Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin
2010; Elsevier BV; Volume: 285; Issue: 38 Linguagem: Inglês
10.1074/jbc.m109.095455
ISSN1083-351X
AutoresAlexandre R. Gingras, Neil Bate, Benjamin T. Goult, B. Patel, Petra Kopp, Jonas Emsley, Igor Barsukov, Gordon C. K. Roberts, David R. Critchley,
Tópico(s)RNA Research and Splicing
ResumoTalin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein. Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein. IntroductionTalin is a large (2541 amino acids) dimeric cytoskeletal protein that provides a direct link between the integrin family of cell adhesion molecules and the actin cytoskeleton (1Critchley D.R. Gingras A.R. J. Cell Sci. 2008; 121: 1345-1347Crossref PubMed Scopus (169) Google Scholar, 2Critchley D.R. Annu. Rev. Biophys. 2009; 38: 235-254Crossref PubMed Scopus (211) Google Scholar, 3Roberts G.C. Critchley D.R. Biophys. Rev. 2009; 1: 61-69Crossref PubMed Scopus (52) Google Scholar), and it has a pivotal role in integrin activation (4Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (386) Google Scholar, 5Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. 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Monkley S.J. Kanaho Y. Critchley D.R. Sasaki T. Birnbaum M.J. Weisel J.W. Hartwig J. Abrams C.S. J. Clin. Invest. 2008; 118: 812-819PubMed Google Scholar), and deletion of both the talin1 and talin2 isoforms in skeletal muscle leads to disruption of the myotendinous junction and inhibition of myoblast fusion (14Conti F.J. Felder A. Monkley S. Schwander M. Wood M.R. Lieber R. Critchley D. Miller U. Development. 2008; 135: 2043-2053Crossref PubMed Scopus (40) Google Scholar, 15Conti F.J. Monkley S.J. Wood M.R. Critchley D.R. Miller U. Development. 2009; 136: 3597-3606Crossref PubMed Scopus (87) Google Scholar).The N-terminal talin head (47 kDa) (Fig. 1) contains a FERM domain composed of F1, F2, and F3 domains, although it is atypical in that F1 contains a large unstructured insert and is preceded by a previously unrecognized domain, F0 (16Goult B.T. Bouaouina M. Harburger D.S. Bate N. Patel B. Anthis N.J. Campbell I.D. Calderwood D.A. Barsukov I.L. Roberts G.C. Critchley D.R. J. Mol. Biol. 2009; 394: 944-956Crossref PubMed Scopus (68) Google Scholar, 17Goult B.T. Bouaouina M. Elliott P.R. Bate N. Patel B. Gingras A.R. Grossmann J.G. Roberts G.C. Calderwood D.A. Critchley D.R. Barsukov I.L. EMBO J. 2010; 29: 1069-1080Crossref PubMed Scopus (116) Google Scholar). The F3 domain has a phosphotyrosine-binding domain-like fold and binds to both the membrane proximal NPXY motif in β3-integrin tails and the membrane proximal helix (18Wegener 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 (519) Google Scholar, 19García-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 20Anthis N.J. Wegener K.L. Ye F. Kim C. Goult B.T. Lowe E.D. Vakonakis I. Bate N. Critchley D.R. Ginsberg M.H. Campbell I.D. EMBO J. 2009; 28: 3623-3632Crossref PubMed Scopus (237) Google Scholar). This is thought to disrupt the inter-subunit interactions between the α- and β-integrin cytoplasmic tails and also their trans-membrane helices (21Lau T.L. Kim C. Ginsberg M.H. Ulmer T.S. EMBO J. 2009; 28: 1351-1361Crossref PubMed Scopus (266) Google Scholar) leading to integrin activation, although it is now clear that integrin activation also requires cooperation between talin and the kindlin family of FERM domain proteins (22Meves A. Stremmel C. Gottschalk K. Fässler R. Trends Cell Biol. 2009; 19: 504-513Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar).The talin rod (220 kDa) is composed of 62 α-helices organized into a series of amphipathic helical bundles (Fig. 1). It contains a second integrin-binding site (IBS2) (23Gingras 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 (66) Google Scholar, 24Moes M. Rodius S. Coleman S.J. Monkley S.J. Goormaghtigh E. Tremuth L. Kox C. van der Holst P.P. Critchley D.R. Kieffer N. J. Biol. Chem. 2007; 282: 17280-17288Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), at least two actin-binding sites (25Hemmings 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), the best characterized of which is at the C terminus (26Smith S.J. McCann R.O. Biochemistry. 2007; 46: 10886-10898Crossref PubMed Scopus (34) Google Scholar, 27Gingras 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 (138) Google Scholar), and numerous vinculin-binding sites (VBSs) (28Gingras 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 (146) Google Scholar). Using limited proteolysis and a large series of recombinant polypeptides, we have begun to identify some of the domains that make up the talin rod and to determine their structures (19García-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 20Anthis N.J. Wegener K.L. Ye F. Kim C. Goult B.T. Lowe E.D. Vakonakis I. Bate N. Critchley D.R. Ginsberg M.H. Campbell I.D. EMBO J. 2009; 28: 3623-3632Crossref PubMed Scopus (237) Google Scholar, 23Gingras 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 (66) Google Scholar, 27Gingras 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 (138) Google Scholar, 29Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure. 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 30Gingras 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 (61) Google Scholar, 31Goult B.T. Bate N. Anthis N.J. Wegener K.L. Gingras A.R. Patel B. Barsukov I.L. Campbell I.D. Roberts G.C. Critchley D.R. J. Biol. Chem. 2009; 284: 15097-15106Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 32Papagrigoriou 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 (131) Google Scholar, 33Goult B.T. Gingras A.R. Bate N. Barsukov I.L. Critchley D.R. Roberts G.C. FEBS Lett. 2010; 584: 2237-2241Crossref PubMed Scopus (17) Google Scholar) and their mode of interaction with integrins, vinculin, and F-actin (2Critchley D.R. Annu. Rev. Biophys. 2009; 38: 235-254Crossref PubMed Scopus (211) Google Scholar). The rod starts with a 5-helix bundle (residues 482–655) that packs against a 4-helix bundle (residues 656–786) via an extensive hydrophobic interface (32Papagrigoriou 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 (131) Google Scholar). The C-terminal region of the rod (residues 1655–2482) is composed of five 5-helix bundles coupled by short flexible linkers and ends with a single helix that is responsible for talin dimer formation (27Gingras 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 (138) Google Scholar). An intramolecular interaction between the F3 FERM domain and one of the C-terminal rod domains (residues 1655–1822) masks the integrin-binding site in F3 (31Goult B.T. Bate N. Anthis N.J. Wegener K.L. Gingras A.R. Patel B. Barsukov I.L. Campbell I.D. Roberts G.C. Critchley D.R. J. Biol. Chem. 2009; 284: 15097-15106Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 34Goksoy 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 (194) Google Scholar) and is thought to contribute to the regulation of talin activity and to the compact form of the molecule seen in electron microscopy (35Winkler J. Linsdorf H. Jockusch B.M. Eur. J. Biochem. 1997; 243: 430-436Crossref PubMed Scopus (45) Google Scholar, 36Molony L. McCaslin D. Abernethy J. Paschal B. Burridge K. J. Biol. Chem. 1987; 262: 7790-7795Abstract Full Text PDF PubMed Google Scholar).However, the domain organization of the central region of the rod has been difficult to resolve. Interestingly, residues 1359–1659 show significant homology to the gene referred to as mesoderm development candidate 1 (Mesdc1) (37Wines M.E. Lee L. Katari M.S. Zhang L. DeRossi C. Shi Y. Perkins S. Feldman M. McCombie W.R. Holdener B.C. Genomics. 2001; 72: 88-98Crossref PubMed Scopus (15) Google Scholar), which is predicted to encode a 364-amino acid protein of unknown function. We therefore expressed this region of talin in Escherichia coli and found that the protein was soluble, stable, and easy to crystallize. We now report the structure of this region of talin and show that it is made up of a 5- and a 4-helix bundle with unique topology. It contains an actin-binding site, two VBSs, and a binding site for the intermediate filament protein α-synemin, which colocalizes with talin in the costameres of skeletal muscle (38Sun N. Critchley D.R. Paulin D. Li Z. Robson R.M. Exp. Cell Res. 2008; 314: 1839-1849Crossref PubMed Scopus (46) Google Scholar). Moreover, we demonstrate that the MESDc1 protein is a novel actin-binding protein.DISCUSSIONHere, we report the crystal structure of talin residues 1359–1659 that contains nine α-helices that are organized into a unique fold with two distinct domains. The 5-helix bundle is formed by nonsequential helices α1, α2, and α3 followed by α8 and α9, whereas the 4-helix bundle is composed of consecutive helices and is inserted between helices 3 and 4 of the 5-helix bundle. The two bundles are connected by a linker that forms a two-stranded anti-parallel β-sheet-like structure. This is a completely novel feature of the talin rod that is otherwise composed of a linear arrangement of 5- and 4-helix bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to the adjacent helical bundles in the talin rod via end-to-end packing, whereas the 4-helix bundle protrudes from the talin rod (Fig. 1) (33Goult B.T. Gingras A.R. Bate N. Barsukov I.L. Critchley D.R. Roberts G.C. FEBS Lett. 2010; 584: 2237-2241Crossref PubMed Scopus (17) Google Scholar).Another novel feature of the module is that it contains a constitutively active VBS that binds vinculin Vd1 at room temperature. Analysis of the isolated 4-helix bundle shows that it also binds Vd1 at room temperature, whereas the VBS in the 5-helix bundle is cryptic, like most other VBSs in the talin rod (23Gingras 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 (66) Google Scholar, 32Papagrigoriou 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 (131) Google Scholar, 52Patel B. Gingras A.R. Bobkov A.A. Fujimoto L.M. Zhang M. Liddington R.C. Mazzeo D. Emsley J. Roberts G.C. Barsukov I.L. Critchley D.R. J. Biol. Chem. 2006; 281: 7458-7467Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To date, the only other region of the talin rod that binds Vd1 at room temperature is the 4-helix bundle spanning residues 755–889 (29Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure. 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar); both these 4-helix bundles are unique in that they have a cluster of threonine residues within their hydrophobic core (supplemental Fig. S6), and this likely modulates the stability of the bundle, and hence the availability of the VBS. Indeed, we have shown that replacing the threonine cluster in talin(755–889) with hydrophobic residues markedly suppresses vinculin binding (52Patel B. Gingras A.R. Bobkov A.A. Fujimoto L.M. Zhang M. Liddington R.C. Mazzeo D. Emsley J. Roberts G.C. Barsukov I.L. Critchley D.R. J. Biol. Chem. 2006; 281: 7458-7467Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, within the 9-helix module, the termini of the 4-helix bundle are kept together by the 5-helix bundle, and CD melting experiments show this stabilizes the fold; because unfolding the bundle is required for vinculin binding, this will decrease its affinity for vinculin, and this may explain why Vd1 binding to the 9-helix module has a lower affinity than binding to the isolated 4-helix bundle. How the various VBS behave within the context of full-length talin remains to be explored.We have previously suggested that activation of the cryptic VBSs in the talin rod might be triggered by force exerted on integrin-talin-actin complexes (52Patel B. Gingras A.R. Bobkov A.A. Fujimoto L.M. Zhang M. Liddington R.C. Mazzeo D. Emsley J. Roberts G.C. Barsukov I.L. Critchley D.R. J. Biol. Chem. 2006; 281: 7458-7467Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and experimental evidence in support of the idea of force-induced activation of the VBSs in talin has recently come from elegant in vitro studies using magnetic tweezers (56del Rio A. Perez-Jimenez R. Liu R. Roca-Cusachs P. Fernandez J.M. Sheetz M.P. Science. 2009; 323: 638-641Crossref PubMed Scopus (1052) Google Scholar). Vinculin stabilizes FAs by cross-linking talin to F-actin or to membrane phospholipids (57Saunders R.M. Holt M.R. Jennings L. Sutton D.H. Barsukov I.L. Bobkov A. Liddington R.C. Adamson E.A. Dunn G.A. Critchley D.R. Eur. J. Cell Biol. 2006; 85: 487-500Crossref PubMed Scopus (147) Google Scholar, 58Chandrasekar I. Stradal T.E. Holt M.R. Entschladen F. Jockusch B.M. Ziegler W.H. J. Cell Sci. 2005; 118: 1461-1472Crossref PubMed Scopus (100) Google Scholar), and perhaps the fact that two of the VBSs in talin are activated more readily than others allows for a graduated recruitment of vinculin in response to progressive increases in force. However, expression of vinculin Vd1 alone in vinculin null fibroblasts has been shown to lead to larger and more stable FAs even in the absence of actomyosin contraction, an effect that was dependent on the ability of Vd1 to bind talin (59Humphries J.D. Wang P. Streuli C. Geiger B. Humphries M.J. Ballestrem C. J. Cell Biol. 2007; 179: 1043-1057Crossref PubMed Scopus (626) Google Scholar). This suggests that mechanisms that regulate exposure of the talin-binding site in vinculin may also drive the interaction of vinculin with talin, and it will be interesting to establish whether the two domains containing constitutively active VBSs in talin are involved in this response.The 9-helix module also binds F-actin, although neither the 4- nor 5-helix bundles alone are able to bind. This raises the possibility that vinculin might bind to the VBS in the 4-helix bundle via its N-terminal Vd1 domain leaving the C-terminal vinculin tail to bind to the F-actin bound to the talin 9-helix module. This may stabilize the interaction between the talin 9-helix bundle and F-actin, which on its own is rather weak. Although this remains to be investigated, what is clear is that several regions of talin, including the talin head (60Lee H.S. Bellin R.M. Walker D.L. Patel B. Powers P. Liu H. Garcia-Alvarez B. de Pereda J.M. Liddington R.C. Volkmann N. Hanein D. Critchley D.R. Robson R.M. J. Mol. Biol. 2004; 343: 771-784Crossref PubMed Scopus (78) Google Scholar), and at least two regions in the talin rod have the ability to bind F-actin (Fig. 1) (25Hemmings 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). Much attention has focused on the C-terminal actin-binding site in talin (26Smith S.J. McCann R.O. Biochemistry. 2007; 46: 10886-10898Crossref PubMed Scopus (34) Google Scholar, 27Gingras 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 (138) Google Scholar), which is homologous to the Hip1R family of actin-binding proteins (61Senetar M.A. Foster S.J. McCann R.O. Biochemistry. 2004; 43: 15418-15428Crossref PubMed Scopus (60) Google Scholar), and our recent data show that it plays an essential role in the assembly of FAs (65Kopp P.M. Bate N. Hansen T.M. Brindle N.P. Praekelt U. Debrand E. Coleman S. Mazzeo D. Goult B.T. Gingras A.R. Pritchard C.A. Critchley D.R. Monkley S.J. Eur. J. Cell Biol. 2010; 89: 661-673Crossref PubMed Scopus (59) Google Scholar). However, we have shown that the talin C-terminal actin-binding site, which is dimeric, binds along a single actin filament and does not cross-link F-actin (27Gingras 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 (138) Google Scholar). Therefore, it seems unlikely that the C-terminal actin-binding site accounts for the ability of talin to cross-link F-actin (62Schmidt J.M. Zhang J. Lee H.S. Stromer M.H. Robson R.M. Arch. Biochem. Biophys. 1999; 366: 139-150Crossref PubMed Scopus (34) Google Scholar), and actin-binding sites elsewhere in the talin molecule, including that in the 9-helix module, may be relevant in this regard. Similarly, recent studies on filamin show that apart from the well characterized actin-binding site in the N-terminal calponin homology domain, Ig repeats 9–15 in the filamin rod also bind F-actin and increase the overall avidity of filamin for F-actin (63Nakamura F. Osborn T.M. Hartemink C.A. Hartwig J.H. Stossel T.P. J. Cell Biol. 2007; 179: 1011-1025Crossref PubMed Scopus (205) Google Scholar).Previous studies have shown that the central part of the talin rod (residues 1327–1948) binds the 312-amino acid insert (SNTIII) present within the muscle variant of the intermediate filament protein α-synemin (38Sun N. Critchley D.R. Paulin D. Li Z. Robson R.M. Exp. Cell Res. 2008; 314: 1839-1849Crossref PubMed Scopus (46) Google Scholar). Using the same overlay assay used above, we have shown that both the 4- and 5-helix bundles present in the talin 9-helix module bind SNTIII indicating that there are two α-synemin-binding sites in this region of talin (see supplemental “Results” and supplemental Fig. S7). It is noteworthy that SNTIII also binds the vinculin tail, but this is competitive with talin binding (64Sun N. Critchley D.R. Paulin D. Li Z. Robson R.M. Biochem. J. 2008; 409: 657-667Crossref PubMed Scopus (37) Google Scholar).Talin residues 1359–1659 show homology to residues 44–352 of the protein encoded by the MESDc1 gene, and this region of MESDc1 is predicted to have a similar secondary structure to talin. Indeed we have generated a homology model of MESDc1 using the crystal structure of the talin residues 1359–1659. Proteins with similar folds often have very different functions, but it is striking that both proteins bind to F-actin, and GFP-tagged MESDc1 colocalized with actin stress fibers when expressed in NIH3T3 cells. However, MESDc1 does not bind to vinculin, and analysis of the MESDc1 sequence shows that helices 7 and 10 (equivalent to the VBS helices in talin) diverge from the VBS consensus sequence (28Gingras 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 (146) Google Scholar). Interestingly, MESDc1 binds to F-actin better than does the talin 9-helix module. Gel filtration experiments indicate that MESDc1 is dimeric in solution, and the NMR HSQC shows broad signals (data not shown), consistent with this conclusion. The C-terminal actin-binding site in talin is also dimeric, and dimerization is important for high affinity actin binding (26Smith S.J. McCann R.O. Biochemistry. 2007; 46: 10886-10898Crossref PubMed Scopus (34) Google Scholar, 27Gingras 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 (138) Google Scholar). Thus, the fact that MESDc1 is also dimeric may contribute to its higher affinity for F-actin. However, although the MESDc1 mRNA appears to be widely expressed, there is no literature on the role of this novel actin-binding protein in cells.In summary, we show that residues 1359–1659 from the central region of the talin rod have a novel structure quite different from that of other regions of the talin rod or any other protein in the Protein Data Bank. It contains binding sites for vinculin, F-actin, and the muscle-specific isoform of the intermediate filament protein α-synemin, and this and its unusual fold suggest that it plays an important role in the function of talin. Moreover, we predict that the previously uncharacterized protein MESDc1 will have a similar structure, and we demonstrate that it also binds F-actin. IntroductionTalin is a large (2541 amino acids) dimeric cytoskeletal protein that provides a direct link between the integrin family of cell adhesion molecules and the actin cytoskeleton (1Critchley D.R. Gingras A.R. J. Cell Sci. 2008; 121: 1345-1347Crossref PubMed Scopus (169) Google Scholar, 2Critchley D.R. Annu. Rev. Biophys. 2009; 38: 235-254Crossref PubMed Scopus (211) Google Scholar, 3Roberts G.C. Critchley D.R. Biophys. Rev. 2009; 1: 61-69Crossref PubMed Scopus (52) Google Scholar), and it has a pivotal role in integrin activation (4Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (386) Google Scholar, 5Tadokoro 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 (961) Google Scholar) and clustering (6Saltel F. Mortier E. 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Critchley D.R. Fässler R. Dev. Dyn. 2000; 219: 560-574Crossref PubMed Scopus (172) Google Scholar). Use of a conditional talin1 allele confirms that talin1 is essential to integrin activation in platelets (11Petrich 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 (216) Google Scholar, 12Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fässler R. J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (198) Google Scholar) and the stability of the membrane cytoskeletal interface in megakaryocytes (13Wang Y. Litvinov R.I. Chen X. Bach T.L. Lian L. Petrich B.G. Monkley S.J. Kanaho Y. Critchley D.R. Sasaki T. Birnbaum M.J. Weisel J.W. Hartwig J. Abrams C.S. J. Clin. Invest. 2008; 118: 812-819PubMed Google Scholar), and deletion of both the talin1 and talin2 isoforms in skeletal muscle leads to disruption of the myotendinous junction and inhibition of myoblast fusion (14Conti F.J. Felder A. Monkley S. Schwander M. Wood M.R. Lieber R. Critchley D. Miller U. Development. 2008; 135: 2043-2053Crossref PubMed Scopus (40) Google Scholar, 15Conti F.J. Monkley S.J. Wood M.R. Critchley D.R. Miller U. Development. 2009; 136: 3597-3606Crossref PubMed Scopus (87) Google Scholar).The N-terminal talin head (47 kDa) (Fig. 1) contains a FERM domain composed of F1, F2, and F3 domains, although it is atypical in that F1 contains a large unstructured insert and is preceded by a previously unrecognized domain, F0 (16Goult B.T. Bouaouina M. Harburger D.S. Bate N. Patel B. Anthis N.J. Campbell I.D. Calderwood D.A. Barsukov I.L. Roberts G.C. Critchley D.R. J. Mol. Biol. 2009; 394: 944-956Crossref PubMed Scopus (68) Google Scholar, 17Goult B.T. Bouaouina M. Elliott P.R. 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This is thought to disrupt the inter-subunit interactions between the α- and β-integrin cytoplasmic tails and also their trans-membrane helices (21Lau T.L. Kim C. Ginsberg M.H. Ulmer T.S. EMBO J. 2009; 28: 1351-1361Crossref PubMed Scopus (266) Google Scholar) leading to integrin activation, although it is now clear that integrin activation also requires cooperation between talin and the kindlin family of FERM domain proteins (22Meves A. Stremmel C. Gottschalk K. Fässler R. Trends Cell Biol. 2009; 19: 504-513Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar).The talin rod (220 kDa) is composed of 62 α-helices organized into a series of amphipathic helical bundles (Fig. 1). It contains a second integrin-binding site (IBS2) (23Gingras 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. 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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 (146) Google Scholar). Using limited proteolysis and a large series of recombinant polypeptides, we have begun to identify some of the domains that make up the talin rod and to determine their structures (19García-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 20Anthis N.J. Wegener K.L. Ye F. Kim C. Goult B.T. Lowe E.D. Vakonakis I. Bate N. Critchley D.R. Ginsberg M.H. Campbell I.D. EMBO J. 2009; 28: 3623-3632Crossref PubMed Scopus (237) Google Scholar, 23Gingras 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. 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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 (131) Google Scholar, 33Goult B.T. Gingras A.R. Bate N. Barsukov I.L. Critchley D.R. Roberts G.C. FEBS Lett. 2010; 584: 2237-2241Crossref PubMed Scopus (17) Google Scholar) and their mode of interaction with integrins, vinculin, and F-actin (2Critchley D.R. Annu. Rev. Biophys. 2009; 38: 235-254Crossref PubMed Scopus (211) Google Scholar). The rod starts with a 5-helix bundle (residues 482–655) that packs against a 4-helix bundle (residues 656–786) via an extensive hydrophobic interface (32Papagrigoriou 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 (131) Google Scholar). The C-terminal region of the rod (residues 1655–2482) is composed of five 5-helix bundles coupled by short flexible linkers and ends with a single helix that is responsible for talin dimer formation (27Gingras 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 (138) Google Scholar). An intramolecular interaction between the F3 FERM domain and one of the C-terminal rod domains (residues 1655–1822) masks the integrin-binding site in F3 (31Goult B.T. Bate N. Anthis N.J. Wegener K.L. Gingras A.R. Patel B. Barsukov I.L. Campbell I.D. Roberts G.C. Critchley D.R. J. Biol. Chem. 2009; 284: 15097-15106Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 34Goksoy 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 (194) Google Scholar) and is thought to contribute to the regulation of talin activity and to the compact form of the molecule seen in electron microscopy (35Winkler J. Linsdorf H. Jockusch B.M. Eur. J. Biochem. 1997; 243: 430-436Crossref PubMed Scopus (45) Google Scholar, 36Molony L. McCaslin D. Abernethy J. Paschal B. Burridge K. J. Biol. Chem. 1987; 262: 7790-7795Abstract Full Text PDF PubMed Google Scholar).However, the domain organization of the central region of the rod has been difficult to resolve. Interestingly, residues 1359–1659 show significant homology to the gene referred to as mesoderm development candidate 1 (Mesdc1) (37Wines M.E. Lee L. Katari M.S. Zhang L. DeRossi C. Shi Y. Perkins S. Feldman M. McCombie W.R. Holdener B.C. Genomics. 2001; 72: 88-98Crossref PubMed Scopus (15) Google Scholar), which is predicted to encode a 364-amino acid protein of unknown function. We therefore expressed this region of talin in Escherichia coli and found that the protein was soluble, stable, and easy to crystallize. We now report the structure of this region of talin and show that it is made up of a 5- and a 4-helix bundle with unique topology. It contains an actin-binding site, two VBSs, and a binding site for the intermediate filament protein α-synemin, which colocalizes with talin in the costameres of skeletal muscle (38Sun N. Critchley D.R. Paulin D. Li Z. Robson R.M. Exp. Cell Res. 2008; 314: 1839-1849Crossref PubMed Scopus (46) Google Scholar). Moreover, we demonstrate that the MESDc1 protein is a novel actin-binding protein.
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