Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions
2009; Elsevier BV; Volume: 284; Issue: 23 Linguagem: Inglês
10.1074/jbc.m109.003673
ISSN1083-351X
AutoresIoannis Vakonakis, David Staunton, Ian Ellis, Peter Sarkies, Aleksandra Flanagan, Ana M. Schor, Seth L. Schor, Iain D. Campbell,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoFibronectin (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. 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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 responsible for a conformational transition that masks the activity sites in this construct and probably in full-length FN. To identify the mechanism behind this transition, we performed structural studies by solution NMR spectroscopy and identified a specific long range interaction between domains 4–5FnI and 3FnIII as essential for this masking effect. Interestingly, this interaction does not involve direct contacts with the GBD but possibly represses motogenic activity through chain compaction, evident in analytical size exclusion assays. Intramolecular interactions thus provide a mechanism by which conformational rearrangement induced, for example, by tension or splicing variation can result in cellular activity differences. Expression and purification of type I domain pairs of FN (2–3FnI, FN residues 93–182; 4–5FnI, residues 183–275; and 8–9FnI, residues 516–608) were described previously (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, 28.Rudiño-Piñera E. Ravelli R.B. Sheldrick G.M. Nanao M.H. Korostelev V.V. Werner J.M. Schwarz-Linek U. Potts J.R. Garman E.F. J. Mol. Biol. 2007; 368: 833-844Crossref PubMed Scopus (31) Google Scholar). 6FnI1–2FnII7FnI (residues 305–515) and GBD (residues 305–608) were produced as described (29.Erat M.C. Slatter D.A. Lowe E.D. Millard C.J. Farndale R.W. Campbell I.D. Vakonakis I. Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 4195-4200Crossref PubMed Scopus (70) Google Scholar). Expression and purification of 1FnIII and 2FnIII have been described previously (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar). Domain 3FnIII (residues 810–900) and 2–3FnIII were produced in a similar manner. In all cases, protein purity was evaluated by SDS-PAGE and NMR as being in excess of 95%. 2–5FnI was integrated in Pichia pastoris in a manner analogous to that described previously (30.Vakonakis I. Langenhan T. Prömel S. Russ A. Campbell I.D. Structure. 2008; 16: 944-953Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Expression in media buffered at pH 3.5 at 25 °C produced small amounts of protein and a number of proteolytic degradation products. 2–5FnI was concentrated from the media by retention in a cation exchange column, and protein-containing fractions were pooled and purified by size exclusion chromatography. SDS-PAGE under reducing and nonreducing conditions produced the expected protein band essentially free of degradation by-products (supplemental Fig. 6). 1H-15N HSQC spectra of this protein showed substantial heterogeneity and significant presence of an unfolded population of molecules (supplemental Fig. 6). The different species were separated using a shallow NaCl gradient in a high resolution anion exchange column (MonoQ, GE Healthcare) at pH 10.6 (supplemental Fig. 6). Approximately 0.2 mg of correctly folded purified protein could be obtained from 0.5 liters of P. pastoris culture under high density fermentation conditions. Recombinant MSF was produced in Escherichia coli, as described previously (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), from a transcript that included the complete N-terminal FN sequence to the middle of 1FnIII (residue 647) and a unique C-terminal amino acid tail. FN fragments consisting of residues 48–608 (Fn70kDa) or 48–900 (Fn100kDa, wild-type or with residue substitutions) were cloned into vector pHLsec resulting in final proteins with three vector-derived residues at the N terminus (ETG) and nine, including a His tag, at the C terminus (GTKHHHHHH). These proteins were produced in human cell line HEK293T by transient expression as described previously (31.Aricescu A.R. Lu W. Jones E.Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 1243-1250Crossref PubMed Scopus (544) Google Scholar). Proteins were expressed for 4 days and then purified directly from the media by metal affinity chromatography followed by size exclusion in phosphate-buffered saline. A typical yield of purified Fn100kDa was 20 mg per liter of conditioned media. Protein purity was evaluated by SDS-PAGE as 85–90%. The proteolytic Fn30kDa fragment was purchased from Sigma. The molecular mass of Fn100kDa was determined by sedimentation equilibrium analysis using an Optima XL-A analytical ultracentrifuge (Beckman). Fn100kDa samples (5 and 11 μm) in phosphate-buffered saline were centrifuged in double sector 12-mm centerpieces at 10,000, 12,000, and 16,000 rpm at 20 °C. Protein sedimentation was monitored at 280 nm. Although expected to be glycosylated at three separate sites, Fn100kDa gave a molecular mass of ∼95 kDa suggesting little glycosylation of the final protein. Small (0.1 ml) samples of FN fragments were passed through an analytical Superdex 200 size exclusion column (GE Healthcare) equilibrated in phosphate-buffered saline. An Äkta FPLC system with 0.5-cm path length (GE Healthcare) was used for data recording of UV absorption at 280 nm. Type I collagen was extracted from rat tail tendons and used to make 2-ml collagen gels in 35-mm plastic tissue culture dishes as described previously (32.Schor S.L. J. Cell Sci. 1980; 41: 159-175Crossref PubMed Google Scholar). Collagen gels were overlaid with 1 ml of either serum-free minimum Eagle's medium or serum-free minimum Eagle's medium containing four times the final concentration of recombinant proteins. Confluent stock cultures of fibroblasts were trypsinized, pelleted by centrifugation, and resuspended in growth medium containing 4% donor calf serum at 2 × 105 cells/ml, and 1-ml aliquots were added to the overlaid gels for a final concentration of 1% serum in both control and recombinant proteins containing cultures. The assay cultures were incubated for 4 days, and the percentage of fibroblasts found within the three-dimensional gel matrix was then ascertained by microscopic observation of 10 randomly selected fields in each of two duplicate cultures, as described previously (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). Each experiment was repeated a minimum of two times. All experiments were performed at 30 °C using home-built spectrometers with 11.7–22.3 tesla field strengths in a 20 mm sodium phosphate, pH 6.0, 20 mm NaCl, 2 mm EDTA, 0.1 mm 2,2-dimethyl-2-silapentanesulfonic acid, 0.02% NaN3, and 5% v/v D2O sample buffer unless otherwise noted. Sequential chemical shift assignments were performed using standard triple-resonance experiments. Analysis of spectral perturbations upon protein interactions and determination of equilibrium parameters were performed as described (30.Vakonakis I. Langenhan T. Prömel S. Russ A. Campbell I.D. Structure. 2008; 16: 944-953Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The chemical shift assignments of 2–3FnI, 4–5FnI, and 3FnIII have been deposited in the BioMagResBank under accession numbers 15756, 15758, and 15759, respectively. The amino acid sequences and numbering schemes used here correspond to the human FN UniProt accession number P02751. The 2–5FnI model was constructed from the 2–3FnI and 4–5FnI crystal structures (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) assuming that the 3–4FnI interface is similar to that of other FnI domain pairs. The 2–3FnIII model was constructed by threading the amino acid sequence through the 9–10FnIII structure (Protein Data Bank code 2MFN) using the Phyre web service (33.Bennett-Lovsey R.M. Herbert A.D. Sternberg M.J. Kelley L.A. Proteins. 2008; 70: 611-625Crossref PubMed Scopus (362) Google Scholar) and then substituting 2FnIII with the high resolution NMR structure of that domain excluding the flexible 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 previously identified alternatively spliced mRNA variant of FN (26.Zhao Q. Liu X. Collodi P. Exp. Cell Res. 2001; 268: 211-219Crossref PubMed Scopus (29) Google Scholar, 27.Liu X. Zhao Q. Collodi P. Matrix Biol. 2003; 22: 393-396Crossref PubMed Scopus (16) Google Scholar) encodes the FN N terminus up to the complete domain 3FnIII and a highly variable C-terminal amino acid tail. We produced a recombinant version of this fragment excluding the tail (Fig. 1a) using transient expression in HEK293T cell line; we refer to this fragment as Fn100kDa, based on its apparent mobility in denaturing gels. Fn100kDa is highly soluble, and analytical ultracentrifugation experiments showed that it is monomeric at ∼11 μm (1.0 mg/ml) concentration (supplemental Fig. 1). This construct includes domains 7FnI and 9FnI that harbor the two IGD tripeptides necessary for stimulation of cell migration; however, assays using adult skin fibroblasts showed no effect on cell motility (Fig. 1a), a result similar that obtained with 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). Inclusion of the C-terminal amino acid tail in this construct did not alter this result (data not shown). In contrast, a recombinant fragment that lacks the first three FnIII domains (Fn70kDa) displays full activity in the same assays, in agreement with previous results (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). Hence, we hypothesized that domains 1–3FnIII are responsible for a structural rearrangement in Fn100kDa that masks the sites of motogenic activity. A likely cause of this rearrangement would be long range interactions between the FnIII domains and the remainder of Fn100kDa. We therefore sought to identify any such interactions using NMR. We performed assays for interdomain interactions using solution NMR spectroscopy by monitoring chemical shift perturbations in 1H-15N HSQC spectra during titrations of FN fragments. Domains 1FnIII, 2FnIII, and 3FnIII were tested against fragments 1–2FnI, 2–3FnI, 4–5FnI, 6FnI1–2FnII7FnI, and 8–9FnI in all pairwise combinations at 30 °C and 20 mm NaCl, 20 mm sodium phosphate pH 6.0 buffer; the choice of buffer pH reflects a compromise between solubility of FnI domains (high at lower pH), and solubility and stability of FnIII domains (high at physiological pH). Under these conditions FnIII domains remained folded and soluble to at least 1 mm concentration, whereas FnI domains are less soluble (∼0.2 mm for 4–5FnI). As shown in Table 1, a total of four specific interactions were detected between 2–3FnI or 4–5FnI and 2FnIII or 3FnIII (Fig. 2); the interaction between 4–5FnI and 3FnIII has an equilibrium dissociation constant (Kd) of 146 ± 9 μm under these conditions, whereas others are weaker (Kd of 700–1200 μm). FN conformation is sensitive to ionic strength (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, 34.Alexander Jr., S.S. Colonna G. Edelhoch H. J. Biol. Chem. 1979; 254: 1501-1505Abstract Full Text PDF PubMed Google Scholar), and thus we tested for the persistence of these four interactions under physiological conditions (150 mm NaCl). Chemical shift perturbations were still observed, although we were unable to estimate dissociation constants accurately in some cases because of low fractional saturation in the respective titrations. The 4–5FnI-3FnIII titration yielded a Kd of 800 ± 46 μm (data not shown); the 5-fold reduction in affinity is consistent with the increase in ionic strength, assuming a linear anticorrelation between these phenomena (35.Schreiber G. Curr. Opin. Struct. Biol. 2002; 12: 41-47Crossref PubMed Scopus (270) Google Scholar). Combined analysis of 1H and 15N perturbations shows good correlation between low and physiological NaCl concentrations (supplemental Fig. 2), suggesting that the domains interact in a similar fashion over this range of ionic strength.TABLE 1Dissociation constants from inter-domain interactions (Kd, mm)FN fragments1FnIII2FnIII3FnIII1–2FnINo interaction detectedaBased on the extent of perturbations observed in other titrations, and the stoichiometric ratios used, we estimate a lower limit for these interactions as in excess of 7.5–10 mm.–bSmall perturbations were detected on2FnI resonances but not on1FnI; hence we consider these interactions in the context of2–3FnI.–bSmall perturbations were detected on2FnI resonances but not on1FnI; hence we consider these interactions in the context of2–3FnI.2–3FnI0.71 ± 0.020.82 ± 0.074–5FnI1.2 ± 0.20.146 ± 0.0096FnI1–2FnII7FnINo interaction detectedaBased on the extent of perturbations observed in other titrations, and the stoichiometric ratios used, we estimate a lower limit for these interactions as in excess of 7.5–10 mm.8–9FnIa Based on the extent of perturbations observed in other titrations, and the stoichiometric ratios used, we estimate a lower limit for these interactions as in excess of 7.5–10 mm.b Small perturbations were detected on2FnI resonances but not on1FnI; hence we consider these interactions in the context of2–3FnI. Open table in a new tab Previously, we reported an interaction Kd under physiological ionic strength conditions of ∼85 μm between wild-type 1–2FnIII and 1–5FnI (Fn30kDa), determined by surface plasmon resonance (5.Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar). This is substantially tighter than the interactions reported here between isolated FnIII domains and FnI pairs under similar conditions. However, the nature of the two techniques used, especially the difference of limited diffusion in surface plasmon resonance versus three-dimensional diffusion in NMR, makes comparisons across techniques difficult. The 4–5FnI-3FnIII interaction did not prove amenable to surface plasmon resonance analysis (supplemental Fig. 3); thus, we tested the 1–2FnIII-Fn30kDa interaction by NMR (supplemental Fig. 4). As shown, only few and very small perturbations are detected in the NMR spectra under physiological conditions, compared with similar spectra of 4–5FnI-3FnIII. Similarly, a recent study of wild-type 1–2FnIII and Fn70kDa did not show substantial interactions between these components using fluorescence (36.Karuri N.W. Lin Z. Rye H.S. Schwarzbauer J.E. J. Biol. Chem. 2009; 284: 3445-3452Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Hence, we infer that 4–5FnI-3FnIII is the strongest among these interactions of wild-type proteins. Mapping the chemical shift perturbations on the amino acid sequence (supplemental Fig. 5) and protein structure highlights the surfaces involved in the 2–5FnI and 2–3FnIII contacts (Fig. 3). 2FnIII perturbs both FnI fragments along the domain-domain (2FnI/3FnI, 4FnI/5FnI) interfaces, which probably reflects a small change in the preferred orientation within the domain pairs; similar changes in FnI pairs have been noted earlier as a result of changing local contacts (28.Rudiño-Piñera E. Ravelli R.B. Sheldrick G.M. Nanao M.H. Korostelev V.V. Werner J.M. Schwarz-Linek U. Potts J.R. Garman E.F. J. Mol. Biol. 2007; 368: 833-844Crossref PubMed Scopus (31) Google Scholar). In addition, 5FnI is perturbed along the β-strand A/E interface and 2FnI along strand E. The pattern of perturbations by 3FnIII is similar to that of 2FnIII; however, the perturbation on 2FnI is more pronounced, and an additional interaction surface is present on 4FnI along the β-strand D/E loop. It is likely that this additional interaction surface accounts for the higher affinity of 3FnIII for 4–5FnI compared with 2FnIII (146 ± 9 μm versus 820 ± 70 μm Kd). Neither 2FnIII nor 3FnIII significantly affect domain 3FnI. Large scale mapping of the 2FnIII and 3FnIII interactions to a 2–5FnI model shows that the vast majority of perturbed areas localize on a single side of the model (Fig. 3, a and b). Similar interface mapping of the 2–3FnI and 4–5FnI titrations on FnIII domains (Fig. 3, c and d) shows strong perturbations along 3FnIII β-strand D and the C-terminal end of strand E for both interactions, although this effect is more highly pronounced in the 4–5FnI titration. In contrast, perturbations on 2FnIII differ for the two interactions as 2–3FnI primarily affects the C terminus of β-strand G and the strand C/D loop (the part of the molecule proximal to 3FnIII), whereas 4–5FnI affects the N terminus of strand G and the strand F/G loop (the distal part of the molecule compared with 3FnIII). Inspection of the modeled 2–3FnIII indicates that, in both cases, the perturbed interfaces of 2FnIII and 3FnIII orient in opposite directions (Fig. 3, c and d); however, chains of FnIII domains are known to be prone to domain reorientation in solution (37.Copié V. Tomita Y. Akiyama S.K. Aota S. Yamada K.M. Venable R.M. Pastor R.W. Krueger S. Torchia D.A. J. Mol. Biol. 1998; 277: 663-682Crossref PubMed Scopus (133) Google Scholar). The flexibility inherent in these multiple module fragments suggested that the observed interactions between adjacent domains could act cooperatively to enhance the binding affinity. 2–3FnIII can be easily produced, but we were initially unable to express 2–5FnI. Optimization of expression and purification procedures yielded only small amounts of this fragment (supplemental Fig. 6); these were, however, sufficient to test the effects of increasing fragment size. 15N-enriched 2–3FnI at low concentrations (20 μm) was titrated with 50 μm unenriched 2–3FnIII (su
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