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Protein Sci. Jun 2007; 16(6): 1223–1229.
PMCID: PMC2206669

Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions

Abstract

Tensin is a cytoskeletal protein that links integrins to the actin cytoskeleton at sites of cell-matrix adhesion. Here we describe the crystal structure of the phosphotyrosine-binding (PTB) domain of tensin1, and show that it binds integrins in an NPxY-dependent fashion. Alanine mutagenesis of both the PTB domain and integrin tails supports a model of integrin binding similar to that of the PTB-like domain of talin. However, we also show that phosphorylation of the NPxY tyrosine, which disrupts talin binding, has a negligible effect on tensin binding. This suggests that tyrosine phosphorylation of integrin, which occurs during the maturation of focal adhesions, could act as a switch to promote the migration of tensin–integrin complexes into fibronectin-mediated fibrillar adhesions.

Keywords: structure/function studies, crystallography, calorimetry, mutagenesis (site-directed and general), docking proteins, surface plasmon resonance

Cells bind to the extracellular matrix (ECM) by means of the integrin family of plasma membrane receptors (Hynes 2002). Integrins make direct bonds to ECM proteins and indirect bonds to the actin cytoskeleton via a number of cytoskeletal proteins, including tensin, talin, vinculin, and paxillin (Zamir et al. 1999). These complexes provide a scaffold for signaling enzymes that regulate many aspects of cellular behavior. Geiger (Zaidel-Bar et al. 2003) has described three stages in the development of cell-ECM contacts: “focal contacts” are short-lived structures containing integrin αVβ3, which transform into classical “focal adhesions” upon activation of RhoA, and contain both integrins αVβ3 and α5β1; a third type of contact, “fibrillar adhesions,” is formed when integrin α5β1 (the fibronectin receptor) translocates from focal adhesions (Pankov et al. 2000).

The cytoskeletal protein tensin is absent from focal contacts but is recruited to focal adhesions and plays a key role in the establishment of fibrillar adhesions (Zaidel-Bar et al. 2003). Tensin contains binding sites for actin in the N terminus that crosslink actin filaments, and a central domain that caps the barbed ends of F-actin (Lo et al. 1994; Chuang et al. 1995). The C-terminal region contains a Src homology 2 (SH2) domain that mediates binding to tyrosine-phosphorylated proteins, such as phosphoinositide 3-kinase, Cas, and focal adhesion kinase (Auger et al. 1996), and a PTB domain that is required for focal adhesion targeting (Chen and Lo 2003). Three isoforms of tensin have been identified—tensin1, tensin2, and tensin3—which are homologous in their N- and C-terminal domains but divergent in their central regions (Chen et al. 2002; Cui et al. 2004). In addition, a fourth family member, cten, lacks the N-terminal actin-binding domain (Lo and Lo 2002).

The factors governing the dynamics and maturation of cell-ECM contacts are poorly understood. Once activated, talin plays a key role in the early stages, by engaging integrin β-subunit cytoplasmic tails (Garcia-Alvarez et al. 2003; Wegener et al. 2007). This interaction disrupts αβ tail association and promotes a conformational change in the extracellular domains of integrins that leads to enhanced affinity for ECM proteins (Vinogradova et al. 2002; Kim et al. 2003; Tadokoro et al. 2003; Xiao et al. 2004), a process termed “inside-out” signaling. A second major player is the tyrosine kinase, Src (or a close relative), which binds constitutively to the C terminus of integrin β-tails, and is activated by intermolecular phosphorylation when integrins cluster at cell-ECM adhesion sites (Arias-Salgado et al. 2003), leading to a variety of downstream events termed “outside-in” signaling. The activity of Src also disrupts the linkage between talin and integrin via tyrosine phosphorylation of the integrin NPxY motif (Tapley et al. 1989), and promotes the binding of talin to the enzyme, phosphatidylinositol phosphate kinase type 1-γ (PIPKIγ), a key activator of proteins involved in focal adhesion assembly, including talin (Ling et al. 2003).

We present here the crystal structure of the tensin1 PTB domain, together with in vitro binding studies and mutagenesis showing that tensin1 binds to integrins in a talin-like but phosphorylation-independent fashion, and we propose a model in which the migration of tensin from focal adhesions to form fibrillar adhesions is promoted by a switch in integrin-binding preferences upon Src phosphorylation.

Results and Discussion

Structure of the tensin1 PTB domain

Crystallization trials with residues 1605–1744 of chicken tensin1 yielded crystals that diffracted to low resolution. Limited proteolysis identified a fragment, residues 1605–1738, which produced high-quality crystals, and its structure was determined at 1.5 Å resolution using multiple anomalous diffraction (MAD) from a selenomethionine derivative (Table 1). The tensin1 PTB domain adopts a mostly canonical PTB fold (Fig. 1). It is most similar to the PTB domain of X11: thus, they both have an inserted β1′ strand and α1 helix between strands β1 and β2, and the central β sandwiches overlap closely (RMSD = 0.93 Å for Cα atoms). In common with other PTB domains, tensin1 lacks the loop found in talin between strands β1 and β2 that is critical for membrane association and integrin activation (Wegener et al. 2007). A unique feature of the tensin1 domain is an insertion of 14 residues between the β5 and β6 strands, which forms one side of a pocket that we propose binds to a tryptophan residue from the integrin tail (see below). The loops connecting strands β3-β4, β5-β6, and β6-β7 are partially disordered (the last is implicated in phosphotyrosine binding; see below) with relatively high B values, indicating that these are flexible regions within the domain.

Table 1.
Data collection and refinement statistics
Figure 1.
Structure of the tensin PTB domain. (A) Stereo Cα plot of the tensin1 PTB domain with every 10th residue, and N and C termini, labeled. The unstructured β6-β7 loop is shown schematically in green. (B) Ribbon diagram of the tensin1 ...

The tensin1 PTB domain binds integrin cytoplasmic tails

PTB domains characteristically recognize ligands bearing an NPxY motif, and show specificity for residues that lie upstream of this motif. Tensin1 colocalizes predominantly with ECM-bound α5β1 integrins in fibrillar adhesions, as well as with focal adhesions containing αVβ3 integrin (Katz et al. 2000). Integrin β subunit cytoplasmic tails contain two NPxY motifs, and tensin2 has previously been shown to bind integrin β-tails in vitro (Calderwood et al. 2003). Using surface plasmon resonance (SPR), we investigated the binding of the tensin1 PTB domain to peptides derived from the β1A and β3 integrin tails (Table 2; Supplemental Fig. 1). We used the longer domain containing the authentic C terminus (residues 1605–1744), which we found to bind integrin ~10-fold more strongly than the shorter domain used for crystallization. We found that tensin1 has a clear preference for the first, membrane-proximal, NPxY motif (over the membrane-distal motif) for both the β1A and β3 tails (residues 779–790 and 738–749, respectively). The affinities, in the low micromolar range, are similar for the β1A and β3 tails, and are comparable with other PTB-peptide interactions, such as Numb-Nak (Zwahlen et al. 2000), IRS-1-IL4 (Zhou et al. 1996), SNT-FGFR1 (Dhalluin et al. 2000), Dok1-RET (Shi et al. 2004), and Dab1-ApoER2 (Stolt et al. 2003). Binding is, however, significantly weaker than the value of Kd = 100 nM estimated for talin-integrin binding (Calderwood et al. 1999).

Table 2.
Binding affinities of β1 and β3 integrin peptides for the tensin1 PTB domain

The NPxY motif typically adopts a reverse β-turn that is critical for binding PTB domains. It was previously shown that mutating the NPxY tyrosine to alanine eliminated binding of integrin to talin (Calderwood et al. 2002), and we found the same result with tensin1 (Table 2). Some PTB domains, such as Shc and IRS-1 (Zhou et al. 1995a; Eck et al. 1996), require phosphorylation of the NPxY tyrosine for strong binding, while others, notably talin, are inhibited by phosphorylation (Tapley et al. 1989). A third group, including X11, Numb, and Dab1 (Zhang et al. 1997; Zwahlen et al. 2000; Stolt et al. 2003), is indifferent to the state of phosphorylation. We found that the tensin1 PTB domain falls into the third group: thus, it binds to unphosphorylated and phosphorylated β1 and β3 integrin tails with nearly identical affinity (Table 2). PTB domains that bind phosphotyrosine typically possess a pocket lined with basic residues that coordinate the phosphate moiety. A structure-based alignment of PTB domains (Fig. 1C) shows that two basic residues within the β6-β7 loop of tensin1, Arg1704 and Lys1705 (conserved in tensin2 and tensin3), are in similar positions to the phosphotyrosine-coordinating residues in the Shc, IRS-1 and Dok1, and X11 PTB domains. The β6-β7 loop is disordered in the tensin1 PTB crystal structure, indicating that it is mobile, which may contribute to the ability of the domain to accommodate either phosphorylated or unphosphorylated tyrosine. The analogous loop in talin is acidic (Garcia-Alvarez et al. 2003), consistent with disruption of integrin binding by tyrosine phosphorylation.

PTB domains display ligand-binding specificities for hydrophobic residues upstream of the NPxY motif. For example, Shc, Numb, and Dab1 PTB domains require a hydrophobic residue at position –5 with respect to the tyrosine (Zhou et al. 1995a; Zwahlen et al. 2000; Stolt et al. 2003), and the IRS-1 PTB domain favors hydrophobic residues at position –6 and –8 (Eck et al. 1996). Trp at position −8 in the β3 integrin tail has been shown to form a critical interaction with the talin PTB domain (Garcia-Alvarez et al. 2003), and a model of the tensin1–integrin interaction (see below) pointed to the existence of an appropriate pocket for the tryptophan. We therefore mutated this Trp to Ala in both the β1 and β3 tails, and found in both cases a substantial (30- to 50-fold) loss of binding to the tensin1 PTB domain (Table 2), supporting an important role for Trp (−8) in the interaction.

Model of tensin1 PTB domain binding integrin tail

Based on our crystal structure and peptide binding studies, we built a model of the tensin1–integrin complex (Figs. 1B, ,2A).2A). As templates we used two crystal structures: the talin–β3 integrin complex (Protein Data Bank [PDB] code 1MK9) (Fig. 2B) and, owing to the structural similarity of their PTB domains, the X11–βAPP complex (PDB code 1X11). We built the β3-integrin NPIY motif with a typical β-turn conformation, placing the Tyr (+0) in a pocket adjacent to the basic β6-β7 loop. This enables the NPxY Asn, whose side-chain stabilizes the integrin β-turn, to form H-bonds with the main-chain nitrogens of two residues in the β4-β5 loop (Leu1672 and Val1675), while the aromatic ring of Tyr (+0) interacts with Thr1676. To test this model, we mutated Thr1676 to alanine; SPR measurements (at a single protein concentration) indicated a substantial loss of binding to the β1A tail (Supplemental Fig. 2). As noted above, the C-terminal residues of tensin (1739–1744) enhance integrin binding; it is likely that these residues become ordered in the presence of the integrin tail and create a pocket for the NPIY Ile, as has been observed with the PTB domains of IRS-1 and Shc (Eck et al. 1996; Farooq et al. 2003).

Figure 2.
PTB-integrin models. Surface representations of the tensin1-integrin model (A) and the talin-integrin crystal structure (B) (Garcia-Alvarez et al. 2003), colored by electrostatic potential (blue for positive, red for negative). The integrin peptides are ...

We next built the three residues upstream of the NPIY motif such that they make canonical main-chain H-bonds to augment the central β-sheet. The side-chain of tensin1 Asp1679 disrupts further upstream β-sheet interactions, although in the other tensin homologs, Asp1679 is replaced by smaller residues (Ser and Gly) that may be less disruptive. Moving further upstream, a hydrophobic pocket is evident at an appropriate location to accept the bulky side-chain of Trp (−8). The pocket is formed by residues from the inserted helix, α1 (Pro 1624 and Ile1627), from the end of the β5-strand (Phe1677 and Asp1679), and by Pro1682 from the beginning of the loop, β5-β6, that is unique to tensin. To test the role of this pocket, we made alanine mutations to three residues lining the pocket (Pro1624, Pro1682, and Phe1677). Consistent with our model, all mutations significantly reduced integrin binding as judged by SPR (Supplemental Fig. 2). However, in the case of F1682A, although the mutant behaved normally during purification, its melting profile measured by differential scanning calorimetry (DSC) indicated a substantial reduction in stability, which may contribute to the observed reduction in peptide binding (Supplemental Fig. 3). The pocket is invariant (except for Asp1679) in tensin homologs, as is most of the integrin-binding surface, suggesting that all three tensin isoforms will bind integrin. Unlike tensin1, the tensin2 PTB domain has a preference for β3 over β1A (Calderwood et al. 2003). Although we cannot immediately explain the structural basis for the different integrin preferences, we note they are consistent with the distinct cellular localizations of the two proteins: thus, tensin2 localizes to fibrillar adhesions (which predominantly contain integrin α5β1 rather than αvβ3) less efficiently than does tensin1 (Chen et al. 2002). Finally, we note that the distance between the NPxY binding pocket and the tryptophan binding pocket in our tensin–integrin model requires that the intervening residues are in a fully extended conformation (Fig. 2A). In the case of talin, the two pockets are closer together, and the intervening residues bulge out from the side of the PTB domain; i.e., there is slack in the system (Fig. 2B). This would explain why the β2 integrin tail, whose intervening sequence is one residue shorter than other integrin tails, can bind to talin but not to tensin (Calderwood et al. 2003).

Implications for cell-matrix dynamics

Our analysis reveals similar but distinct structural determinants for the binding of integrin to tensin vis-à-vis talin. The relatively low binding affinity of tensin1 for integrin (Kd~μM) is consistent with the finding that the PTB domain is necessary but not sufficient for focal adhesion targeting (Chen and Lo 2003); that is, high-affinity attachment to cell adhesion sites presumably requires the combinatorial binding of distinct elements of the adhesion site to other domains within the full-length protein. Although we have shown that tensin and talin bind to the same site on the integrin, direct competition is initially unlikely since talin binds with much higher affinity in the absence of phosphorylation (Calderwood et al. 1999). However, upon phosphorylation of the NPxY tyrosine, while the affinity for talin is greatly reduced, we showed that the affinity for tensin is unaffected; that is, we predict that the relative affinities will be reversed. As noted above, during the maturation of focal contacts, integrin tails are phosphorylated by Src, which should trigger the release of talin from integrins (Tapley et al. 1989; Ling et al. 2003). This would make integrin available for tensin binding and thus potentially facilitate the translocation of tensin-bound α5β1 integrin to fibrillar adhesions. In support of this model, we note that in Src-null cells, which have low levels of phosphorylation at focal adhesions, tensin fails to segregate into fibrillar adhesions (Volberg et al. 2001).

Materials and Methods

Protein expression and purification

DNA sequences corresponding to residues 1605–1744 and 1605–1738 of chicken tensin1 were amplified by PCR and cloned into pET-15b (Novagen) vectors and confirmed by di-deoxy sequencing. Protein was expressed in the Escherichia coli strain BL21 (DE3). Following induction of protein expression with 0.5 mM IPTG for 3 h at 37°C, cell harvest, and lysis by sonication, the protein was purified using a Ni-affinity column (Pharmacia). The His tag was cleaved with thrombin and removed by dialysis against 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT. The purified proteins contain four additional residues (Gly-Ser-His-Met) from the vector at the N terminus. Selenomethionine (SeMet)-labeled protein (residues 1605–1738) was expressed and purified as for the native protein. Single amino acid mutations were generated in the PTB domain (residues 1605–1744) using the QuikChange site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing. All mutants were expressed and purified as for the wild type. To test the effects of the mutations on protein folding, melting profiles proteins were determined using DSC (Supplemental Fig. 3). All peptides were synthesized by United Biochemical Research.

Crystallography

Tensin1 PTB domain (residues 1605–1738) was concentrated to 15 mg/mL in an ultrafiltration cell (Amicon) prior to crystallization. Crystals grew at 4°C by sitting-drop vapor diffusion, using 2 μL protein and 2 μL precipitant (40% PEG 600 and 0.1 M CHES at pH 9.5), and belong to space group I4 with unit cell dimensions a = 75.7 Å, c = 48.7 Å. The asymmetric unit contains one PTB domain and 48% solvent. Diffraction data were collected at 100 K using flash-cooled crystals stabilized in 42% PEG 600 and 0.1 M CHES (pH 9.5). Data from a single native crystal and multiple wavelength anomalous diffraction (MAD) data at three wavelengths near the Se absorption edge from a SeMet-substituted crystal were collected at the SSRL beamline 9-2 (Table 1). All data were processed with DENZO and SCALEPACK (Otwinowski and Minor 1997). The scaled data were input into SOLVE (Terwilliger and Berendzen 1999), and two selenium atoms were located in the asymmetric unit. Phases (calculated to 1.8 Å resolution using SOLVE) were refined by solvent flattening with RESOLVE (Terwilliger and Berendzen 1999). An atomic model (~75% complete) was built with RESOLVE and completed manually using TURBO-FRODO (Roussel and Cambillau 1991). The model was refined with CNS (Brünger et al. 1998) to 1.5 Å resolution (Table 1). After initial simulated annealing, the model was further refined with iterative cycles of manual fitting, conjugate gradient minimization, and isotropic B factor refinement. Further refinement included addition of 107 solvent molecules. The final model converged to an RWORK of 0.21 and RFREE value of 0.23 (calculated with 5% of reflections omitted from refinement). Poor electron density was observed for Gly1605 at the N terminus, residues Arg1662–Phe1666 in the β3-β4 loop, Lys1689–G1694 in the β5-β6 loop, and Arg1704–Thr1710 in the β6-β7 loop. The final model was evaluated with PROCHECK (Laskowski et al. 1993): 88.3% of the main-chain torsion angles (non-glycine) lie in the most favored regions of the Ramachandran plot, with none in disallowed regions. The coordinates and structure factors have been deposited in the PDB with accession code 1WVH. Coordinates of the tensin–integrin model are available from the authors.

Peptide binding studies

SPR studies were carried out on a Biacore 3000 Biosensor (Biacore AB). Peptides were immobilized on a CM5 sensor chip (Biacore) via the amine coupling method using N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linking, as directed by the manufacturer. A blank flow cell that had been EDC/NHS-activated and ethanolamine-blocked was used as a reference. Tensin1 PTB domain was dialyzed extensively into running buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM DTT, and 0.005% polysorbate 20. Steady-state experiments were performed by injecting the protein over the chip at 10 μL/min for 4 min. The chip surface was regenerated with 1.0 M NaCl. Typically, sensorgrams were obtained at six different concentrations for each experiment, and each set of titrations included a running buffer injection as an additional blank (Supplemental Fig. 1). All data were analyzed using BIAevaluation 3.0, subtracting binding to the blank flow cell and the blank buffer injection to account for any nonspecific binding. For each sensorgram, the peak response levels achieved at steady-state were plotted against analyte concentration. This plot was fitted to a single site binding equation (Langmuir isotherm) to determine dissociation constant (KD) values. All data are the average of at least three independent experiments. Tensin1 mutant binding experiments (Supplemental Fig. 2) were carried out using immobilized integrin β1A peptide (residues 779–790) at a single fixed protein concentration (10 μM) and thus give only a qualitative measure of binding affinity.

Acknowledgments

We thank the NIH Cell Migration Consortium (to R.C.L.) and the NIH GM22289 (S.L.) for financial support, and the beamline support team at the SSRL, a national synchrotron facility supported by the NIH and DOE.

Footnotes

Supplemental material: see www.proteinscience.org

Reprint requests to: Robert C. Liddington, Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: gro.mahnrub@gniddilr; fax: (858) 713-9925.

Abbreviations: PTB, phosphotyrosine-binding (domain); ECM, extracellular matrix; SH2, Src homology 2; PIPKIγ, phosphatidylinositol phosphate kinase type Iγ ; EDC, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; RMSD, root mean square deviation; SPR, surface plasmon resonance; DSC, differential scanning calorimetry.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072798707.

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