice versa , the vinculin head is thermodynamically less stable in the absence of a swapped talin helix [30] and thus forms the auto-inhibitory complex with its tail [23–26] .', 'We further suggest a biphasic vinculin binding behavior: the maximum probability of the α-helix swapping is reached once the VB helix has broken away from the other talin helices and exposes the hydrophobic residues of the otherwise structurally intact VB helices to water ( Figure 6 ) .', 'Once a VB helix is sufficiently stretched and starts to lose its secondary structure , helix swapping might again be inhibited .', 'Of major physiological importance is furthermore the insight that the talin rod is engineered such that the activation of vinculin binding is force and time dependent , thus acting as a hierarchical force-time integrator .', 'Not all VB helices are activated at the same time since the VB helices are located in sub-bundles that break up sequentially due to their differential mechanical stabilities ( Figures 3 and 5 ) .', 'A hierarchy thus exists in which the VBSs are force-activated ( as indicated by asterixes in Figure 3D ) : the earliest to be exposed are the VB helices H6 and H12 , which are located in the weakest helix bundle interface .', 'The next to be exposed are the VB helices H9 and H11 located in the C-terminal H9–H12 bundle , and the last one is VB helix H4 , which is located in the sub-bundle H1–H5 .', 'If the talin rod is kept under constant force , the number of VB helices that have been activated will initially increase with time and the amount of force applied .', 'Once a vinculin complex has been formed during the lifetime of the activated helix , the complexed helix might be protected against complete unraveling due to the additional interactions formed between a VBH and the unstrained vinculin head .', 'In contrast , when applying force to the terminal ends of H1–H12 , the VB helices H11 and H12 lose already their secondary structure before the bundle is completely fractured , potentially leading to their early deactivation .', 'Vinculin recruitment to talin thus initially increases if talin is incorporated into a force-bearing network formed when a cell adheres to a surface or matrix fibrils [15–18 , 20 , 29 , 30] .', 'However , since each VB helix has its unique biphasic time response , increased force thus accelerates the sequence of these events , but not necessarily the total number of activated VBSs at later time points , particularly at low vinculin concentrations .', 'The comparative analysis carried out here for the rod fragment H1–H12 of talin1 and talin2 shows that they share a similar sequence of early unfolding events that lead to the fragmentation of the N-terminal part of the talin rod .', 'Talin1 and talin2 are not only differentially expressed in various tissues but also localize in different parts of a cell [49] .', 'While we do not see major changes between the mechanical stabilities of the H1–H12 fragments of two talins , i . e . , in their likelihood of being fragmented by tensile force , it is important to note that the talin rod has six additional VBSs in the structurally unresolved C-terminus and that we do not have any information so far regarding the differential mechanical stresses to activate them ( see Figures 1B and S1 ) .', 'Unclear is also to what extend functional differences of talin1 and talin2 might result from mutations in their binding sites to other proteins .', 'The mechanism described here might not be unique to the talin-vinculin bond but might be more widespread among proteins that are composed of α-helical bundles .', 'First of all , once an amphiphatic helix is broken off from a helix bundle by the application of tensile force , it might be stabilized by insertion into either hydrophobic pockets of other proteins or even into the lipid bilayer [27] .', 'Alternatively , other proteins that form helix bundles might also bind vinculin in a force-regulated manner .', 'α-actinin , for example , has also a VB helix that can form a similar structural complex with vinculin [23 , 26] .', 'Similarly to talin , the VBS in α-actinin is buried in the native structure [50] .', 'Identifying the repertoire of mechanisms by which forces can upregulate adhesive interactions has led to the recent discovery of catch bonds where a receptor-ligand interaction is enhanced when tensile mechanical force is applied between a receptor and its ligand ( for review see [51 , 52] ) .', 'In contrast , the force-activated helix swapping mechanism proposed here requires that the force is applied to just one of the binding partners , thereby activating bond formation with a free ligand .', 'Also in contrast to catch bonds , the ligand does not necessarily have to be part of the force-bearing protein network at the time the swap is initiated .', 'While force-induced helix swapping thus primarily upregulates the bond formation rate , the catch bond mechanism primarily extends the lifetime of an already existing complex .'], ['The talin rod domain H1–H12 ( residues 486–889 ) of mouse talin was obtained from the Protein Data Bank ( http://www . pdb . org/ [PDB theoretical models section: 1XWX] ) [28] .', 'This structure is an energy-minimized model derived from combining two talin fragments derived from NMR ( H9–H12 , residues 755–889 [PDB: 1U89] [28] ) and X-ray ( H1–H9 , residues 482–789 [PDB: 1SJ8] [27] ) .', 'Another talin structure was also studied ( H1–H9 [PDB: 1SJ8] ) .', 'Talin fragments H2–H8 ( residues 523–757 ) and H9–H12 ( residues 755–882 ) were obtained from the 1XWX talin rod structure by removing the excess atoms .', 'Homology model for talin2 H1–H12 was generated based on talin1 [PDB: 1XWX] using SWISS-MODEL protein structure homology-modeling server [53] and subjected to WhatCheck analysis .', 'The human talin2 sequence was used; human and mouse talin2 are virtually identical in the region of H1–H12 , and there are only 2 sequence differences: the residue corresponding to human talin2 Glu604 is Asp in mouse , and the residue corresponding human to talin2 Thr762 is serine in mouse talin2 .', 'The sequence alignment was prepared using ClustalW ( http://www . ebi . ac . uk/clustalw/ ) .', 'The H1–H12 sequences of talin1 and talin2 have 74% identity .', 'All the simulations were carried out in explicit water .', 'The periodical TIP3 water box was created using the program VMD plugin solvate [54] The system was neutralized by adding Na+- or Cl−-ions to the system using VMD autoionize-plugin .', 'The systems used in the simulations are summarized in Table S1 .', 'Since efficient arrangement of water is a pre-exquisite for validity of the results , we used Solvate 1 . 0 ( http://www . mpibpc . mpg . de/groups/grubmueller/start/software/solvate/docu . html ) to find possible hydration sites inside the protein , and energetic evaluation of those positions was performed using Dowser [55] .', 'Positions energetically favoring hydration were analyzed in the H1–H12 which was subjected to a 1 ns equilibration after solvation with the VMD solvate plugin .', 'A further analysis revealed that 1 ns equilibrations were sufficiently long to allow water to penetrate into all energetically favored locations .', 'A 12 Å cutoff was used for the van der Waals interactions , switching function starting from 10 Å .', 'The PME ( Particle Mesh Ewald ) method was used to calculate long-range electrostatics without a cutoff using grid spacing lower than 1/Å3 .', 'Each system was minimized with a conjugate gradient method using NAMD [56] .', 'Initially , only the solvent and ions were allowed to move for 4 , 000 steps .', 'Next , the entire system was allowed to move for another 4 , 000 steps .', 'After minimization , the system was heated from 0 K to 300 or 310 K in 30 ps under Berendsen pressure control ( 1 atm ) and subsequently equilibrated for 1 ns under constant pressure ( Berendsen pressure control at 1 atm ) and temperature ( tCouple method ) .', 'Structures equilibrated for 1 ns were used in all SMD simulations .', 'Constant force was applied either to both termini , or alternatively to Cα-atoms of the terminal helices ( residues 495–514 and 853–872 ) and the force vector was then calculated using residues in the middle of the helices ( residues 504 and 865 ) .', 'The system coordinates were saved every picosecond , and the system energies were recorded every 0 . 1 ps .', 'Constant velocity SMD simulations were performed by applying moving springs with a spring constant of 5 kcal/mol/Å2 [30] to the Cα-atoms of residues 853–872 in the C-terminal helix H12 .', 'The springs were moved along the force vector calculated according to the vec
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Upon cell adhesion , talin physically couples the cytoskeleton via integrins to the extracellular matrix , and subsequent vinculin recruitment is enhanced by locally applied tensile force .', 'Since the vinculin binding ( VB ) sites are buried in the talin rod under equilibrium conditions , the structural mechanism of how vinculin binding to talin is force-activated remains unknown .', 'Taken together with experimental data , a biphasic vinculin binding model , as derived from steered molecular dynamics , provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment ( residues 486–889 constituting helices H1–H12 ) might activate the VB sites .', 'Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water .', 'Finally , unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin .', 'The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar .', 'The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds , including catch bonds .']
['For cell survival , most eukaryotic cells need to be mechanically anchored to their environment .', 'This is done by transmembrane proteins , including integrins , which externally bind to the extracellular matrix and on the cell interior to the contractile cytoskeleton via scaffolding proteins .', 'One essential scaffolding protein is talin , which binds to integrins via its head and to the cytoskeletal filament f-actin via its rodlike tail .', 'As cells apply tensile forces to newly formed adhesion sites , proteins that are part of such force-bearing networks get stretched and might change their structure and thus function .', 'One of many proteins that are recruited to newly formed adhesions is vinculin , and vinculin recruitment is upregulated by tensile mechanical force—but how ?', "Since talin's vinculin binding sites are buried in its native structure , we used steered molecular dynamics here to derive a high resolution structural model of how tensile mechanical forces might activate talin's vinculin binding sites .", 'Once tensile forces break up the talin rod into helix subbundles , an event that we find here to constitute the main energy barrier , we propose how the strain-induced gradual exposure of the vinculin-binding helices finally allows for their activation and enables helix swapping with the vinculin head .']
['biophysics', 'cell biology', 'mammals', 'computational biology']