The Structure of an Interdomain Complex That Regulates Talin Activity
2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês
10.1074/jbc.m900078200
ISSN1083-351X
AutoresBenjamin T. Goult, Neil Bate, Nicholas J. Anthis, Kate L. Wegener, Alexandre R. Gingras, B. Patel, Igor Barsukov, Iain D. Campbell, Gordon C. K. Roberts, David R. Critchley,
Tópico(s)RNA Research and Splicing
ResumoTalin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane. Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane. The cytoskeletal protein talin has emerged as a key player, both in regulating the affinity of the integrin family of cell adhesion molecules for ligand (1Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (401) Google Scholar) and in coupling integrins to the actin cytoskeleton (2Critchley D.R. Gingras A.R. J. Cell Sci. 2008; 121: 1345-1347Crossref PubMed Scopus (174) Google Scholar). Thus, depletion of talin results in defects in integrin activation (3Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (999) Google Scholar), integrin signaling through focal adhesion kinase, the maintenance of cell spreading, and the assembly of focal adhesions in cultured cells (4Zhang X. Jiang G. Cai Y. Monkley S.J. Critchley D.R. Sheetz M.P. Nat. Cell Biol. 2008; Google Scholar). In the whole organism, studies on the single talin gene in worms (5Cram E.J. Clark S.G. Schwarzbauer J.E. J. Cell Sci. 2003; 116: 3871-3878Crossref PubMed Scopus (93) Google Scholar) and flies (6Tanentzapf G. Martin-Bermudo M.D. Hicks M.S. Brown N.H. J. Cell Sci. 2006; 119: 1632-1644Crossref PubMed Scopus (51) Google Scholar) show that talin is essential for a variety of integrin-mediated events that are crucial for normal embryonic development. In vertebrates, there are two talin genes, and mice carrying a talin1 null allele fail to complete gastrulation (7Monkley S.J. Zhou X.H. Kinston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fassler R. Dev. Dyn. 2000; 219: 560-574Crossref PubMed Scopus (180) Google Scholar). Tissue-specific inactivation of talin1 results in an inability to activate integrins in platelets (8Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fassler R. J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (207) Google Scholar, 9Petrich B.G. Marchese P. Ruggeri Z.M. Spiess S. Weichert R.A. Ye F. Tiedt R. Skoda R.C. Monkley S.J. Critchley D.R. Ginsberg M.H. J. Exp. Med. 2007; 204: 3103-3111Crossref PubMed Scopus (233) Google Scholar), defects in the membrane-cytoskeletal interface in megakaryocytes (10Wang Y. Litvinov R.I. Chen X. Bach T.L. Lian L. Petrich B.G. Monkley S.J. Critchley D.R. Sasaki T. Birnbaum M.J. Weisel J.W. Hartwig J. Abrams C.S. J. Clin. Investig. 2008; 118: 812-819PubMed Google Scholar), and disruption of the myotendinous junction in skeletal muscle (11Conti F.J. Felder A. Monkley S. Schwander M. Wood M.R. Lieber R. Critchley D. Muller U. Development (Camb.). 2008; 135: 2043-2053Crossref PubMed Scopus (42) Google Scholar). In contrast, mice homozygous for a talin2 gene trap allele have no phenotype, although the allele may be hypomorphic (12Chen N.T. Lo S.H. Biochem. Biophys. Res. Commun. 2005; 337: 670-676Crossref PubMed Scopus (20) Google Scholar). Recent structural studies have provided substantial insights into the molecular basis of talin action. Talin is composed of an N-terminal globular head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The talin head contains a FERM 2The abbreviations used are: FERM, four-point-one, ezrin, radixin, moesin; HADDOCK, high ambiguity driven biomolecular docking; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser enhancement spectroscopy; PIPKI, phosphatidylinositol-4-phosphate 5-kinase type 1. domain (made up of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0 (2Critchley D.R. Gingras A.R. J. Cell Sci. 2008; 121: 1345-1347Crossref PubMed Scopus (174) Google Scholar). Studies by Wegener et al. (30Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar) have shown how the F3 FERM subdomain, which has a phosphotyrosine binding domain fold, interacts with both the canonical NPXY motif and the membrane-proximal helical region of the cytoplasmic tails of integrin β-subunits (13Wegener K.L. Campbell I.D. Mol. Membr. Biol. 2008; 25: 376-387Crossref PubMed Scopus (100) Google Scholar). The latter interaction apparently activates the integrin by disrupting the salt bridge between the integrin α- and β-subunit tails that normally keeps integrins locked in a low affinity state. The observation that the F0 region is also important in integrin activation (14Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) may be explained by our recent finding that F0 binds, albeit with low affinity, Rap1-GTP, 3B. T. Goult, P. R. Elliott, N. Bate, B. Patel, A. R. Gingras, J. G. Grossmann, G. C. K. Roberts, D. R. Critchley, and I. L. Barsukov, manuscript in preparation. a known activator of integrins (15Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (401) Google Scholar, 16Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The talin rod is made up of a series of amphipathic α-helical bundles (17Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (135) Google Scholar, 18Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb.). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19Gingras A.R. Vogel K.P. Steinhoff H.J. Ziegler W.H. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Biochemistry. 2006; 45: 1805-1817Crossref PubMed Scopus (62) Google Scholar, 20Gingras A.R. Bate N. Goult B.T. Hazelwood L. Canestrelli I. Grossmann J.G. Liu H. Putz N.S. Roberts G.C. Volkmann N. Hanein D. Barsukov I.L. Critchley D.R. EMBO J. 2008; 27: 458-469Crossref PubMed Scopus (144) Google Scholar) and contains a second integrin binding site (IBS2) (21Gingras A.R. Ziegler W.H. Bobkov A.A. Joyce M.G. Fasci D. Himmel M. Rothemund S. Ritter A. Grossmann J.G. Patel B. Bate N. Goult B.T. Emsley J. Barsukov I.L. Roberts G.C. Liddington R.C. Ginsberg M.H. Critchley D.R. J. Biol. Chem. 2009; 284: 8866-8876Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), numerous binding sites for the cytoskeletal protein vinculin (22Gingras A.R. Ziegler W.H. Frank R. Barsukov I.L. Roberts G.C. Critchley D.R. Emsley J. J. Biol. Chem. 2005; 280: 37217-37224Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), at least two actin binding sites (23Hemmings L. Rees D.J. Ohanian V. Bolton S.J. Gilmore A.P. Patel B. Priddle H. Trevithick J.E. Hynes R.O. Critchley D.R. J. Cell Sci. 1996; 109: 2715-2726Crossref PubMed Google Scholar), and a C-terminal helix that is required for assembly of talin dimers (20Gingras A.R. Bate N. Goult B.T. Hazelwood L. Canestrelli I. Grossmann J.G. Liu H. Putz N.S. Roberts G.C. Volkmann N. Hanein D. Barsukov I.L. Critchley D.R. EMBO J. 2008; 27: 458-469Crossref PubMed Scopus (144) Google Scholar, 24Smith S.J. McCann R.O. Biochemistry. 2007; 46: 10886-10898Crossref PubMed Scopus (35) Google Scholar). Both biochemical (25Martel V. Racaud-Sultan C. Dupe S. Marie C. Paulhe F. Galmiche A. Block M.R. Albiges-Rizo C. J. Biol. Chem. 2001; 276: 21217-21227Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and cellular studies (16Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) suggest that the integrin binding sites in full-length talin are masked, and both phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in exposing these sites. It is well established that some members of the FERM domain family of proteins are regulated by a head-tail interaction (26Bretscher A. Chambers D. Nguyen R. Reczek D. Annu. Rev. Cell Dev. Biol. 2000; 16: 113-143Crossref PubMed Scopus (328) Google Scholar); gel filtration, sedimentation velocity, and electron microscopy studies all show that talin is globular in low salt buffers, although it is more elongated (∼60 nm in length) in high salt (27Molony L. McCaslin D. Abernethy J. Paschal B. Burridge K. J. Biol. Chem. 1987; 262: 7790-7795Abstract Full Text PDF PubMed Google Scholar). By contrast, the talin rod liberated from full-length talin by calpain-II cleavage is elongated in both buffers, indicating that the head is required for talin to adopt a more compact state. Direct evidence for an interaction between the talin head and rod has recently emerged from NMR studies by Goksoy et al. (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), who demonstrated binding of 15N-labeled talin F3 to a talin rod fragment spanning residues 1654–2344, an interaction that was confirmed by surface plasmon resonance (Kd = 0.57 μm) (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Chemical shift data also showed that this segment of the talin rod partially masked the binding site in F3 for the membraneproximal helix of the β3-integrin tail (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), directly implicating the talin head-rod interaction in regulating the integrin binding activity of talin. Goksoy et al. (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) subdivided the F3 binding site in this rod fragment into two sites with higher affinity (Kd ∼3.6 μm; residues 1654–1848) and lower affinity (Kd ∼78 μm; residues 1984–2344). Here, we define the rod domain boundaries and determine the NMR structure of residues 1655–1822, a five-helix bundle. We further show that this domain binds F3 predominantly via surface-exposed residues on helix 4, with an affinity similar to the high affinity site reported by Goksoy et al. (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). We also report the structure of the complex between F3 and the rod domain and show that the latter masks the known binding site in F3 for the β3-integrin tail and is expected to inhibit the association of the talin FERM domain with the membrane. Peptide Preparation—The preparation of U-15N-labeled β3-integrin tail was performed as described previously (29Oxley C.L. Anthis N.J. Lowe E.D. Vakonakis I. Campbell I.D. Wegener K.L. J. Biol. Chem. 2008; 283: 5420-5426Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Expression of Recombinant Talin Polypeptides—The cDNAs encoding murine talin1 residues 309–400 (F3) and 1655–1822 were synthesized by PCR using a mouse talin1 cDNA as template and cloned into the expression vector pet-151TOPO (Invitrogen). Talin polypeptides were expressed in Escherichia coli BL21 STAR (DE3) cultured either in LB for unlabeled protein or in M9 minimal medium for preparation of isotopically labeled samples for NMR. Recombinant His-tagged talin polypeptides were purified by nickel-affinity chromatography following standard procedures. The His tag was removed by cleavage with AcTEV protease (Invitrogen), and the proteins were further purified by anion-exchange (Domain E) or cation-exchange (F3). U-15N-labeled F3 (309–405) and F3 point mutants were made as described previously (30Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Protein concentrations were determined using their respective extinction coefficient at 280 nm. Protein concentrations were based on absorption coefficients calculated from the aromatic content according to ProtParam as follows: talin 1655–1822, 3105 m-1cm-1; talin F3, 16,960 m-1cm-1; PIPKIγ peptide, 8480 m-1cm-1; integrin peptide, 8480 m-1cm-1. NMR Spectroscopy—NMR experiments for the resonance assignment and structure determination of talin 1655–1822 were carried out with 1 mm protein in 20 mm sodium phosphate, pH 6.5, 50 mm NaCl, 2 mm dithiothreitol, 10% (v/v) 2H2O. NMR spectra of all the proteins were obtained at 298 K using Bruker AVANCE DRX 600 or AVANCE DRX 800 spectrometers both equipped with CryoProbes. Proton chemical shifts were referenced to external 2,2-dimethyl-2-silapentane-5-sulfonic acid, and 15N and 13C chemical shifts were referenced indirectly using recommended gyromagnetic ratios (31Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2104) Google Scholar). Spectra were processed with TopSpin (Bruker Corp.) and analyzed using Analysis (32Vranken W.F. Boucher W. Stevens T.J. Fogh R.H. Pajon A. Llinas M. Ulrich E.L. Markley J.L. Ionides J. Laue E.D. Proteins. 2005; 59: 687-696Crossref PubMed Scopus (2455) Google Scholar). Three-dimensional HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB experiments were used for the sequential assignment of the backbone NH, N, CO, Cα, and Cβ resonances. Side chain assignments were obtained using three-dimensional HBHA(CO)NH, HBHANH, H(C)CH-TOCSY, and (H)CCH-TOCSY experiments. Aromatic side chain assignments were obtained using 13C-resolved three-dimensional NOESY-HSQC. The resonance assignments of 1655–1822 have been deposited in the BioMagResBank with the accession number 15457. Experiments to examine the competition between talin 1655–1822 and the β3-integrin tail were performed on a spectrometer equipped with a 500-MHz Oxford Instruments superconducting magnet and GE-Omega computer. Samples were prepared in 50 mm phosphate buffer, pH 6.1, containing 100 mm NaCl, 1 mm dithiothreitol, 5% 2H2O, and Complete protease inhibitors (Roche Applied Science). Data were processed using NMRPipe (33Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11828) Google Scholar), and spectra were visualized using SPARKY. Spectra were referenced as described above (31Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2104) Google Scholar). The 1H and 15N resonances of U-15N-labeled β3-integrin tail were assigned previously (29Oxley C.L. Anthis N.J. Lowe E.D. Vakonakis I. Campbell I.D. Wegener K.L. J. Biol. Chem. 2008; 283: 5420-5426Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Structure Calculations—Distance restraints were obtained from the following experiments: three-dimensional 15N-edited NOESY-HSQC (800 MHz, 100 ms), 13C-edited NOESY-HSQC (800 MHz, 100 ms), and 13C-edited NOESY-HSQC (800 MHz, 80 ms) on aromatics. All NOESY peaks were picked semiautomatically in Analysis with noise and artifact peaks removed manually. Cross-peak intensities were used to evaluate target distances. Dihedral restraints (Φ/Ψ) were obtained from the TALOS data base (34Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2744) Google Scholar). Hydrogen-bond restraints within secondary structure elements identified from initial rounds of structure calculation were incorporated based on the temperature dependence of NMR chemical shifts (35Baxter N.J. Hosszu L.L. Waltho J.P. Williamson M.P. J. Mol. Biol. 1998; 284: 1625-1639Crossref PubMed Scopus (72) Google Scholar) using a series of 1H,15N HSQC spectra collected from 15 to 35 °C. Initial models were generated with CYANA using the CANDID (36Herrmann T. Guntert P. Wuthrich K. J. Biomol. NMR. 2002; 24: 171-189Crossref PubMed Scopus (434) Google Scholar) method for NOESY cross-peak assignment and calibration. These models were used as initial structures in structure calculations by Aria (37Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (335) Google Scholar). The acceptance tolerances in the standard protocol of Aria 1.2 were modified to set violation tolerances to 5.0, 2.0, 1.0, 0.5, 2.0, 0.5, and 0.1 Å for iterations 2–8, respectively, with iteration 1 containing the initial models. Any cross-peaks rejected by Aria were checked manually, and those found to be reliable were added to the calculation. 200 structures were calculated at each iteration, the 20 lowest energy structures retained and 10 used for final restraint analysis. The 30 lowest energy structures from iteration 8 were further refined in the presence of explicit water molecules. Molecular models were generated using PyMOL (38DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA2004Google Scholar). The structural statistics for each domain are presented in Table 1. The set of 20 lowest energy structures has been submitted to the Protein Data Bank with the accession number 2KBB.TABLE 1Solution structure determination of talin 1655–1822RestraintsUnique/Ambiguous NOEs4488/520Intraresidue1707/116Sequential948/109Short range (1 < [i – j] < 5)970/145Long range ([i – j] > 4)863/150φ/ψ dihedral anglesaFrom chemical shifts using Talos257Energies (kcal mol–1)bCalculated in ARIA 1.2 for the 20 lowest energy structures refined in water. r.m.s., root mean squareTotal–6688.29 ± 86.70Van Der Waals–1626.14 ± 10.38NOE33.50 ± 5.38r.m.s. deviationsbCalculated in ARIA 1.2 for the 20 lowest energy structures refined in water. r.m.s., root mean squareNOEs (Å) (no violations > 0.5 Å)0.011 ± 0.001Dihedral restraints (°) (no violations > 5°)0.39 ± 0.03Bonds (Å)0.0017 ± 0.0001Angles (°)0.34 ± 0.01Impropers (°)0.244 ± 0.01Ramachandran map analysiscObtained using PROCHECK-NMRAllowed regions94.1%Additional allowed regions5.2%Generously allowed regions0.4%Disallowed regions0.3%Pairwise r.m.s. differences (Å)dFor backbone atoms; value for all heavy atoms in brackets. r.m.s., root mean squareResidues 1662–18200.78 (1.18)Secondary structure0.33 (0.76)a From chemical shifts using Talosb Calculated in ARIA 1.2 for the 20 lowest energy structures refined in water. r.m.s., root mean squarec Obtained using PROCHECK-NMRd For backbone atoms; value for all heavy atoms in brackets. r.m.s., root mean square Open table in a new tab NMR Titrations—All titrations were carried out in 20 mm phosphate, pH 6.5, 50 mm NaCl, 2 mm dithiothreitol using 1H,15N HSQC. Weighted combined 1H and 15N amide secondary shifts (Δ(H,N)) were calculated using the equationΔ(H,N)=ΔHWH2+ΔNWN2(Eq. 1) where WH and WN are weighting factors for the 1H and 15N amide shifts, respectively (WH = 1 and WH = 0.15) (39Ayed A. Mulder F.A. Yi G.S. Lu Y. Kay L.E. Arrowsmith C.H. Nat. Struct. Biol. 2001; 8: 756-760Crossref PubMed Scopus (238) Google Scholar), and Δ= δbound – δfree. Dissociation constants (Kd) were determined by fitting the changes in secondary shift with concentration to the following equationΔ(H,N)=Δ(H,N)0[P]+[L]+Kd−([P]+[L]+Kd−4[P][L])2[P](Eq. 2) where Δ(H,N) is the weighted secondary shift, Δ(H,N)0 is the shift at saturation, and [P] and [L] are the protein and ligand concentrations, respectively. Data from peaks with the largest shift changes were fitted to this equation using Analysis. Competition experiments were conducted by acquiring 1H,15N HSQC spectra of 50 μm β3-tail in the presence of 0.25 mm talin F3 domain, 1 mm talin 1655–1822, or both. Weighted combined 1H and 15N amide shifts (Δ(H,N)) were calculated as above. Molecular Docking Calculations—Docking of talin 1655–1822 to F3 was performed with the software HADDOCK2 (40Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2291) Google Scholar, 41de Vries S.J. van Dijk A.D. Krzeminski M. van Dijk M. Thureau A. Hsu V. Wassenaar T. Bonvin A.M. Proteins. 2007; 69: 726-733Crossref PubMed Scopus (493) Google Scholar). The starting structures were model 1 of the F3 complex with the β3/PIPKIγ-chimeric peptide (Protein Data Bank (PDB) ID code 2H7D) (30Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar) and the lowest energy NMR structure of talin 1655–1822. Mutagenesis data, chemical shift mapping, and surface accessibility data were used to define the active residues according to HADDOCK definitions. The well defined filtered NOEs between Tyr-377 in F3 and Leu-1680 and Val-1683 of talin 1655–1822 were also used in the calculation. In all, 1000 initial complex structures were generated by rigid body energy minimization, and the best 200 by total energy were selected for torsion angle dynamics and subsequent Cartesian dynamics in an explicit water solvent. Default scaling for energy terms is as described previously (40Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2291) Google Scholar). Mapping the Domain Boundaries of the High Affinity F3 Binding Site in the Talin Rod—A fragment of the talin rod, spanning residues 1654–2344, has been shown to contain two non-overlapping binding sites for the talin F3 FERM subdomain (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). To further investigate the molecular basis of the interaction, we first determined the domain boundaries of the relevant section of the talin rod, which is made up of a total of 62 α-helices (Fig. 1A) arranged into ∼13 compact bundles or domains. We have previously established that the most C-terminal bundle (residues 2300–2482), which contains an actin binding site (20Gingras A.R. Bate N. Goult B.T. Hazelwood L. Canestrelli I. Grossmann J.G. Liu H. Putz N.S. Roberts G.C. Volkmann N. Hanein D. Barsukov I.L. Critchley D.R. EMBO J. 2008; 27: 458-469Crossref PubMed Scopus (144) Google Scholar), is preceded by two five-helix domains (residues 1974–2140 and 2137–2294) that together make up the integrin binding site referred to as IBS2 (21Gingras A.R. Ziegler W.H. Bobkov A.A. Joyce M.G. Fasci D. Himmel M. Rothemund S. Ritter A. Grossmann J.G. Patel B. Bate N. Goult B.T. Emsley J. Barsukov I.L. Roberts G.C. Liddington R.C. Ginsberg M.H. Critchley D.R. J. Biol. Chem. 2009; 284: 8866-8876Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Working toward the N terminus, the next domain, which contains a vinculin binding site (VBS3), was thought to be a four-helix bundle (residues 1843–1973) (19Gingras A.R. Vogel K.P. Steinhoff H.J. Ziegler W.H. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Biochemistry. 2006; 45: 1805-1817Crossref PubMed Scopus (62) Google Scholar), but we have recently found that it is more stable as a five-helix bundle (residues 1815–1973). 4B. T. Goult, A. R. Gingras, N. Bate, G. C. K. Roberts, I. L. Barsukov, and D. R. Critchley, unpublished work. This domain overlaps, by one helix, the F3 binding fragment identified by Goksoy et al. (28Goksoy E. Ma Y.Q. Wang X. Kong X. Perera D. Plow E.F. Qin J. Mol Cell. 2008; 31: 124-133Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) (residues 1654–1848). To establish whether the preceding domain is also a five-helix bundle, we expressed residues 1655–1822, a region that is highly conserved in both talin1 and talin2 and across species (Fig. 1B). The protein was soluble, and its melting temperature, as determined by CD, was 65 °C, providing evidence for a stable fold. Moreover, the 1H,15N HSQC spectrum showed good dispersion indicative of a well folded domain and with peak line widths consistent with a monomeric state. Structure of Talin Residues 1655–1822—The solution structure of talin 1655–1822 was calculated from 5502 distance, and 257 dihedral angle restraints were determined using 13C,15N-labeled protein. The structure consists of five antiparallel amphipathic α-helices forming a bundle stabilized by hydrophobic interactions (Fig. 1, C and D). The loops between helices 1 and 2 and helices 4 and 5 are relatively long (9–10 residues), whereas those between helices 2 and 3 and 3 and 4 are shorter (4–5 residues). Two of the helices contain proline residues (Pro-1715 in helix 2 and Pro-1740 in helix 3) that introduce kinks in the helices; interestingly, these prolines are conserved among talin sequences (Fig. 1B). The topology of the bundle is similar to that seen in talin 482–655 (17Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (135) Google Scholar) and the two bundles in talin 1974–2293, which make up IBS2 (21Gingras A.R. Ziegler W.H. Bobkov A.A. Joyce M.G. Fasci D. Himmel M. Rothemund S. Ritter A. Grossmann J.G. Patel B. Bate N. Goult B.T. Emsley J. Barsukov I.L. Roberts G.C. Liddington R.C. Ginsberg M.H. Critchley D.R. J. Biol. Chem. 2009; 284: 8866-8876Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The core is almost exclusively hydrophobic with a conserved phenylalanine (Phe-1738) surrounded by the hydrophobic side chains of Leu, Ile, and Val residues. The only non-hydrophobic residue in the core is a conserved threonine, Thr-1765. Unlike other talin bundles (17Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (135) Google Scholar, 18Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb.). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19Gingras A.R. Vogel K.P. Steinhoff H.J. Ziegler W.H. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Biochemistry. 2006; 45: 1805-1817Crossref PubMed Scopus (62) Google Scholar, 20Gingras A.R. Bate N. Goult B.T. Hazelwood L. Canestrelli I. Grossmann J.G. Liu H. Putz N.S. Roberts G.C. Volkmann N. Hanein D. Barsukov I.L. Critchley D.R. EMBO J. 2008; 27: 458-469Crossref PubMed Scopus (144) Google Scholar), there is no aromatic residue capping the end of the bundle. An interesting feature of the domain is that there is a hydrophobic patch on helices 2 and 3, including Leu-1698 and Leu-1743, that is masked by the long loop between helices 1 and 2, which contains several hydrophobic residues (including Ile-1693 and Leu-1687) that dock into this patch. It remains to be established whether this hydrophobic patch is a site for domain-domain interaction in the intact rod (17Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (135) Google Scholar). Talin 1655–1822 Interacts with the Talin F3 Domain—The interaction between the talin rod 1655–1822 five-helix bundle and the F3 FERM subdomain of the talin head was studied by collecting 1H,15N HSQC spectra of 15N-labeled talin 1655–1822 in the presence of increasing concentrations of unlabeled F3 (Fig. 2A). A number of resonances showed progressive changes in chemical shift
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