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Originally published In Press as doi:10.1074/jbc.M508060200 on September 21, 2005 Originally published In Press as doi:10.1074/jbc.M508060200 on September 21, 2005 Originally published In Press as doi:10.1074/jbc.M508060200 on August 30, 2005

J. Biol. Chem., Vol. 280, Issue 44, 37217-37224, November 4, 2005
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Mapping and Consensus Sequence Identification for Multiple Vinculin Binding Sites within the Talin Rod*{boxs}

Alexandre R. Gingras{ddagger}1, Wolfgang H. Ziegler§1, Ronald Frank¶, Igor L. Barsukov{ddagger}, Gordon C. K. Roberts{ddagger}, David R. Critchley{ddagger}, and Jonas Emsley||2

From the {ddagger}Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom, the §Interdisciplinary Centre for Clinical Research (IZKF) Leipzig, Faculty of Medicine, University of Leipzig, D-04103 Leipzig, Germany, the Department of Chemical Biology, German Research Centre for Biotechnology, D-38124 Braunschweig, Germany, and the ||Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG72RD, United Kingdom

Received for publication, July 22, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the {alpha}-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS {alpha}-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinculin (1066 amino acids) is associated with integrin-containing cell-extracellular matrix and cadherin-based cell-cell junctions (1). Electron microscopy images of chicken vinculin show a trilobar head 80 Å in diameter connected to a long flexible "tail" (2), and a recent crystal structure of the full-length vinculin molecule shows a circular five-domain autoinhibited conformation in which the N-terminal head domain (Vd1)3 forms an extensive interaction with the C-terminal tail domain (Vt) (3). The Vd1 domain contains binding sites for talin (4) and {alpha}-actinin (5), whereas Vt binds to paxillin (6), F-actin (7), and acidic phospholipids (8). The intramolecular Vd1-Vt interaction regulates vinculin activity by masking the binding sites for talin (9) and {alpha}-actinin (5) in Vd1, the VASP binding site in the proline-rich domain (10, 11), and the F-actin binding site in Vt (7).

Talin (2541 amino acids) is an elongated (60 nm) flexible anti-parallel dimer, with a small globular head connected to an extended rod (2). The talin head contains a FERM domain (residues 86-400) with binding sites for several {beta}-integrin cytodomains (12) as well as the type 1{gamma} 661 isoform of phosphatidylinositol-4-phosphate 5-kinase (13-15), and the protein-tyrosine kinase FAK (16) both of which are important in focal adhesion dynamics. The talin rod contains a second lower affinity integrin binding site (17, 18), a highly conserved C-terminal actin binding site (residues 2345-2541) (19, 20), and also several binding sites for vinculin (19). A yeast two-hybrid assay was used to map three of these vinculin binding sites (VBS1, -2, and -3) to short peptide sequences 25-30 residues in length, spanning residues 607-636, 852-876, and 1944-1969 (21).

Crystal structures of complexes formed between peptides corresponding to talin VBS1, -2, and -3 and the N-terminal Vd1 have identical features (22-24). In each case, the VBS is composed of six turns of an {alpha}-helix, which is 50% buried upon binding, forming extensive hydrophobic interactions with the Vd1 four-helix bundle. A comparison of the structures of the ligand-bound Vd1 domain with those of the autoinhibited form of Vd1 shows that talin provokes marked conformational changes in vinculin (3, 24). The talin binding site does not overlap with that of Vt, but instead VBS binding distorts the Vd1-Vt interface resulting in displacement of Vt. This raises the possibility that a VBS from talin might contribute to vinculin activation in focal adhesions, in combination with the phospholipid phosphatidylinositol 4,5-bisphosphate, which binds to Vt and weakens the interaction between Vt and the vinculin head (3, 25), although the involvement of phosphatidylinositol diphosphate in vinculin activation within the cell has recently been challenged (26).

We have previously described the structure of the N-terminal domain from the talin rod (residues 482-655) (22), which contains VBS1 (21). This revealed a five-helix bundle structure with all the key VBS1 side chains involved in Vd1 binding buried within the hydrophobic core of the fold. Biochemical studies demonstrated that this five-helix bundle represents an inactive conformation, and either mutations that disrupt the hydrophobic core or deletion of the C-terminal helix were required to induce an active conformation in which the VBS is exposed. An NMR structure for talin residues 755-889 reveals a left-handed four-helix bundle (23). The VBSs contained therein are also buried within this structure, but in contrast to talin 482-655, the recombinant talin 755-889 polypeptide does bind vinculin constitutively, and in so doing undergoes a major conformational change to expose the hydrophobic side chains involved in Vd1 binding (23).

Defining the specificity of the talin VBS-vinculin Vd1 interaction is fundamental to the understanding of ligand recognition by vinculin and its activation. The size of the talin rod (2059 residues) and occurrence of many predicted amphipathic helical sequences within it (27) intimates that further VBS might exist (23). By analyzing vinculin binding to a large series of talin synthetic peptides, we have been able to resolve both of these issues and (i) define the specificity of Vd1 binding to a talin VBS helix and (ii) establish that there are ~11 VBSs within the talin rod.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Vinculin Head Binding to SPOT-Peptide Arrays—Peptides (25-mer) based on the mouse talin rod sequence were spot-synthesized on a cellulose membrane (28) with 0.5 nmol of peptide per spot. Membranes were blocked overnight in Tris-buffered saline (50 mM Tris-HCl, pH 7.0) with 1% fetal bovine serum. Glutathione S-transferase (GST)-tagged mouse vinculin Vd1-(1-258) or Vd1-3-(1-716), was expressed using pGEX-4T (Amersham Biosciences) and purified as described previously (26). The purified GST-Vd1-3 was transferred into phosphate-buffered saline or 50 mM Tris-HCl, pH 8.0, with 2 mM sucrose and lyophilized. Membranes were overlaid for 1 h with GST-Vd1-3 (50 nM) in Tris-buffered saline with 1% bovine serum albumin. Bound GST-Vd1-3 was detected using a polyclonal GST antibody (Sigma) and alkaline phosphatase-coupled anti-rabbit Ig (Sigma) as described previously (22).

Crystal Structure Determination of VBS Peptide·Vd1 Complexes—Recombinant His-tagged vinculin Vd1 domain (residues 1-258) was expressed in pET-15b and purified as described previously (22). Vd1 was dialyzed against 20 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 5 mM dithiothreitol, concentrated to 9.0 mg/ml, aliquoted, and stored at -80 °C. Peptides corresponding to predicted talin rod helices 11, 36, and 58 (residues 820-844, 1628-1652, and 2345-2369) were synthesized and high performance liquid chromatography-purified by Werner Tegge (German Research Centre for Biotechnology, Braunschweig). For crystallization experiments, the peptides were dissolved in 50 mM Tris-HCl, pH 8.0, to a final concentration of 5 mM, and added to Vd1 at a final ratio of 2:1. For all three Vd1 complexes, data were collected at European Synchrotron Radiation Facility, beamline 14-1 ({lambda} = 0.934E). Structures were solved by molecular replacement using Phaser (CCP4 suite) with the structure of the VBS3·Vd1 peptide complex (1XWJ) as a search model. Models were built using XtalView (29), and refinement was carried out using REFMAC (CCP4 suite). The structures have been submitted to the Protein Data Bank with the Vd1 complexes with peptides 11, 36, and 58 having codes 1ZVZ, 1ZW3, and 1ZW2, respectively.

Crystals of Vd1 in complex with peptide 11 were grown from 0.1 M Tris-HCl, pH 7.0, 23% polyethylene glycol 2000 monomethylether, at 20 °C. The crystals belonged to the space group P212121 with cell dimensions a = 36.0Å, b = 52.2Å, c = 153.8Å, {alpha} = 90°, {beta} = 90°, and {gamma} = 90°. The model was refined to a final Rcryst of 0.239 for all data between 30 and 1.80 Å and Rfree of 0.276. The main-chain torsion angles of 95.2% of the residues lie within most favored regions, 3.2% in additional favored regions, and 0.8% in generously allowed regions. The crystals of Vd1 in complex with peptide 36 were grown from 1.2 M NaH2PO4/0.8 M K2HPO4, 0.1 M CAPS, pH 10.5, at 20 °C. The crystals belonged to the space group P21212 with cell dimensions a = 51.6Å, b = 72.2Å, c = 96.7Å, {alpha} = 90°, {beta} = 90°, and {gamma} = 90°. The model was refined to a final Rcryst of 0.224 for all data between 30 and 3.30 Å and Rfree of 0.307. The main-chain torsion angles of 88.8% of the residues lie within most favored regions, 8.8% in additional favored regions, and 1.6% in generously allowed regions. The crystals of Vd1 in complex with peptide 58 were grown from 0.1 M Tris-HCl, pH 7.0, 23% polyethylene glycol 2000 MME at 20 °C. The crystals belonged to the space group P21212 with cell dimensions a = 52.0Å, b = 70.0Å, c = 96.0Å, {alpha} = 90°, {beta} = 90°, and {gamma} = 90°. The model was refined to a final Rcryst of 0.254 for all data between 20 and 2.10 Å and Rfree of 0.295. The main-chain torsion angles of 94.4% of the residues lie within most favored regions, 3.6% in additional favored regions, and 1.2% in generously allowed regions.

Molecular Modeling—A homology search of the Protein Data Bank (pdb, www.rcsb.org) revealed that talin residues 2295-2481 align with the crystal structure of the F-actin binding thatch domain from Huntingtin Interacting Protein 1-related protein (hip1r, pdb code 1R0D) with 29% identity extending over 206 residues. Only a single one residue insertion occurs in the alignment, and the modeling of talin 2295-2481 was performed using SWISSMODEL (30) first approach mode and subsequently energy minimized.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Vinculin Binding to a Series of Talin Rod Peptides—The talin rod is 2059 residues in length and is predicted to have predominantly {alpha}-helical secondary structure (27). To extend our previous search for vinculin binding sites (VBSs) (21), we synthesized a series of 25-mer peptides corresponding to all talin rod {alpha}-helices predicted using the secondary structure algorithm PSIPRED (31). These peptides, which cover 1575 residues (76%) of the rod, were spot-synthesized on a cellulose membrane (28) and overlaid with vinculin N-terminal domains 1-3 (Vd1-3) fused to GST (50 nM). Bound GST-Vd1-3 was detected using a polyclonal GST antibody and alkaline phosphatase-coupled anti-rabbit Ig. In agreement with the original yeast two-hybrid data (21), talin peptides 4, 12, and 46, corresponding to VBS1, -2, and -3 showed strong vinculin binding (Fig. 1). However, a further 7 peptides 6, 9, 11, 27, 33, 50, and 58 showed a similar level of binding. There were also 9 peptides that bound more weakly (peptides 20, 25, 26, 36, 40, 51, 55, 57, and 60). Analysis of the sequence of these latter peptides and crystallographic data presented below provides evidence that only peptide 36 is likely to be a vinculin binder, giving an overall total of 11 VBS in the talin rod.

The relative position of the 63 peptides along the length of the talin rod is illustrated in Fig. 2 with the 11 vinculin binding peptides highlighted in blue. The distribution of the VBS along the rod is discontinuous. Within the first 400 residues, there is a prominent cluster of 5 VBS (peptides 4, 6, 9, 11, and 12) two of which (peptides 4 and 12) were previously characterized as VBS1 and -2 (21). It is notable that two of the VBS (peptides 11 and 12) are arranged in tandem. Following this there is a gap with 13 non-binding peptides spanning a total of 450 residues. The next 320 residues (1330-1652) contain a further group of three vinculin binding peptides (27, 33, and 36), but there is only one VBS (VBS3, residues 1945-1969) between residues 1653 and 2050. The final 400 residues contain two VBS peptides, 50 and 58. Interestingly, this region of the talin rod contains an integrin binding site (1984-2113) (18), which overlaps with peptide 50. Peptide 58 lies within one of most conserved regions of the rod, which contains the principal actin-binding site and is homologous to the Huntingtin Interacting Protein 1 (hip1) family of actin-binding proteins (32).

Alignment of the 11 VBS Sequences—The 11 VBS peptides can be aligned based on sequence similarity using ClustalW (33) (Fig. 3A). Residues highlighted in blue align with those that are >75% buried in the VBS1·Vd1 complex crystal structure (22) (Fig. 3B), and the alignment shows that their hydrophobic/non-polar character is conserved in all VBS peptides. The preferences for residue types and classes at each position are illustrated in the 50 and 90% consensus sequences shown at the top of the alignment, calculated using program CONSENSUS.4



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  		FIGURE 1.
Talin SPOT-peptide analysis. The 63 talin rod peptide sequences are arranged in order from the N terminus to the C terminus. Binding of GST vinculin domains 1-3 to each peptide was determined using a polyclonal GST antibody and alkaline phosphatase-coupled anti-rabbit Ig. The results of the binding assay are shown on the right of the corresponding peptide sequence. Strong signals equivalent to those for the known vinculin binding sites VBS1, -2, and -3 (peptides 4, 12, and 46) are numbered from 1 to 10. Less intense spots, which may indicate potential weak vinculin binders, are indicated with a `w.' Of this subset, additional evidence suggests that only peptide 36 (underlined) may be an authentic VBS. The complete sequence of the talin rod with the secondary structure predictions for the 63 helices is illustrated in Fig. S1 (supplementary data).

 



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  		FIGURE 2.
Relative distribution of the VBS peptides among the 63 predicted {alpha}-helices which make up the talin rod. Vinculin binding helices are shown in blue and non-binders in gray. VBS1-3 (peptides 4, 12, and 46) were previously mapped using a yeast two-hybrid assay (21). The black line indicates the position of an integrin binding site (residues 1984-2113) (18), and the red line the C-terminal actin binding site (residues 2295-2541) (19, 20).

 
The consensus sequence at the 50% level spans 23 residues and shows that 8 of these are conserved in ≥50% of the sequences. Examining the buried residues across the six turns of the VBS helix, we see that Ll predominates in the first helical turn (where `l' represents an aliphatic residue); AA in the second, VA in the third, Al in the fourth, Ll in the fifth, and finally a small side chain is preferred in the sixth. The strongest preferences occur for Ala at position 5 on the second helical turn (9/11 peptides), Val at position 8 on the third (7/11), and Leu at position 15 on the fifth turn (9/11). The consensus at the 90% level shows that no side chain is absolutely conserved across the 11 peptides, but many of the buried positions have size and charge constraints. In particular residue positions 5 and 9 are conserved as small and uncharged in character (i.e. Ala, Ser, Thr, and Val). Turn 4 has position 11 conserved as a small residue with all peptides having either Ala or Ser with the exception of peptide 46 (VBS3). Other highly constrained positions occur at 8, 15, and 16 on turns 3 and 5 where only aliphatic side chains occur. Whereas Ala is the most common residue within the central turns, in the flanking buried positions of the first and fifth helical turns, small side chains like Ala and Gly are absent and aliphatics, in particular Leu, predominate.



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  		FIGURE 3.
Alignment of the 11 VBS sequences in the talin rod. A, VBS peptide sequences were aligned using ClustalW. Residues highlighted in blue align with the highly buried (>75%) hydrophobic side chains from the VBS1·Vd1 complex crystal structure shown in B. Basic residues are highlighted in green and acidic in red. The 50 and 90% consensus sequence is shown at the top. Uppercase letters indicate conserved residues (single-letter amino acid code). Lowercase letters indicate conserved classes of amino acids as follows: h, hydrophobic residues (A, C, F, G, H, I, K, L, M, P, T, V, W, and Y); p, polar residues (C, D, E, H, K, N, Q, R, S, and T); c, charged resides (D, E, H, K, and R); s, small residues (A, C, D, G, N, P, S, T, and V); +, positive residues (H, K, and R); l, aliphatic residues (I, L, and V); and u, tiny (A, G, S, and C). B, schematic diagram showing the VBS1·Vd1 domain complex with the Vd1 {alpha}1 and {alpha}2 helices shown as solid gray ribbons and helices {alpha}3, {alpha}4 as transparent cylinders. Buried side chains are indicated in blue.

 
Residues that are only partially buried in the VBS1·Vd1 complex generally show greatly reduced or no preferences. The clear exception to this rule is the clustering of basic residues (shown in green in Fig. 3A) on turns 2 and 6, where there is a striking preference for Arg or Lys at position 6 (9/11 peptides) and position 20 (7/11). There is some tendency for negatively charged residues (red in Fig. 3A) to occur at positions 10 (4/11) and 14 (4/11). The structural consequences of this clustering are discussed later.

A Complete Substitution Analysis of the VBS1 Sequence—The data from the above SPOT-peptide analysis provides a useful description of the common features of those talin sequences, which bind tightly to vinculin. However, careful examination of the 52 non-binding sequences reveals several examples of a reasonable VBS consensus. For example, peptide 5 has a very similar sequence to VBS1 and contains all the buried conserved hydrophobic positions, yet it does not bind to vinculin. Modeling of the peptide 5 sequence onto the VBS1·Vd1 structure does not predict any steric clashes that might preclude binding. Because a VBS lacks any critical residues absolutely required for binding (Fig. 3), more subtle effects must account for the specificity.

To address the above issues and to further define the specificity of vinculin binding, we performed a SPOT-peptide analysis substituting each of the commonly occurring amino acids into each of the 16 central positions of the VBS1 sequence. This generates a series of 25-mer peptides that were spot-synthesized and then assayed for binding to GSTVd1-3. The data are represented as a matrix with the VBS1 sequence arranged vertically on the far left, and the different amino acid substitutions at each position shown horizontally (Fig. 4). Looking along the horizontal lines reveals the specificity at each position, and this is indicated at the far right. Residues giving a spot intensity similar to wild-type are indicated in bold, whereas those giving a reduced but still clearly detectable signal are indicated in gray. Thus, horizontal lines with significant gaps or runs of low intensity spots occur for the buried side chains, which have restrictions of size and charge, whereas horizontal lines of intense spots occur for positions that are not buried in the VBS1·Vd1 structure, and for which substitutions do not result in a marked reduction in binding. For the exposed positions 6, 11, 13, and 14, where many side chains can substitute, the disallowed residues, such as proline, are shown at the right in italics.

The most restrictive positions are those where the side chains are highly buried in the complex. They can be divided into (i) five highly restrictive positions, 1, 8, 12, 15, and 16, which exclude Ala; (ii) three more relaxed positions, 4, 5, and 9, which will allow Ala; and (iii) position 11, which allows charged side chains. The centrally located position 8 on turn 3 is the most restricted of all, where only three residues, Ile, Leu, or Val, support binding. Val clearly has a stronger spot than Ile or Leu, and a strong preference for Val at this position is revealed in the alignment of the talin rod VBS peptides (Fig. 3). Positions 15 and 16 are slightly less stringent, being restricted to Phe, Ile, Leu, or Val. Finally positions 1 and 12 are slightly more relaxed again, with Phe, Ile, Leu, or Val being allowed and in addition Tyr in position 1 and Thr in position 12. More relaxed specificity occurs at the buried positions 4, 5, and 9 where Ala and polar uncharged side chains are allowed. Position 11 is the most relaxed of the buried positions, allowing charged residues such as Lys and Arg and only excluding Trp, Asp, Gly, and Pro.

As expected, the requirements for the more exposed side chains are greatly reduced with binding being observed with any residue at positions 2, 3, 7, and 10 and any residue with the exception of Pro at positions 6, 13, and 14. Even at these positions distinct side-chain preferences can be observed by examining the spot intensities. For example, only a very weak spot appears for Gly in position 2, whereas stronger spots are observed for Leu, Gln, Val, and Tyr; the alignment of the 11 VBS peptides shows hydrophobic side chains present at position 2 in all cases (Fig. 3). The preference for Arg or Lys side chains at position 6 in the sequence comparison (Fig. 3) is not, however, mirrored in Fig. 4, with a variety of side chains resulting in a similar spot intensity at this position. In exposed positions 7 and 10, flanking turn 3, some of the substitutions result in spot intensities greater than wild-type VBS1. These positions are both Gly in the VBS1 sequence, and it is likely that Gly has a negative effect on vinculin binding either by disfavoring the {alpha}-helical conformation, or by failing to contribute significant van der Waals contacts.



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  		FIGURE 4.
VBS1 SPOT-peptide substitution analysis. A series of VBS1 peptides were spot-synthesized in which each residue was substituted in turn with all of the commonly occurring amino acids. The effects of each substitution on GST-Vd1-3 binding was then determined as described in Fig. 1. The VBS1 sequence is shown vertically on the left (single amino acid code) with boxed residues indicating highly buried (>75%) hydrophobic side chains (based on the talin VBS1·vinculin Vd1 complex crystal structure). The top row indicates the amino acids substituted into each position of the VBS1 sequence. For example, the top left represents Ala substituted into position 1, and the bottom right represents Tyr substituted into position 16. Boxed spots within this matrix have the wild-type sequence. On the right, substitutions that do not appreciably decrease binding compared with wild-type are indicated in black capitals; those that lead to a significantly reduced binding are shown in brackets as gray capitals. Substitutions shown in lowercase italic are not allowed.

 



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  		FIGURE 5.
Analysis of talin VBS rule violations. A, alignment of talin peptides with sequences that do not conform with the position-specific preferences for vinculin binding defined in Fig. 4. Residues on a gray background align with the highly buried (>75%) hydrophobic side chains from the talin VBS1·vinculin Vd1 complex crystal structure. Residues that would be predicted to knock out binding of VBS1 to vinculin (Fig. 4) are indicated in white. Included are weak/borderline vinculin binding VBS peptides and also two non-binding peptides 5 and 21, which appear to conform to the VBS consensus. All of the strong binders conform to the rules except peptides 11 and 12 (VBS2). From the weak binders, only peptides 36 and 60 have no rule violations. B, conversion of VBS2 (peptide 12) into a strong vinculin binder by substituting residues from VBS1 (peptide 4). Note the negative influence of Thr and Met in positions 12 and 15.

 
Examination of the vertical lines in the matrix (Fig. 4) illustrates the overall VBS1 side-chain preferences for Vd1 binding. The least preferred is proline, which gives a non-binding peptide when present at twelve of the 16 positions, with only a very weak spot present for the remaining four (exposed) positions. A second class of unfavored side chains includes Gly, Asp, and Asn, which again only give reasonable spot intensities in four to seven of the exposed positions. It is interesting to note that in all positions except position 8 a Phe residue clearly allows binding to vinculin, yet in the talin VBS peptides (Fig. 3), there is only one example of a Phe residue (position 1 in peptide 33). For reasons of steric hindrance, Trp is only allowed at 7 exposed positions (2, 3, 6, 7, 10, 13, and 14) and does not appear at any position within the central region of the 11 talin VBS peptides, with only one occurrence in peptide 36 at the N terminus. By contrast Leu, Ile, and Val are the most highly favored side chains and can occupy any position within a VBS.

Comparison of the Two SPOT-Peptide Array Datasets—Comparison of the datasets reveals remarkably good overall agreement. Although there is little sequence identity across the 11 talin peptides, they all fulfill the position-specific side-chain constraints defined in the VBS1 substitution analysis with the exception of peptides 11 and 12. Fig. 5A shows the sequences of the vinculin binding peptides VBS1 (peptide 4), peptide 11, and peptide 12 aligned with those of the weakly binding peptides and two of the non-binding peptides, 5 and 21. The buried positions are outlined in gray, with residues from the whole talin analysis (Figs. 1 and 3) that do not conform to the VBS1 substitution matrix (Fig. 4) highlighted in white. In this alignment, the non-binding peptides 5 and 21 both have a single "rule violation" at position 12, which is a Ser in peptide 5 and an Ala in peptide 21, in contrast to the preferred Phe, Ile, Leu, Thr, and Val residues (Fig. 4). This is also the case for the weakly binding peptides 20, 25, 26, and 40, which all have a similar substitution of Ser or Ala at position 12. The weakly binding peptides 25, 51, 55, and 57 have "disallowed" side chains at one or several of the key buried positions, notably at position 8, the most restrictive position. Thus, despite showing a weak binding in the SPOT-peptide analysis (Fig. 1), none of these peptides are likely to be vinculin binders. On the other hand, peptides 36 and 60 show only weak binding in Fig. 1, but the data in Fig. 4 does not allow one to identify a clear-cut reason for their weak binding. In the subsequent section we describe a crystal structure of the peptide 36·Vd1 complex showing that this peptide does bind to vinculin in a manner reminiscent of VBS1, and peptide 60 may also be a VBS.

Both the vinculin-binding peptides 11 and 12 have Met at positions (1 and 12) where it would be predicted to abolish binding in VBS1. Although Met is a hydrophobic side chain, its substitution into seven of the eight restricted VBS1 positions abolishes binding. In the case of peptide 11, a crystal structure of the complex, described in the following section, reveals that this peptide induces a subtly different conformation within the Vd1 domain to accommodate the extra bulk of the Met side chain. In the case of peptide 12 (VBS2), examination of the VBS2·Vd1 structure reveals that at the adjacent position 2 the less bulky Val in VBS2 compared with Leu at this position in VBS1 (23) creates more space for the Met; in addition, VBS2 binds vinculin significantly less tightly than VBS1 (21). To understand why VBS2 binds less well, we used the SPOT-peptide approach to examine the effects of replacing the buried residues in VBS2 with the corresponding residues from VBS1 (Fig. 5B). This experiment showed that indeed substituting Met at position 15 by a Leu led to a strong increase in binding, giving a spot intensity comparable to that for wild-type VBS1.



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  		FIGURE 6.
Crystal structures of talin VBS peptides 11, 36, and 58 bound to vinculin Vd1. Topology diagrams for the complex crystal structures of the vinculin Vd1 domain (N-terminal helices {alpha}1-{alpha}4 shown in yellow) and the talin VBS peptides 11, 36, and 58 (shown in purple). Hydrogen bonding and water mediated interactions are illustrated as dotted red lines. A, the VBS peptide 11·Vd1 complex shows that three basic side chains make contact with the Vd1 domain; (i) on turn 1 an Arg packs against and forms an electrostatic interaction with {alpha}2, (ii) on turn 2 an Arg forms a hydrogen bond to Ser-10 from Vd1 helix {alpha}1 (a similar interaction is observed for peptide 36, VBS1, and VBS2), and (iii) on turn 6, a Lys side-chain hydrogen bonds to a main chain carbonyl and a His side chain from {alpha}1. B, peptide 36·Vd1 complex structure. C, peptide 58·Vd1 complex has basic Lys residues on turn 2 and 5 similar to VBS1. In this case no direct interaction with the Vd1 domain helix {alpha}1 is observed for the Lys on turn 2 as this is affected by a cis interaction with a Glu on turn 1. Conformational differences between the peptide 11·Vd1 structure and the VBS1-like structures of peptides 36 and 58 can be observed as changes in the relative positions of helices {alpha}1 and {alpha}2 and the Phe-125 side chain shown in gray. D, charged molecular surface representation of the peptide 11·Vd1 structure showing the overall negative charge (red) of the vinculin Vd1 helix {alpha}1. The peptide 11 helix is shown in green.

 
This detailed analysis thus resolves essentially all the apparent discrepancies between the two approaches to defining the specificity of vinculin binding to talin peptides. Combining the consistently preferred side chains from the two SPOT-peptide datasets allows one to define the optimal consensus sequence for vinculin binding (the optimal VBS) as LXXAAXXVAXXVXXLIXXA.

Structures of Complexes of Vd1 with Peptides 11, 36, and 58—We have co-crystallized the VBS peptides 11, 36, and 58 with the recombinant vinculin Vd1 domain, determining the structures to 1.8, 3.2, and 2.1 Å resolution, respectively. The overall organization of the new VBS·Vd1 structures is topologically identical to that of VBS1·Vd1 (22) and consists of two subdomains. At the N terminus four helices ({alpha}1-{alpha}4) from Vd1 surround residues from the talin VBS. This sub-domain is connected to the second Vd1 sub-domain, comprising the C-terminal four-helix bundle, through an elongated helix {alpha}4. The structures of the N-terminal Vd1 sub-domain complexed with peptides 11, 36, and 58 are illustrated in Fig. 6.

Peptide 11 has a Met side chain at position 1 and an unfavorable Gln residue at position 4. When the structure of the peptide 11·Vd1 complex is superposed onto that of VBS1·Vd1, it can be seen that to accommodate these two bulkier side chains Vd1 has changed its conformation by a 1.5-Å shift of Vd1 helix {alpha}2 away from the VBS helix. The VBS Met packs against the Vd1 Gly-58 peptide bond and the VBS Gln packs against the Vd1 Val-57 side chain. Furthermore, the side chain of Vd1 Phe-125 alters its conformation about {chi}1 by 180° to pack against the VBS Met (Fig. 6A).

Peptide 36 binds vinculin only weakly under the conditions of the SPOT-peptide analysis (Fig. 1) but does not have any "violations" of the consensus sequence described by the VBS1 substitution analysis. The crystal structure of its complex with Vd1 shows that it binds in an almost identical fashion to VBS1 (Fig. 6B). One possible reason for its weak binding is that in contrast to the strong binding peptides, it has a Met at position 19. This far end of the VBS has yet to be subjected to a full substitution analysis, but the corresponding position is occupied by Ala in VBS1, and by similar small uncharged side chains in the other 10 VBS peptides. This is likely to represent a further instance of Met side chains resulting in a negative effect on VBS binding to vinculin. The Met is accommodated in the peptide 36·Vd1 complex by simple rotations of side chains and a small uplift of the end of the peptide 36 helix. The peptide 58·Vd1 structure superposes very well with VBS1·Vd1, and all the buried hydrophobic side chains form essentially identical interactions with Vd1 (Fig. 6C).

Examination of these structures and comparison with previous crystal structures of VBS complexes illustrates how the positions of charged residues on the exposed face of the VBS helix can affect vinculin binding. The most important of these are the basic residues Arg and Lys. All the eight available VBS·Vd1 complex crystal structures demonstrate either one or two Arg or Lys side chains from the peptide forming an interaction with Vd1, primarily with helix {alpha}1. The structure of the peptide 11·Vd1 complex shown in Fig. 6A illustrates this with an Arg at position 6 and Lys at position 20 making hydrogen bonding contacts to main chain and side-chain groups on helix {alpha}1 of the Vd1 domain. By contrast, the negatively charged Glu and Asp side chains do not form any interactions with Vd1 in the available VBS·Vd1 crystal structures. These side chains tend to be found at positions 3, 7, 10, and 14 toward the center and N-terminal part of the VBS helix, placing them further away from the negatively charged Vd1 helix {alpha}1, to interact with other VBS side chains (Fig. 6D). Examples are shown in Fig. 6, where an Asp on turn 4 of peptide 11 and a Glu on turn 1 of peptide 58 each interact with other side chains of the peptide. These observations correlate well with both SPOT-peptide datasets which show a preference for acidic residues at positions 3, 7, 10, and 14.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Talin is a large, ubiquitously expressed multifunctional adaptor protein that couples the integrin family of cell adhesion molecules to cytoskeletal and signaling proteins (35-38). It is therefore not surprising that knock-out of the talin1 isoform compromises the assembly of cell-extracellular matrix junctions (focal adhesions) in cells in culture (39), as well as embryonic development in worms (40), flies (41), and mice (42). One of the best characterized talin-binding proteins is vinculin (1), which is recruited to focal adhesions in response to applied force (43) and may serve to stabilize the initial weak linkage between integrins, talin, and F-actin (44). We have previously identified three vinculin binding sites (VBS1-3) in the talin rod using a series of overlapping recombinant talin polypeptides and a vinculin SDS-gel blot assay (19, 45), and these were further localized to ~25 residue peptide sequences using a yeast two-hybrid assay (21). In the present study, we have used synthetic peptides representing each of the 63 predicted {alpha}-helices in the talin rod and identified an additional 8 VBS sequences giving a total of 11. The new VBSs were missed in the original study probably due to the fact VBSs tend to be clustered within the talin rod, and so deletion of one VBS would not necessarily abolish vinculin binding.



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  		FIGURE 7.
Native structural context of VBS sequences. Structures of isolated domains from the talin rod are illustrated as ribbon diagrams with buried side chains from the VBS consensus highlighted. A, crystal structure of talin 482-789 (pdb 1SJ8) with peptides 4, 6, and 9 illustrated. B, NMR structure of talin 755-889 (pdb 1U89) with peptides 9, 11, and 12 illustrated. C, homology model of talin residues 2295-2481 with peptide 58 illustrated. Talin 2295-2481 is modeled using the crystal structure determined for the homologous Huntingdon Interacting Protein 1-related protein (hip1r) C-terminal domain (pdb 1R0D). In all cases the VBS peptides lie within {alpha}-helical parts of the structure with the hydrophobic faces buried by interactions with other helices contributing to the hydrophobic core. Peptide 9 (765-789) at the C terminus of the 482-789 crystal structure appears to be exposed but is buried in the overlapping NMR structure of 755-889. Collectively this region has been predicted to form a 7-helix bundle fold spanning residues 655-889 (pdb 1XWX) and containing VBS peptides 6, 9, 11, and 12.

 
Based on VBS sequence alignments, analysis of vinculin binding to substituted VBS1 peptides, and the data from VBS·Vd1 complex crystal structures, we have been able to define the specificity of the talin-vinculin interaction. Binding is driven by position-specific contacts made predominantly by hydrophobic side chains leucine, isoleucine, valine, and alanine lying on one face of an amphipathic {alpha}-helix spanning six turns, with the consensus LXXAAXXVAXXVXXLIXXA. The VBS sequence is reminiscent of helical motifs such as leucine zippers from DNA-binding proteins (46) and LXXLL motifs in transcription factors (47), although a distinctive feature of the VBS is the absence of any individual absolutely essential side chain, which makes it difficult to identify a VBS based on sequence alone. Nevertheless the rules derived from the VBS1 peptide substitution analysis have considerable predictive power in this respect. Across the 63 peptides and using sequence alone, we can successfully discriminate between vinculin binders and non-binders with the sole exceptions of peptides 11 and 12. These would both be predicted to be non-binders, because they include Met in positions predominantly occupied by Leu in the other talin VBS. The crystal structures reveal that Met is accommodated by the vinculin Vd1 domain either as a consequence of changes in other side chains in the vicinity or as a result of changes in Vd1 conformation.

A further distinctive feature of the talin rod sequence is the common occurrence of Ala. Runs of Ala side chains are observed in the VBS peptides 6, 9, 27, and 58 and in many of the non-binding peptides. Because Ala is not accepted at five of the buried VBS positions, the common occurrence of the Ala side chain abrogates vinculin binding to many talin helices that might otherwise be VBS. The role of alanine in talin rod function is unclear. It may play a structural role, because Ala is established as a helix-stabilizing residue (48) and motifs such as AXXXA are characterized as mediating close packing of {alpha}-helices (49, 50).

Several of the new VBS peptides lie within regions of talin for which structural information is already available. These structures are illustrated in Fig. 7 and include peptides 4 (VBS1), 6, and 9, which are described by the crystal structure of N-terminal talin residues 482-789 (22), and peptides 9, 11, and 12, which are described by the structure of residues 755-889 determined by NMR (23). Peptide 58 can be modeled using the crystal structure determined for the homologous C-terminal domain of the Huntingtin Interacting Protein 1-related protein.5 In all these cases, the hydrophobic faces of the VBS peptides are buried by interactions with other helices. However, it is clear that one or more of the three VBS in the vicinity of residues 755-889 (equivalent to peptides 9, 11, and 12) in talin purified from tissue do bind vinculin.6 Experiments with recombinant talin 755-889 showed that this four-helix bundle is capable of binding more than one Vd1 molecule simultaneously, and NMR data reveal that this leads to unraveling of the bundle, with the single non-VBS helix unfolding completely. It remains to be determined whether a four-helix bundle with a single VBS would constitutively bind vinculin. The common four-helix bundle topology does have inherent flexibility, particularly for the terminal helices. This is seen, for example, in the focal adhesion kinase FAT domain crystal structure where the N-terminal helix is "domain swapped," contributing to the hydrophobic core of the adjacent four-helix bundle in the crystal (51). We have recently demonstrated a clear negative correlation between the stability of isolated talin helical bundles and their ability to bind vinculin (22). However, quaternary contacts in the full-length talin molecule may also further restrict the availability of VBS.

Activation of the cryptic VBS could potentially greatly increase the number of vinculin molecules bound simultaneously to talin. Because the vinculin tail binds F-actin (34, 52), a progressive increase in vinculin binding to talin could provide a mechanism for a graduated strengthening of the link between integrins and the actin cytoskeleton. How cryptic VBS in talin might be activated remains to be explored, although phosphorylation or the effects of mechanical force on the structure of the talin rod are attractive possibilities.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1ZW2 and 1ZW3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was funded by grants from Biotechnology and Biological Sciences and Research Council, the Wellcome Trust, and the German Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 44-1-158-467-092; Fax: 44-1-158-468-002; E-mail: jonas.emsley{at}nottingham.ac.uk.

3 The abbreviations used are: Vd1, vinculin domain 1; VBS, vinculin binding site; Vt, vinculin tail domain; VASP, vasodilator-stimulated phosphoprotein; FERM, protein 4.1/ezrin/radixin/moesin family; GST, glutathione S-transferase; MME, monomethylether; CAPS, 3-cyclohexylamino-1-propanesulfonic acid; Hip1r, Huntingdon Interacting Protein 1-related protein; SPOT-peptide, spot-synthesized peptide; FAK, focal adhesion kinase. Back

4 N. Brown and L. Lai (1996) CONSENSUS, available at www.bork.embl-heidelberg.de/cgi/consensus. Back

5 T. J. Brett, V. Legendre-Guillemin, P. S. McPherson, and D. H. Fremont, unpublished data. Back

6 B. C. Patel, A. Gingras, A. A. Bobkov, L. M. Fujimoto, D. Mazzeo, J. Emsley, G. C. K. Roberts, I. L. Barsukov, and D. R. Critchley, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We are grateful to Werner Tegge and Susanne Daenicke (German Research Centre for Biotechnology, Braunschweig) for peptide synthesis and Eva Saxinger (Technical University of Braunschweig) for technical help.



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D. M. Cohen, B. Kutscher, H. Chen, D. B. Murphy, and S. W. Craig
A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions
J. Biol. Chem., June 9, 2006; 281(23): 16006 - 16015.
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B. Patel, A. R. Gingras, A. A. Bobkov, L. M. Fujimoto, M. Zhang, R. C. Liddington, D. Mazzeo, J. Emsley, G. C. K. Roberts, I. L. Barsukov, et al.
The Activity of the Vinculin Binding Sites in Talin Is Influenced by the Stability of the Helical Bundles That Make Up The Talin Rod
J. Biol. Chem., March 17, 2006; 281(11): 7458 - 7467.
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P. R. J. Bois, B. P. O'Hara, D. Nietlispach, J. Kirkpatrick, and T. Izard
The Vinculin Binding Sites of Talin and {alpha}-Actinin Are Sufficient to Activate Vinculin
J. Biol. Chem., March 17, 2006; 281(11): 7228 - 7236.
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