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• W3038248358 abstract "Integrin receptors regulate normal cellular processes such as signaling, cell migration, adhesion to the extracellular matrix, and leukocyte function. Talin recruitment to the membrane is necessary for its binding to and activation of integrin. Vertebrates have two highly conserved talin homologs that differ in their expression patterns. The F1–F3 FERM subdomains of cytoskeletal proteins resemble a cloverleaf, but in talin1, its F1 subdomain and additional F0 subdomain align more linearly with its F2 and F3 subdomains. Here, we present the talin2 crystal structure, revealing that its F0-F1 di-subdomain displays another unprecedented constellation, whereby the F0-F1-F2 adopts a new cloverleaf-like arrangement. Using multiangle light scattering (MALS), fluorescence lifetime imaging (FLIM), and FRET analyses, we found that substituting the corresponding residues in talin2 that abolish lipid binding in talin1 disrupt the binding of talin to the membrane, focal adhesion formation, and cell spreading. Our results provide the molecular details of the functions of specific talin isoforms in cell adhesion. Integrin receptors regulate normal cellular processes such as signaling, cell migration, adhesion to the extracellular matrix, and leukocyte function. Talin recruitment to the membrane is necessary for its binding to and activation of integrin. Vertebrates have two highly conserved talin homologs that differ in their expression patterns. The F1–F3 FERM subdomains of cytoskeletal proteins resemble a cloverleaf, but in talin1, its F1 subdomain and additional F0 subdomain align more linearly with its F2 and F3 subdomains. Here, we present the talin2 crystal structure, revealing that its F0-F1 di-subdomain displays another unprecedented constellation, whereby the F0-F1-F2 adopts a new cloverleaf-like arrangement. Using multiangle light scattering (MALS), fluorescence lifetime imaging (FLIM), and FRET analyses, we found that substituting the corresponding residues in talin2 that abolish lipid binding in talin1 disrupt the binding of talin to the membrane, focal adhesion formation, and cell spreading. Our results provide the molecular details of the functions of specific talin isoforms in cell adhesion. Talin plays pivotal roles in integrin activation as well as in cell migration, invasion, and cancer metastasis (1Desiniotis A. Kyprianou N. Significance of talin in cancer progression and metastasis.Int. Rev. Cell Mol. Biol. 2011; 289 (21749900): 117-14710.1016/B978-0-12-386039-2.00004-3Crossref PubMed Scopus (63) Google Scholar, 2Huang C. Rajfur Z. Yousefi N. Chen Z. Jacobson K. Ginsberg M.H. Talin phosphorylation by Cdk5 regulates Smurf1-mediated talin head ubiquitylation and cell migration.Nat. Cell Biol. 2009; 11 (19363486): 624-63010.1038/ncb1868Crossref PubMed Scopus (157) Google Scholar, 3Jin J.K. Tien P.C. Cheng C.J. Song J.H. Huang C. Lin S.H. Gallick G.E. Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.Oncogene. 2015; 34 (24793790): 1811-182110.1038/onc.2014.116Crossref PubMed Scopus (84) Google Scholar, 4Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Talin binding to integrin β tails: a final common step in integrin activation.Science. 2003; 302 (14526080): 103-10610.1126/science.1086652Crossref PubMed Scopus (976) Google Scholar). Talin activates integrin by binding to the β cytoplasmic integrin tail domain (4Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Talin binding to integrin β tails: a final common step in integrin activation.Science. 2003; 302 (14526080): 103-10610.1126/science.1086652Crossref PubMed Scopus (976) Google Scholar, 5Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. The Talin head domain binds to integrin β subunit cytoplasmic tails and regulates integrin activation.J. Biol. Chem. 1999; 274 (10497155): 28071-2807410.1074/jbc.274.40.28071Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar), which controls focal adhesion dynamics and invadopodium formation (6Bate N. Gingras A.R. Bachir A. Horwitz R. Ye F. Patel B. Goult B.T. Critchley D.R. Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics.PLoS ONE. 2012; 7 (22496808): e3446110.1371/journal.pone.0034461Crossref PubMed Scopus (51) Google Scholar, 7Beaty B.T. Condeelis J. Digging a little deeper: the stages of invadopodium formation and maturation.Eur. J. Cell Biol. 2014; 93 (25113547): 438-44410.1016/j.ejcb.2014.07.003Crossref PubMed Scopus (121) Google Scholar, 8Franco S.J. Rodgers M.A. Perrin B.J. Han J. Bennin D.A. Critchley D.R. Huttenlocher A. Calpain-mediated proteolysis of talin regulates adhesion dynamics.Nat. Cell Biol. 2004; 6 (15448700): 977-98310.1038/ncb1175Crossref PubMed Scopus (422) Google Scholar, 9Nayal A. Webb D.J. Horwitz A.F. Talin: an emerging focal point of adhesion dynamics.Curr. Opin. Cell Biol. 2004; 16 (15037311): 94-9810.1016/j.ceb.2003.11.007Crossref PubMed Scopus (137) Google Scholar), and thus invasion (7Beaty B.T. Condeelis J. Digging a little deeper: the stages of invadopodium formation and maturation.Eur. J. Cell Biol. 2014; 93 (25113547): 438-44410.1016/j.ejcb.2014.07.003Crossref PubMed Scopus (121) Google Scholar, 10Saykali B.A. El-Sibai M. Invadopodia, regulation, and assembly in cancer cell invasion.Cell Commun. Adhes. 2014; 21 (24930891): 207-21210.3109/15419061.2014.923845Crossref PubMed Scopus (31) Google Scholar, 11Webb D.J. Parsons J.T. Horwitz A.F. Adhesion assembly, disassembly and turnover in migrating cells—over and over and over again.Nat. Cell Biol. 2002; 4 (11944043): E97-E10010.1038/ncb0402-e97Crossref PubMed Scopus (603) Google Scholar, 12Wehrle-Haller B. Assembly and disassembly of cell matrix adhesions.Curr. Opin. Cell Biol. 2012; 24 (22819514): 569-58110.1016/j.ceb.2012.06.010Crossref PubMed Scopus (157) Google Scholar). Talin is crucial for initial mechanical force generation (13Giannone G. Jiang G. Sutton D.H. Critchley D.R. Sheetz M.P. Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation.J. Cell Biol. 2003; 163 (14581461): 409-41910.1083/jcb.200302001Crossref PubMed Scopus (221) Google Scholar, 14Zhang X. Jiang G. Cai Y. Monkley S.J. Critchley D.R. Sheetz M.P. Talin depletion reveals independence of initial cell spreading from integrin activation and traction.Nat. Cell Biol. 2008; 10 (19160486): 1062-106810.1038/ncb1765Crossref PubMed Scopus (343) Google Scholar) and is also involved in calpain-induced focal adhesion disassembly (6Bate N. Gingras A.R. Bachir A. Horwitz R. Ye F. Patel B. Goult B.T. Critchley D.R. Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics.PLoS ONE. 2012; 7 (22496808): e3446110.1371/journal.pone.0034461Crossref PubMed Scopus (51) Google Scholar, 8Franco S.J. Rodgers M.A. Perrin B.J. Han J. Bennin D.A. Critchley D.R. Huttenlocher A. Calpain-mediated proteolysis of talin regulates adhesion dynamics.Nat. Cell Biol. 2004; 6 (15448700): 977-98310.1038/ncb1175Crossref PubMed Scopus (422) Google Scholar). Furthermore, talin binds to and activates phosphatidylinositol phosphate kinase γ, which then generates phosphatidylinositol 4,5-bisphosphate (PIP2) that controls focal adhesion dynamics (15Chen C. Wang X. Xiong X. Liu Q. Huang Y. Xu Q. Hu J. Ge G. Ling K. Targeting type Iγ phosphatidylinositol phosphate kinase inhibits breast cancer metastasis.Oncogene. 2015; 34 (25486426): 4635-464610.1038/onc.2014.393Crossref PubMed Scopus (22) Google Scholar, 16Di Paolo G. Pellegrini L. Letinic K. Cestra G. Zoncu R. Voronov S. Chang S. Guo J. Wenk M.R. De Camilli P. Recruitment and regulation of phosphatidylinositol phosphate kinase type 1γ by the FERM domain of talin.Nature. 2002; 420 (12422219): 85-8910.1038/nature01147Crossref PubMed Scopus (374) Google Scholar, 17Li X. Zhou Q. Sunkara M. Kutys M.L. Wu Z. Rychahou P. Morris A.J. Zhu H. Evers B.M. Huang C. Ubiquitylation of phosphatidylinositol 4-phosphate 5-kinase type Iγ by HECTD1 regulates focal adhesion dynamics and cell migration.J. Cell Sci. 2013; 126 (23572508): 2617-262810.1242/jcs.117044Crossref PubMed Scopus (47) Google Scholar, 18Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Type Iγ phosphatidylinositol phosphate kinase targets and regulates focal adhesions.Nature. 2002; 420 (12422220): 89-9310.1038/nature01082Crossref PubMed Scopus (378) Google Scholar, 19Wu J. Chen Y. Lu L.Y. Wu Y. Paulsen M.T. Ljungman M. Ferguson D.O. Yu X. Chfr and RNF8 synergistically regulate ATM activation.Nat. Struct. Mol. Biol. 2011; 18 (21706008): 761-76810.1038/nsmb.2078Crossref PubMed Scopus (79) Google Scholar). Talin is further involved in recruiting moesin in complex with the sodium-hydrogen antiporter 1 (NHE-1) that regulates the pH at invadopodia, thereby providing stability to the generation of the invadopodium and the matrix (7Beaty B.T. Condeelis J. Digging a little deeper: the stages of invadopodium formation and maturation.Eur. J. Cell Biol. 2014; 93 (25113547): 438-44410.1016/j.ejcb.2014.07.003Crossref PubMed Scopus (121) Google Scholar). Talin is a ∼270-kDa cytoskeletal protein characterized by an N-terminal globular head domain (residues 1–400) and a C-terminal tail domain (residues 437–2,541) that are connected by a linker that contains a calpain-II cleavage site (20Goult B.T. Xu X.P. Gingras A.R. Swift M. Patel B. Bate N. Kopp P.M. Barsukov I.L. Critchley D.R. Volkmann N. Hanein D. Structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation.J. Struct. Biol. 2013; 184 (23726984): 21-3210.1016/j.jsb.2013.05.014Crossref PubMed Scopus (82) Google Scholar, 21Nuckolls G.H. Turner C.E. Burridge K. Functional studies of the domains of talin.J. Cell Biol. 1990; 110 (2110569): 1635-164410.1083/jcb.110.5.1635Crossref PubMed Scopus (69) Google Scholar). The talin head domain comprises a four point one, ezrin, radixin, moesin (FERM) domain that binds to the talin C-terminal domain and to the cytoplasmic integrin β tail domain (5Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. The Talin head domain binds to integrin β subunit cytoplasmic tails and regulates integrin activation.J. Biol. Chem. 1999; 274 (10497155): 28071-2807410.1074/jbc.274.40.28071Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). The talin tail domain harbors 13 single amphipathic α-helical vinculin-binding sites (22Bass M.D. Patel B. Barsukov I.G. Fillingham I.J. Mason R. Smith B.J. Bagshaw C.R. Critchley D.R. Further characterization of the interaction between the cytoskeletal proteins talin and vinculin.Biochem. J. 2002; 362 (11879206): 761-76810.1042/0264-6021:3620761Crossref PubMed Scopus (50) Google Scholar, 23Bois P.R. Borgon R.A. Vonrhein C. Izard T. Structural dynamics of α-actinin-vinculin interactions.Mol. 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Chem. 2005; 280 (16135522): 37217-3722410.1074/jbc.M508060200Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 27Izard T. Vonrhein C. Structural basis for amplifying vinculin activation by talin.J. Biol. Chem. 2004; 279 (15070891): 27667-2767810.1074/jbc.M403076200Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Papagrigoriou 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. Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle.EMBO J. 2004; 23 (15272303): 2942-295110.1038/sj.emboj.7600285Crossref PubMed Scopus (132) Google Scholar, 29Tran Van Nhieu G. Izard T. Vinculin binding in its closed conformation by a helix addition mechanism.EMBO J. 2007; 26 (17932491): 4588-459610.1038/sj.emboj.7601863Crossref PubMed Scopus (41) Google Scholar, 30Yogesha S.D. Rangarajan E.S. Vonrhein C. Bricogne G. Izard T. Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin.Protein Sci. 2012; 21 (22334306): 583-58810.1002/pro.2041Crossref PubMed Scopus (10) Google Scholar, 31Yogesha S.D. Sharff A. Bricogne G. Izard T. Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin.Protein Sci. 2011; 20 (21648001): 1471-147610.1002/pro.671Crossref PubMed Scopus (8) Google Scholar, 32Izard T. Evans G. Borgon R.A. Rush C.L. Bricogne G. Bois P.R. Vinculin activation by talin through helical bundle conversion.Nature. 2004; 427 (14702644): 171-17510.1038/nature02281Crossref PubMed Scopus (195) Google Scholar) as well as two actin-binding sites (33Atherton P. Stutchbury B. Wang D.Y. Jethwa D. Tsang R. Meiler-Rodriguez E. Wang P. Bate N. Zent R. Barsukov I.L. Goult B.T. Critchley D.R. Ballestrem C. Vinculin controls talin engagement with the actomyosin machinery.Nat. Commun. 2015; 6 (26634421): 1003810.1038/ncomms10038Crossref PubMed Scopus (130) Google Scholar, 34Gingras 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. The structure of the C-terminal actin-binding domain of talin.EMBO J. 2008; 27 (18157087): 458-46910.1038/sj.emboj.7601965Crossref PubMed Scopus (140) Google Scholar, 35Hemmings 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. Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.J. Cell Sci. 1996; 109 (8937989): 2715-2726Crossref PubMed Google Scholar) in addition to the F-actin–binding site that is located on the F2 and F3 subdomains of the FERM domain (33Atherton P. Stutchbury B. Wang D.Y. Jethwa D. Tsang R. Meiler-Rodriguez E. Wang P. Bate N. Zent R. Barsukov I.L. Goult B.T. Critchley D.R. Ballestrem C. Vinculin controls talin engagement with the actomyosin machinery.Nat. Commun. 2015; 6 (26634421): 1003810.1038/ncomms10038Crossref PubMed Scopus (130) Google Scholar, 34Gingras 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. The structure of the C-terminal actin-binding domain of talin.EMBO J. 2008; 27 (18157087): 458-46910.1038/sj.emboj.7601965Crossref PubMed Scopus (140) Google Scholar, 35Hemmings 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. Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.J. Cell Sci. 1996; 109 (8937989): 2715-2726Crossref PubMed Google Scholar, 36Smith S.J. McCann R.O. A C-terminal dimerization motif is required for focal adhesion targeting of Talin1 and the interaction of the Talin1 I/LWEQ module with F-actin.Biochemistry. 2007; 46 (17722883): 10886-1089810.1021/bi700637aCrossref PubMed Scopus (34) Google Scholar, 37Lee 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. Characterization of an actin-binding site within the talin FERM domain.J. Mol. Biol. 2004; 343 (15465061): 771-78410.1016/j.jmb.2004.08.069Crossref PubMed Scopus (78) Google Scholar). Vertebrates have two highly conserved isoforms with ∼76% sequence identity, but they are not functionally redundant. We know a lot about the talin1 structure and function, whereas much less has been published on talin2 both in terms of structure and function. Talin1 is ubiquitously expressed, whereas talin2 is primarily found in the heart, brain, and skeletal muscle (38Monkley S.J. Pritchard C.A. Critchley D.R. Analysis of the mammalian talin2 gene TLN2.Biochem. Biophys. Res. Commun. 2001; 286 (11527381): 880-88510.1006/bbrc.2001.5497Crossref PubMed Scopus (89) Google Scholar, 39Thul P.J. Akesson L. Wiking M. Mahdessian D. Geladaki A. Ait Blal H. Alm T. Asplund A. Björk L. Breckels L.M. Bäckstrom A. Danielsson F. Fagerberg L. Fall J. Gatto L. et al.A subcellular map of the human proteome.Science. 2017; 356 (28495876): eaal332110.1126/science.aal3321Crossref PubMed Scopus (1384) Google Scholar). Talin1 is also expressed at smaller focal adhesions in the peripheral region, whereas talin2 is mainly found at large focal adhesions and fibrillar adhesions (40Praekelt U. Kopp P.M. Rehm K. Linder S. Bate N. Patel B. Debrand E. Manso A.M. Ross R.S. Conti F. Zhang M.Z. Harris R.C. Zent R. Critchley D.R. Monkley S.J. New isoform-specific monoclonal antibodies reveal different sub-cellular localisations for talin1 and talin2.Eur. J. Cell Biol. 2012; 91 (22306379): 180-19110.1016/j.ejcb.2011.12.003Crossref PubMed Scopus (37) Google Scholar, 41Senetar M.A. Moncman C.L. McCann R.O. Talin2 is induced during striated muscle differentiation and is targeted to stable adhesion complexes in mature muscle.Cell Motil. Cytoskeleton. 2007; 64 (17183545): 157-17310.1002/cm.20173Crossref PubMed Scopus (55) Google Scholar). Upon phosphorylation by Cdk5, talin1 controls focal adhesion dynamics, integrin activation, cell migration, invasion, and metastasis (2Huang C. Rajfur Z. Yousefi N. Chen Z. Jacobson K. Ginsberg M.H. Talin phosphorylation by Cdk5 regulates Smurf1-mediated talin head ubiquitylation and cell migration.Nat. Cell Biol. 2009; 11 (19363486): 624-63010.1038/ncb1868Crossref PubMed Scopus (157) Google Scholar, 3Jin J.K. Tien P.C. Cheng C.J. Song J.H. Huang C. Lin S.H. Gallick G.E. Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.Oncogene. 2015; 34 (24793790): 1811-182110.1038/onc.2014.116Crossref PubMed Scopus (84) Google Scholar), whereas talin2 controls focal adhesion assembly and focal adhesion kinase signaling in cells that are depleted of talin1 (14Zhang X. Jiang G. Cai Y. Monkley S.J. Critchley D.R. Sheetz M.P. Talin depletion reveals independence of initial cell spreading from integrin activation and traction.Nat. Cell Biol. 2008; 10 (19160486): 1062-106810.1038/ncb1765Crossref PubMed Scopus (343) Google Scholar). Talin1 knockout mice are embryonic lethal by E8.5–E9.0 due to gastrulation defects (42Monkley S.J. Zhou X.-H. Kinston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fässler R. Disruption of the talin gene arrests mouse development at the gastrulation stage.Dev. Dyn. 2000; 219 (<::AID-DVDY1079>3.0.CO;2-Y 11084655): 560-57410.1002/1097-0177(2000)9999:9999Crossref PubMed Scopus (176) Google Scholar). Talin2 knockout mice display mild skeletal myopathy at 3 months of age resulting from defects in the myotendinous junction (43Conti F.J. Monkley S.J. Wood M.R. Critchley D.R. Muller U. Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions.Development. 2009; 136 (19793892): 3597-360610.1242/dev.035857Crossref PubMed Scopus (88) Google Scholar). Talin2 can fully rescue the profound defects in focal adhesions and the organization of the cytoskeleton that are manifest in talin1-null cells (44Conti F.J. Felder A. Monkley S. Schwander M. Wood M.R. Lieber R. Critchley D. Müller U. Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle.Development. 2008; 135 (18434420): 2043-205310.1242/dev.015818Crossref PubMed Scopus (41) Google Scholar). Thus, talin1 and talin2 seemed at first glance to be functionally redundant. However, the two isoforms transduce mechanical force differently. Under tension, talin1 recruits vinculin, but with talin2, vinculin recruitment occurs even in the absence of its F-actin–binding domain (45Austen K. Ringer P. Mehlich A. Chrostek-Grashoff A. Kluger C. Klingner C. Sabass B. Zent R. Rief M. Grashoff C. Extracellular rigidity sensing by talin isoform-specific mechanical linkages.Nat. Cell Biol. 2015; 17 (26523364): 1597-160610.1038/ncb3268Crossref PubMed Scopus (217) Google Scholar). Further, cardiac myocyte talin2 is necessary for proper integrin β1D expression, whereas talin1 can preserve heart function in the absence of talin2. Loss of both talin isoforms from the heart muscle results in myocyte instability and dilated cardiomyopathy (46Manso A.M. Okada H. Sakamoto F.M. Moreno E. Monkley S.J. Li R. Critchley D.R. Ross R.S. Loss of mouse cardiomyocyte talin-1 and talin-2 leads to β-1 integrin reduction, costameric instability, and dilated cardiomyopathy.Proc. Natl. Acad. Sci. U. S. A. 2017; 114 (28698364): E6250-E625910.1073/pnas.1701416114Crossref PubMed Scopus (43) Google Scholar). Cell migration and invasion is inhibited by an antibody therapeutic that down-regulates talin2 by targeting human epidermal growth factor 2 for cancer treatment (47Le X.F. Almeida M.I. Mao W. Spizzo R. Rossi S. Nicoloso M.S. Zhang S. Wu Y. Calin G.A. Bast Jr., R.C. Modulation of microRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer.PLoS ONE. 2012; 7 (22829924): e4117010.1371/journal.pone.0041170Crossref PubMed Scopus (60) Google Scholar). However, depletion of talin2 has no effects on integrin β1 activation (3Jin J.K. Tien P.C. Cheng C.J. Song J.H. Huang C. Lin S.H. Gallick G.E. Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.Oncogene. 2015; 34 (24793790): 1811-182110.1038/onc.2014.116Crossref PubMed Scopus (84) Google Scholar). A recent talin2-null study on T-cell exosomes showed reduced binding of integrins αLβ2 and α4β7 to the intercellular adhesion molecule-1 (ICAM-1) and to the mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) (48Soe Z.Y. Prajuabjinda O. Myint P.K. Gaowa A. Kawamoto E. Park E.J. Shimaoka M. Talin-2 regulates integrin functions in exosomes.Biochem. Biophys. Res. Commun. 2019; 512 (30879762): 429-43410.1016/j.bbrc.2019.03.027Crossref PubMed Scopus (11) Google Scholar). Collectively, the talin isoforms do not compensate for the loss of the other, and their functions are distinct. To determine the molecular basis for the difference between the talin isoform functions, we solved the crystal structure of the talin2 head domain (residues 1–403) to 2.56 Å resolution and provide structural and functional insights into the talin isoforms. Despite their ∼76% sequence identity and ∼88% sequence similarity for mammalian isoforms, in talin2, whereas the F0-F1 and F2-F3 domains resemble those seen in talin1, F1 engages in distinct interdomain interactions with F2 to result in a completely distinct head domain constellation that has not been observed for any other FERM domain–containing protein. Our solution studies obtained from size-exclusion chromatography support these findings. Despite their distinct head domain constellation, the mammalian talin isoforms share the same membrane-binding sites. Both full-length proteins are monomeric as determined by multiangle light scattering. This previously disputed observation is now also confirmed by the cryo-EM structure (49Dedden D. Schumacher S. Kelley C.F. Zacharias M. Biertumpfel C. Fassler R. Mizuno N. The architecture of Talin1 reveals an autoinhibition mechanism.Cell. 2019; 179 (31539492): 120-131.e1310.1016/j.cell.2019.08.034Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) that was published while this manuscript was in revision. Our confocal microscopy and in vivo fluorescence lifetime imaging microscopy (FLIM) with FRET analyses revealed the functional relevance of the talin interaction with the cell membrane. Collectively, we provide significant new insights into talin structure and function that pave the way toward a better understanding of the regulation of integrin function and of fundamental processes in cell biology including the cytoskeletal organization. To provide structural insights into the functional differences between the two talin isoforms, we determined the crystal structure of human talin2 (residues 1–403) to 2.56 Å resolution (Table 1, Table 2). Whereas the individual talin2 di-subdomains F0-F1 and F2-F3 are almost identical as seen in the talin1 structure, the F0-F1 motif is rotated by ∼140° in talin2 relative to its orientation in talin1 (Fig. 1A and Fig. S1). This is accomplished by isoform-specific F1-F2 interdomain interactions.Table 1X-ray data reduction statistics for our human talin2 structure (residues 1–403)ParametersValuesSpace groupP 65Unit cell dimensionsa, b, c (Å)58.89, 58.89, 203.8α, β, γ (degrees)90, 90, 120Resolution (Å)101.9–2.56 (2.60–2.56)Total measurements13,1041 (11,611)No. of unique reflections12,969 (631)Wavelength1.0 ÅRpimaRpim is the precision-indicating merging R-factor given as Rpim=∑hkl1n−1∑i|Ihkl,i−Ihkl|∑hkl∑iIhkl,i.0.044 (0.381)Signal/noise I/σ(I)15.7 (2.7)Completeness1.0 (1.0)Multiplicity10.1 (5.7)CC½bCC½ is a Pearson's correlation coefficient calculated between the average intensities of each random half of measurements of unique reflections.0.998 (0.734)a Rpim is the precision-indicating merging R-factor given as Rpim=∑hkl1n−1∑i|Ihkl,i−Ihkl|∑hkl∑iIhkl,i.b CC½ is a Pearson's correlation coefficient calculated between the average intensities of each random half of measurements of unique reflections. Open table in a new tab Table 2Crystallographic refinement statistics for our human talin2 structure (residues 1–403)ParametersValuesResolution (Å)51.00–2.56 (2.80–2.56)No. of reflections, working set12,256No. of reflections, test set648R-factoraR-factor = ∑hkl|Fobs(hkl)−Fcalc(hkl)|∑hklFobs(hkl).0.197 (0.300)R-free (5% of reflections omitted)0.246 (0.353)No. of nonhydrogen atomsProtein3,193Solvent117Average B-factorProtein (Å2)61.39Solvent (Å2)54.08Root mean square deviation from ideal valuesBond lengths (Å)0.05Bond angles (degrees)0.745Ramachandran favored (%)96.90Ramachandran allowed (%)3.10Ramachandran outliersNonea R-factor = ∑hkl|Fobs(hkl)−Fcalc(hkl)|∑hklFobs(hkl). Open table in a new tab We assessed the contribution of crystal contacts in the distinct constellations of the F0-F1 di-domain relative to the F2-F3 di-domains in the two talin isoforms (Figs. S2 and S3 and Table 3, Table 4). Murine talin1 crystallized in P 22121 as a chimera of residues 1–138 fused to 169–400, whereas our human talin2 (residues 1–403) structure crystallized in space group P 65. The volume/mass ratios are comparable, with 2.27 and 2.18 Å3/Da corresponding to a solvent content of 0.49 and 0.436 for talin1 and talin2, respectively. The crystal contacts in the talin1 structure (Fig. S2 and Table 3, Table 4) were already analyzed by the University of Leicester team (50Elliott P.R. Goult B.T. Kopp P.M. Bate N. Grossmann J.G. Roberts G.C. Critchley D.R. Barsukov I.L. The structure of the talin head reveals a novel extended conformation of the FERM domain.Structure. 2010; 18 (20947018): 1289-129910.1016/j.str.2010.07.011Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and Campbell (51Campbell I.D. The talin FERM domain is not so FERM.Structure. 2010; 18 (20947007): 1222-122310.1016/j.str.2010.09.002Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) and were not thought to contribute to the talin1 conformation. In our talin2 crystal, the screw axis packs the talin2 F1 subdomain against F0' from a symmetry-related molecule (Fig. S3A) and allows for F1-F1' (Fig. S3C) or F3-F0' (Fig. S3D) intramolecular interactions, whereas the crystal is held together along the y axis through F0-F2' intramolecular interactions (Fig. S3B).Table 3Analyses of the crystal contacts of the two talin isoforms by the PDBePISA server (RRID:SCR_015749)A. The murine talin1 head domain structure, PDB entry 3IVF, residues 1–400 Δ139–168, has a surface area of 20,705 Å2xyz iNatxyz iNresSymmetry operatorSym iNatSym iNresInterface area (Å2)ΔiG (kcal/mol)ΔiG p valueNhbNsbNdsFigure7223x, −y, −z + 17223726.1−2.90.429860S2A6217−x − 1, y − 1/2, −z + 1/25922562.52.20.737820S2B5318−x − 1, −y + 1/2, z − 1/26015540.0−0.20.535950S2C4713x − 1, y, z4814386.8−1.30.415300S2D21−x, y − 1/2, −z + 1/25251.91.70.909000S2F22x, −y + 1, −z + 12216.40.80.878000S2EB. Our human talin2 head domain structure, residues 1–403, has a surface area of 23,822 Å212939x − y, x − 1, z − 1/6145391,165.2−14.60.160600S3A8327x, y − 1, z8326775.3−4.90.433340S3B6022x − y, x, z − 1/66825547.2−0.80.652200S3C3711x − y + 1, x − 1, z − 1/63310334.6−4.40.258100S3D176x − y + 1, x, z − 1/6216171.1−0.90.531210196−y, x − y − 1, z − 1/3112137.30.10.650200103−y + 2, x − y, z − 1/3155126.8−1.40.386000106x − 1, y − 1, z11486.00.10.57600011x − y − 1, x − 1, z − 1/6112.70.10.763000 Open table in a new tab Table 4Residues involved in crystal contacts in the two talin isoform structures as identified by the PDBePISA server (RRID:SCR_015749)A. Crystal contacts in the talin1 head domain structure (residues 1–400 Δ139–168)i. Fig. S2ADistance (Å)x, −y, −z + 1Tyr-70 OH2.70Asp-20" @default.
• W3038248358 created "2020-07-10" @default.
• W3038248358 creator A5043645948 @default.
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• W3038248358 date "2020-09-01" @default.
• W3038248358 modified "2023-09-28" @default.
• W3038248358 title "A distinct talin2 structure directs isoform specificity in cell adhesion" @default.
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• W3038248358 doi "https://doi.org/10.1074/jbc.ra119.010789" @default.
• W3038248358 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7489915" @default.
• W3038248358 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32605925" @default.
• W3038248358 hasPublicationYear "2020" @default.
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