Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains 4–5FnI and 3FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes 3FnIII and disrupts the observed 4–5FnI-3FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites. Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains 4–5FnI and 3FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes 3FnIII and disrupts the observed 4–5FnI-3FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites. Fibronectin (FN), 4The abbreviations used are: FNfibronectinMSFmigration stimulation factorFnI/II/IIIFN type I/II/III domainsFn30kDafibronectin domains 1FnI-5FnIFn70kDafibronectin domains 1FnI-9FnIFn100kDafibronectin domains 1FnI-3FnIIIGBDfibronectin gelatin binding domainHSQCheteronuclear single quantum coherence. a large multidomain glycoprotein found in all vertebrates, plays a vital role in cell adhesion, tissue development, and wound healing (1.Vakonakis I. Campbell I.D. Curr. Opin. Cell Biol. 2007; 19: 578-583Crossref PubMed Scopus (65) Google Scholar). It exists in soluble form in plasma and tissue fluids but is also present in fibrillar networks as part of the extracellular matrix. The structures of many FN domains of all three types, FnI, FnII, and FnIII, are known, for example (2.Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 3.Pickford A.R. Smith S.P. Staunton D. Boyd J. Campbell I.D. EMBO J. 2001; 20: 1519-1529Crossref PubMed Scopus (69) Google Scholar, 4.Bingham R.J. Rudiño-Piñera E. Meenan N.A. Schwarz-Linek U. Turkenburg J.P. Höök M. Garman E.F. Potts J.R. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 12254-12258Crossref PubMed Scopus (109) Google Scholar). Although interactions between domains that are close in primary sequence have been demonstrated (3.Pickford A.R. Smith S.P. Staunton D. Boyd J. Campbell I.D. EMBO J. 2001; 20: 1519-1529Crossref PubMed Scopus (69) Google Scholar, 5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar), studies of multidomain fragments generally assume a beads-on-string model (2.Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). There is, however, much evidence for the presence of long range order in soluble FN as a number of functional sites, termed cryptic, are not active in the native molecule, until exposed through conformational change. These include self-association sites (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar, 6.Morla A. Ruoslahti E. J. Cell Biol. 1992; 118: 421-429Crossref PubMed Scopus (146) Google Scholar, 7.Hocking D.C. Sottile J. McKeown-Longo P.J. J. Biol. Chem. 1994; 269: 19183-19187Abstract Full Text PDF PubMed Google Scholar, 8.Ingham K.C. Brew S.A. Huff S. Litvinovich S.V. J. Biol. Chem. 1997; 272: 1718-1724Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), sites of protein interactions (9.Ingham K.C. Brew S.A. Erickson H.P. J. Biol. Chem. 2004; 279: 28132-28135Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and sites that control cellular activity (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 11.Ugarova T.P. Zamarron C. Veklich Y. Bowditch R.D. Ginsberg M.H. Weisel J.W. Plow E.F. Biochemistry. 1995; 34: 4457-4466Crossref PubMed Scopus (131) Google Scholar). Low resolution studies of the FN dimer suggest a compact conformation under physiological conditions (12.Lai C.S. Wolff C.E. Novello D. Griffone L. Cuniberti C. Molina F. Rocco M. J. Mol. Biol. 1993; 230: 625-640Crossref PubMed Scopus (46) Google Scholar, 13.Wolff C. Lai C.S. Biochemistry. 1988; 27: 3483-3487Crossref PubMed Scopus (14) Google Scholar, 14.Nelea V. Nakano Y. Kaartinen M.T. Protein J. 2008; 27: 223-233Crossref PubMed Scopus (40) Google Scholar); however, attempts to define large scale structure in FN by small angle scattering or electric birefringence (15.Sjöberg B. Pap S. Osterlund E. Osterlund K. Vuento M. Kjems J. Arch. Biochem. Biophys. 1987; 255: 347-353Crossref PubMed Scopus (21) Google Scholar, 16.Vuillard L. Roux B. Miller A. Eur. J. Biochem. 1990; 191: 333-336Crossref PubMed Scopus (9) Google Scholar, 17.Pelta J. Berry H. Fadda G.C. Pauthe E. Lairez D. Biochemistry. 2000; 39: 5146-5154Crossref PubMed Scopus (46) Google Scholar) have yielded contradictory results. Interpretation of domain stability changes in terms of interaction sites (18.Litvinovich S.V. Ingham K.C. J. Mol. Biol. 1995; 248: 611-626Crossref PubMed Scopus (50) Google Scholar) has also not been straightforward (2.Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), possibly because of domain stabilization through nearest-neighbor effects (19.Spitzfaden C. Grant R.P. Mardon H.J. Campbell I.D. J. Mol. Biol. 1997; 265: 565-579Crossref PubMed Scopus (92) Google Scholar, 20.Altroff H. Schlinkert R. van der Walle C.F. Bernini A. Campbell I.D. Werner J.M. Mardon H.J. J. Biol. Chem. 2004; 279: 55995-56003Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). fibronectin migration stimulation factor FN type I/II/III domains fibronectin domains 1FnI-5FnI fibronectin domains 1FnI-9FnI fibronectin domains 1FnI-3FnIII fibronectin gelatin binding domain heteronuclear single quantum coherence. A FN splicing variant produced in fetal and cancer patient fibroblasts, termed migration stimulation factor (MSF), stimulates migration of adult skin fibroblasts into type I collagen gels (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 21.Schor S.L. Schor A.M. Grey A.M. Rushton G. J. Cell Sci. 1988; 90: 391-399PubMed Google Scholar) and breast carcinoma cells using the Boyden chamber (22.Houard X. Germain S. Gervais M. Michaud A. van den Brûle F. Foidart J.M. Noël A. Monnot C. Corvol P. Int. J. Cancer. 2005; 116: 378-384Crossref PubMed Scopus (21) Google Scholar). MSF comprises FN domains 1FnI to 9FnI, a truncated 1FnIII, and a small C-terminal extension; a recombinant FN fragment corresponding to 1FnI-9FnI (Fn70kDa) displays the same activity (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar). An overview of FN domain structure and nomenclature is presented in Fig. 1a. Further experiments sub-localized full motogenic activity to the gelatin binding domain of FN (GBD, domains 6FnI-9FnI) (23.Schor S.L. Ellis I. Dolman C. Banyard J. Humphries M.J. Mosher D.F. Grey A.M. Mould A.P. Sottile J. Schor A.M. J. Cell Sci. 1996; 109: 2581-2590PubMed Google Scholar) and partial activity to a shorter fragment spanning domains 7–9FnI (24.Millard C.J. Ellis I.R. Pickford A.R. Schor A.M. Schor S.L. Campbell I.D. J. Biol. Chem. 2007; 282: 35530-35535Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Two IGD tripeptides of domains 7FnI and 9FnI were shown to be essential through residue substitutions and reconstitution of partial motogenic activity in synthetic peptides (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 24.Millard C.J. Ellis I.R. Pickford A.R. Schor A.M. Schor S.L. Campbell I.D. J. Biol. Chem. 2007; 282: 35530-35535Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 25.Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar); however, similar IGD tripeptides outside the GBD, on domains 3FnI and 5FnI, appear to have little effect (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 23.Schor S.L. Ellis I. Dolman C. Banyard J. Humphries M.J. Mosher D.F. Grey A.M. Mould A.P. Sottile J. Schor A.M. J. Cell Sci. 1996; 109: 2581-2590PubMed Google Scholar). Full-length adult FN does not affect cell migration in similar assays (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 23.Schor S.L. Ellis I. Dolman C. Banyard J. Humphries M.J. Mosher D.F. Grey A.M. Mould A.P. Sottile J. Schor A.M. J. Cell Sci. 1996; 109: 2581-2590PubMed Google Scholar); thus motogenic activity sites are presumed to be masked in the conformation adopted by soluble FN, although they could be exposed by molecular rearrangement. Here we show that a recombinant fragment, closely matching a truncated form of FN identified in zebrafish (26.Zhao Q. Liu X. Collodi P. Exp. Cell Res. 2001; 268: 211-219Crossref PubMed Scopus (29) Google Scholar), as well as amphibians, birds, and mammals (27.Liu X. Zhao Q. Collodi P. Matrix Biol. 2003; 22: 393-396Crossref PubMed Scopus (16) Google Scholar), does not stimulate cell migration. This fragment is similar to MSF but includes the first three FnIII domains (1–3FnIII), suggesting that these domains are for a conformational that the activity sites in this and in full-length FN. the this we studies by NMR spectroscopy and identified a specific long range interaction between domains 4–5FnI and 3FnIII as essential for this this interaction does not contacts the but possibly motogenic activity through in interactions thus a by which conformational for by or splicing in cellular activity and of type I domain of FN FN and were (24.Millard C.J. Ellis I.R. Pickford A.R. Schor A.M. Schor S.L. Campbell I.D. J. Biol. Chem. 2007; 282: 35530-35535Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, E. M.H. Werner J.M. Schwarz-Linek U. Potts J.R. Garman E.F. J. Mol. Biol. 2007; PubMed Scopus (31) Google Scholar). and were produced as C.J. Campbell I.D. I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (70) Google Scholar). and of and have been (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar). 3FnIII and were produced in a similar all protein by and NMR as in of in in a to that I. S. A. Campbell I.D. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). in produced small of protein and a number of the by in a and were and by under and conditions produced the protein of Fig. of this protein and presence of an of Fig. The were using a in a resolution Fig. of protein could be of P. under MSF produced in as (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar), a that the N-terminal FN sequence to the of and a C-terminal amino acid FN fragments of (Fn70kDa) or or residue were into in three the N terminus and including a the terminus These were produced in cell by as A.R. Jones Biol. PubMed Scopus Google Scholar). were for and the by by in A of of Protein by as The fragment The molecular of by using an and in were in and Protein Although to be three sites, a molecular of suggesting little of the of FN fragments were through an in An used for of I collagen and used to collagen gels in tissue as S.L. J. Cell Sci. PubMed Google Scholar). gels were of or the of recombinant of fibroblasts were by and in and were to the gels for a of in control and recombinant The were for and the of fibroblasts found the matrix by of in of two as (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 24.Millard C.J. Ellis I.R. Pickford A.R. Schor A.M. Schor S.L. Campbell I.D. J. Biol. Chem. 2007; 282: 35530-35535Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). a of two experiments were using in a and were using of protein interactions and of were as I. S. A. Campbell I.D. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The of and 3FnIII have been in the under and The amino acid and used to the FN number The model the and 4–5FnI structures R.J. Rudiño-Piñera E. Meenan N.A. Schwarz-Linek U. Turkenburg J.P. Höök M. Garman E.F. Potts J.R. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 12254-12258Crossref PubMed Scopus (109) Google Scholar) that the is similar to that of domain The model by the amino acid sequence through the structure using the M.J. 2008; PubMed Scopus Google Scholar) and the resolution NMR structure of that domain the N terminus (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar). A identified variant of FN (26.Zhao Q. Liu X. Collodi P. Exp. Cell Res. 2001; 268: 211-219Crossref PubMed Scopus (29) Google Scholar, X. Zhao Q. Collodi P. Matrix Biol. 2003; 22: 393-396Crossref PubMed Scopus (16) Google Scholar) the FN N terminus to the domain 3FnIII and a C-terminal amino acid We produced a recombinant of this fragment the using in cell we to this fragment as on in gels. is and experiments that is Fig. This includes domains 7FnI and 9FnI that the two IGD tripeptides for stimulation of cell however, assays using adult skin fibroblasts effect on cell a similar that full-length FN (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 23.Schor S.L. Ellis I. Dolman C. Banyard J. Humphries M.J. Mosher D.F. Grey A.M. Mould A.P. Sottile J. Schor A.M. J. Cell Sci. 1996; 109: 2581-2590PubMed Google Scholar). of the C-terminal amino acid in this not this not a recombinant fragment that lacks the first three FnIII domains (Fn70kDa) displays full activity in the same in (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar). we that domains are for a in that the sites of motogenic activity. A of this be long range interactions between the FnIII domains and the of We to interactions using We assays for interdomain interactions using NMR spectroscopy by in of FN 1FnIII, and 3FnIII were fragments and in all and the of a between of domains and and stability of FnIII domains physiological these conditions FnIII domains and soluble to domains are soluble for shown in a of specific interactions were between or 4–5FnI and or 3FnIII the interaction between 4–5FnI and 3FnIII has an of under these are of FN conformation is to T.P. Zamarron C. Veklich Y. Bowditch R.D. Ginsberg M.H. Weisel J.W. Plow E.F. Biochemistry. 1995; 34: 4457-4466Crossref PubMed Scopus (131) Google Scholar, G. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and thus we for the of these interactions under physiological conditions were although we were to in because of in the The 4–5FnI-3FnIII yielded a of not the in is the in a between these G. Curr. Opin. Biol. PubMed Scopus Google Scholar). of and between and physiological Fig. suggesting that the domains in a similar this range of interactions interaction on the of observed in and the we a for these interactions as in of were but not we these interactions in the context were but not we these interactions in the context interaction on the of observed in and the we a for these interactions as in of on the of observed in and the we a for these interactions as in of were but not we these interactions in the context in a we an interaction under physiological conditions of between and by (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar). This is the interactions between FnIII domains and under similar the of the two the of in in The 4–5FnI-3FnIII interaction not to Fig. we the interaction by NMR Fig. and small are in the NMR under physiological similar of a of and not show interactions between these using J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that 4–5FnI-3FnIII is the strongest these interactions of the on the amino acid sequence Fig. and protein structure the in the and contacts fragments the which a small in the the domain similar changes in have been as a of contacts E. M.H. Werner J.M. Schwarz-Linek U. Potts J.R. Garman E.F. J. Mol. Biol. 2007; PubMed Scopus (31) Google Scholar). is the and E. The of by 3FnIII is similar to that of however, the on is and an interaction is present on the It is that this interaction for the of 3FnIII for 4–5FnI 3FnIII affect domain scale of the and 3FnIII interactions to a model that the of on a single of the model a and of the and 4–5FnI on FnIII domains and 3FnIII and the C-terminal of for although this effect is in the 4–5FnI on for the two interactions as the terminus of and the part of the to 4–5FnI the N terminus of and the part of the of the in the of and 3FnIII in and however, of FnIII domains are to be to domain in V. Y. S. S. J. Mol. Biol. PubMed Scopus Google Scholar). The in these fragments that the observed interactions between domains could to the binding be but we were to of and yielded small of this fragment Fig. these however, to the effects of fragment Fig. of of produced little not were for and 4–5FnI and of the changes to observed for fragments a of the protein this to an of to that of the 4–5FnI-3FnIII of 3FnIII produced similar to Fig. We the in the as a binding 3FnIII the 4–5FnI in to an in the 3FnIII and a of a interaction. Anastellin, a truncated form of 1FnIII, of plasma FN to a A. Ruoslahti E. 1994; PubMed Scopus Google Scholar). type III K. Ruoslahti E. J. Mol. Biol. 2003; PubMed Scopus Google Scholar) but displays to of 1FnIII, including binding to 3FnIII (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar, Erickson H.P. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). 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The 4–5FnI-3FnIII interaction is the strongest we to through amino acid substitutions to role in long range order in show that this interaction includes a large The and interaction of these a and show a number of or of these were for including and in and and in these substitutions in a range of the of the interaction, observed for the of yielded effect on stimulation of fibroblast migration for the and substitutions the and in produced motogenic in protein and Fig. these variants protein for which that of the conformational for of the stimulation activity is of motogenic activity sites in variants could be the of in between a and conformation which large in could be in assays that on the of the we assays on and shown in Fig. is in the the a compact range FN structure and the of conformational sites have been the of much and the large of the molecule, to and specific have of the soluble plasma FN The a that functional FN variants that are to and displays activity similar to FN, which a model and an in the of FN and matrix of experiments (12.Lai C.S. Wolff C.E. Novello D. Griffone L. Cuniberti C. Molina F. Rocco M. J. Mol. Biol. 1993; 230: 625-640Crossref PubMed Scopus (46) Google Scholar, 13.Wolff C. Lai C.S. Biochemistry. 1988; 27: 3483-3487Crossref PubMed Scopus (14) Google Scholar) in single and are of includes domains that motogenic but does not stimulate fibroblast migration. We have shown that the conformational for this effect on the presence of a specific interaction between domains 3FnIII and this interaction is to a single as this protein much used in It is that a similar interaction is for the of motogenic activity in full-length however, interactions the FN dimer be Although we a model of this because of present in the and the conformation of GBD, suggest that the of are in an compact conformation we the This is to be by interactions in these interactions be but they are the be to to form The of these between 4–5FnI and 3FnIII, is by of 3FnIII and of the interaction the for A. 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P. 2007; PubMed Scopus Google Scholar). MSF (10.Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar) and the (26.Zhao Q. Liu X. Collodi P. Exp. Cell Res. 2001; 268: 211-219Crossref PubMed Scopus (29) Google Scholar, X. Zhao Q. Collodi P. Matrix Biol. 2003; 22: 393-396Crossref PubMed Scopus (16) Google Scholar) are also found as splicing variants of the FN migration stimulation and possibly be the of We have shown that sites for these are present in FN, but specific interactions this FN to splicing by or of protein domains. We that splicing as well as could be for of FN as well as control of We and Boyd for the NMR C. for of the and for the for the by the