The Compact Conformation of Fibronectin Is Determined by Intramolecular Ionic Interactions*

Kamin J. JohnsonDagger §, Harvey Sage, Gina BriscoeDagger , and Harold P. EricksonDagger parallel

From the Departments of Dagger  Cell Biology and  Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibronectin is a large (440-kDa) glycoprotein composed of two nearly identical covalently linked subunits found in the extracellular matrix of many tissues. Each subunit is composed of multiple homologous domains termed fibronectin types I, II, and III in an array analogous to a "string of beads" (1-3). Two major classes of fibronectin have been described, cellular fibronectin and plasma fibronectin, which differ in the inclusion or exclusion of the alternatively spliced domains A, B, and V. Plasma fibronectin is produced by hepatocytes, lacks A and B domains, but possesses the V domain in one subunit; cellular fibronectin is produced by a number of cell types and contains various combinations of A, B, and V (4-6). Fibronectin is thought to be crucial for making the primitive extracellular matrix and for cellular adhesion, migration, and differentiation (7). Underscoring the importance of fibronectin, gene knockout leads to early embryonic lethality (8).

The structure of a fibronectin molecule is diagrammed in Fig. 1 as a scale model with an integrin and the cell membrane. The molecule is composed primarily of 15 fibronectin type III (FN-III) domains plus the alternatively spliced A and B domains, which are also type III. Following the nomenclature of Leahy et al. (3), we will use the abbreviation FN7-10 to designate the segment containing FN-III domains 7 through 10. Other segments will be named accordingly. FN7-14+A will indicate that the segment includes domain A (at its natural location between FN11 and -12).


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Fig. 1.   A model of a fibronectin molecule and an integrin with domains drawn approximately to scale. The 15 FN-III domains are numbered, and the alternatively spliced A and B domains are lettered. The V domain is present in one subunit of plasma fibronectin. The number of positive and negative charged amino acids in each FN-III domain is shown above the domains.

The conformation of plasma fibronectin is dependent upon the pH and ionic environment (9-11). At physiological pH and salt concentrations and at lower ionic strength, plasma fibronectin exists in a compact conformation that sediments at 10-13.5 S. However, at increased pH or salt concentration, plasma fibronectin undergoes a reversible expansion to produce an extended conformation sedimenting at 6-8 S (9-11). This expansion occurs without any significant changes in secondary or tertiary structure (12, 13). Erickson and Carrell (11) used rotary shadowing electron microscopy to show that the fibronectin molecule in the extended conformation appeared as an extended thin strand with a few bends, whereas in the compact conformation the strand was substantially more bent and folded upon itself. These images of the fibronectin molecule in the two conformations provide the basis for understanding the transition.

The transition between fibronectin's extended and compact conformations requires a flexibility in the links between domains as well as ionic interactions to generate the compact conformation. Erickson and Carrell (11) originally proposed that all the links between FN-III domains were more or less flexible and that the important ionic interactions occurred at domain interfaces. An alternative model postulates that most of the links between FN-III domains are rigid but that there are a few specific hinge regions where the molecule can bend, leading to specific bonding of one segment of the fibronectin dimer to another segment on the same or opposite subunit (10, 14-16).

What might be the biological role of plasma fibronectin's compact conformation? Plasma fibronectin contains a number of cryptic activities revealed after proteolysis or through the use of recombinant fragments. For example, a 75-kDa fragment containing the central cell binding domain had a higher apparent affinity for cells (presumably the integrin receptors) than did intact plasma fibronectin (17). Similarly, cell spreading was enhanced for large fragments (146 kDa, 113 kDa, or 75 kDa) from the central region of plasma fibronectin, relative to intact plasma fibronectin (18). These experiments were performed under physiological pH and salt concentrations (150 mM), conditions in which plasma fibronectin is in a compact conformation (19), suggesting that the compact conformation of plasma fibronectin suppresses its biological activity. Given the large amount of fibronectin in blood, perhaps fibronectin's compact conformation curbs the wanton activation of integrins on circulating blood cells and endothelial cells.

Some isoforms of cellular fibronectin contain the alternatively spliced A domain. Expression of A is up-regulated during embryogenesis, wound healing, and tumor progression (7). Manabe et al. (20) showed that inclusion of A augmented the binding of fibronectin to alpha 5beta 1 integrin as well as the spreading of cells on fibronectin. The activity of A was only realized in the context of intact fibronectin because the isolated A domain had no augmentation activity. These data suggested that A produced a conformational change in the compact form of fibronectin that exposed the integrin binding site.

We sought to determine the domain(s) responsible for plasma fibronectin's compact conformation. We show here that FN12-14 is necessary to mediate fibronectin intersubunit dimerization and interacts with FN2-3. Similarly, the A domain is shown to interact with FN12-14, providing a structural mechanism for the functional effects of the A domain.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Thermolysin (protease, Type X), gelatin-Sepharose, and heparin-agarose were obtained from Sigma; ExpandTM High Fidelity PCR1 polymerase and dispase I from Roche Molecular Biochemicals, Pfu polymerase from Stratagene (La Jolla, CA), and Superose 6, Mono S, Mono Q, Resource Q, and cyanogen bromide-activated Sepharose 4B chromatography media from Amersham Pharmacia Biotech (Sweden).

Proteolysis of Bovine Plasma Fibronectin-- Bovine fibronectin was purified from citrated serum by elution from a gelatin-Sepharose column with 4 M urea, according to standard procedures (21). For experiments with nonpurified fragments, fibronectin was incubated with 5 µg/mg thermolysin in 10 mM Tris, pH 7.4, 150 mM NaCl, 0.2 mM CaCl2, and 0.2% NaN3 (digestion buffer) for 4 or 24 h at room temperature. Four-hour digestion resulted in major fragments of 150 kDa (FN2-15+V) and 140 kDa (FN2-14) whereas 24-h digestion gave major fragments of 140 kDa (FN2-14) and 110 kDa (FN2-11) (22). Digestion was terminated by adding EDTA to 5 mM. For purification of FN2-11, plasma fibronectin was digested overnight with thermolysin. This mixture was then applied sequentially to heparin-agarose and gelatin-Sepharose affinity columns, with FN2-11 appearing in the flow-through from both columns. FN2-11 was dialyzed against 1 mM PIPES, pH 7, 20 mM NaCl, frozen in liquid N2, and stored at -70 °C until use. For purification of FN2-14, plasma fibronectin was incubated with 5 µg/mg Dispase I for 1 h in digestion buffer. This mixture was passed over a heparin-agarose column and eluted with 500 mM NaCl. After dialysis against 20 mM Tris, pH 8.0, peak eluted fractions were applied to a Resource Q column and eluted with a 0-500 mM linear NaCl gradient. FN2-14 was dialyzed against 1 mM PIPES, pH 7.0, 20 mM NaCl, frozen in liquid N2, and stored at -70 °C until use. The identities of proteolytic fragments were confirmed by Western blotting.

cDNA and Expression Vector Construction for Recombinant Fibronectin Fragments-- Complete cloning details are available upon request. Standard molecular biological procedures were used (23). All bacterially produced fragments were expressed by inserting PCR fragments into the pET11b expression vector (Novagen, Inc., Madison, WI). PCR reactions used ExpandTM High Fidelity polymerase. A human fibronectin cDNA (hFN-IIIfull), spanning from the NsiI site to the ScaI site in the fibronectin cDNA, was prepared by several ligations of fragments derived from the following cDNAs: pFH1, pFH154, pFH134, and lF10 (24, 25). All ligations and the final hFN-IIIfull construct were verified by restriction analysis. hFN-IIIfull was used as a PCR template for bacterially expressed fibronectin fragments not containing the A domain. All forward primers contained an NdeI site just upstream of the first codon for ligation into pET11b. Reverse primer for FN12-14 contained an in-frame stop codon followed by a BamHI site; because there is a BamHI site in FN9, reverse primers for FN7-14, FN4-14, and FN2-14 contained an in-frame stop codon followed by an NdeI site. Forward primer for FN2-11 also contained a SacII site upstream of the NdeI site, whereas FN2-11 reverse primer contained an in-frame stop codon followed by a SmaI site. Because the reverse primer for FN2-11 did not contain an NdeI site, FN2-11 PCR product was first cloned into pGEM5 as an SacII-SmaI fragment and then recovered from the vector as an NdeI-NdeI fragment. FN12-14 was cloned into pET11b as an NdeI-BamHI PCR fragment. FN7-14, FN4-14, FN2-11, and FN2-14 were cloned as NdeI-NdeI PCR fragments; the correct orientation was selected by restriction mapping. FN7-10 was produced as described previously (3).

Expression vectors containing the A domain were made by replacing a fragment between FN7 and FN12 in the FN7-14 expression vector with a fragment containing the FNA coding sequence. A silent SpeI site was introduced into FN11 during the cloning procedures. To produce FN2-14+A, an FNA-containing fragment from FN7-14+A was cloned into the FN2-14 expression vector. FNA and FN12-14+A expression vectors were produced by PCR amplification using FN2-14+A expression vector as template.

We used a baculovirus expression system to produce full-length wild-type fibronectin (rFN wild-type), fibronectin in which FN-III domains 12-14 were deleted (Delta FN12-14), and fibronectin in which FN12-14 had been replaced by tenascin C FN-III domains A1-A3 (FNTNA1-A3). These constructs were produced from a full-length chimeric rat/human cDNA in pVL1393 baculovirus expression vector (a gift of Dr. Jean Schwarzbauer, Princeton University). In this vector, the first five type I repeats are rat (from nucleotides 186-1152 as in GenBankTM accession number X15906) whereas the remainder of the sequence is human (from nucleotides 864-7031 as in GenBankTM accession number A14133). Production of Delta FN12-14 fibronectin in pVL1393 involved two PCR amplifications using Pfu polymerase to generate an SpeI site (encoding amino acids TS) replacing FN12-14, followed by several ligation steps. Restriction analysis was used to verify each cloning step as well as the final vector. FNTNA1-A3 was constructed by inserting an SpeI PCR fragment (using a human tenascin C template and Pfu polymerase) containing FN-III domains A1-A3 from human tenascin into the SpeI site of Delta FN12-14. This resulted in an insertion of the 6-base pair SpeI sequence at both ends of TNA1-A3.

Purification of Recombinant Fibronectin Fragments-- Two types of sedimentation experiments were performed with recombinant fibronectin fragments. The first type, velocity sedimentation, does not require highly purified preparations because the data are analyzed by examining SDS-PAGE gels. However, the second type, equilibrium sedimentation, does require highly purified samples to obtain reliable data. Some of the proteins in this study (FNA, FN12-14, FN12-14+A, and FN4-14) could not be produced with sufficient purity to use for equilibrium sedimentation analysis.

All proteins expressed in BL21 cells were soluble; however, the highly expressed proteins, FN12-14, FN12-14+A, and FNA, contained significant amounts of insoluble protein after bacterial lysis. Proteins were purified by ammonium sulfate precipitation from bacterial supernatants followed by combinations of Resource or Mono Q, Mono S, and gel filtration chromatography. A complete description of purification procedures is available upon request. The amounts of protein recovered varied with FN12-14 being the highest (yield of 2.5 mg/liter) and FN4-14 the lowest (yield of 0.5 mg/liter).

Fibronectins expressed in baculovirus were prepared by cotransfection of Sf9 cells with the fibronectin-containing pVL1393 and Baculo Gold (Pharmingen, San Diego, CA) vectors. Culture supernatants containing recombinant virus were used to infect High Five cells grown in Express Five media (Life Technologies, Inc.). High Five cells do not produce endogenous fibronectin. After culturing for 3 days, fibronectins were purified from the culture supernatant by gelatin-Sepharose chromatography (21).

Sedimentation Analysis-- Apparent sedimentation coefficients (s) were determined by velocity sedimentation through 5-ml 15-40% glycerol gradients. Gradients were buffered using 1 mM PIPES, pH 7.0, and contained either 20 mM NaCl (low salt) or 200 mM NaCl (high salt). These salt concentrations produce near maximal changes in fibronectin sedimentation through glycerol gradients (11). Each gradient was overlaid with 200-µl samples containing a fibronectin fragment at a concentration between 200 and 500 µg/ml. Ovalbumin (3.5 S), bovine serum albumin (4.6 S), aldolase (7.3 S), and/or catalase (11.3 S) were included in each sample as internal standards. The samples were centrifuged between 34,000 and 50,000 rpm (depending upon the expected s value of the various fibronectin fragments) for 16 h in a Beckman SW-50.1 rotor at 20 °C. Following collection of 13 fractions and SDS-PAGE analysis, s values were determined by comparing the sample peaks to the standard peaks. For proteins expressed in baculovirus, sedimentation fractions were analyzed by Western blotting to detect fibronectins, in addition to silver staining the gel. s values determined in different runs were reproducible to about ± 0.2 S.

Equilibrium sedimentation was performed in a Beckman XL-A analytic ultracentrifuge. 200-µl samples, containing purified fibronectin fragments at concentrations between 200 and 400 µg/ml, in 1 mM PIPES, pH 7.0, with either 20 mM NaCl (low salt) or 500 mM NaCl (high salt), were used. Because glycerol has an effect similar to salt on fibronectin sedimentation (2, 27), the NaCl concentration used for equilibrium sedimentation under high salt conditions (500 mM) was greater than that used for velocity sedimentation (200 mM). Sedimentation was performed between 5000 and 10,000 rpm at 4 or 20 °C. Base lines, obtained by centrifuging samples at high speed, were subtracted from the measurements. The resulting absorbance profiles were fit to a single molecular species using software provided with the centrifuge.

FN2-11 Affinity Column-- 2 mg of purified FN2-11 was coupled to 0.5 ml of cyanogen bromide-activated Sepharose 4B according to the manufacturer's protocol. Purified FN7-10 and FN7-14 were combined in 20 mM Tris, pH 8.0, and passed through the column. The column was washed with 20 mM Tris, pH 8.0, and bound protein eluted with 20 mM Tris, pH 8.0, 500 mM NaCl. Fractions were analyzed by SDS-PAGE, and proteins were visualized by Coomassie Blue staining.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sedimentation of Bovine Plasma Fibronectin Proteolytic Fragments-- In trying to determine which portions of fibronectin mediate its compact conformation, our initial experiments examined the sedimentation of plasma fibronectin proteolytic fragments generated by thermolysin or dispase I. Thermolysin digestion of bovine fibronectin produced major bands of 110 kDa, 140 kDa, and 150 kDa, which represent FN2-11, FN2-14, and FN2-15+V, respectively, whereas dispase I digestion resulted in primarily FN2-14 (22). Table I shows the sedimentation coefficients for bovine fibronectin fragments FN2-11, FN2-14, and FN2-15+V determined in 1 mM PIPES, pH 7.0, buffered 15-40% glycerol gradients containing either 200 mM NaCl (high salt) or 20 mM NaCl (low salt). Purified FN2-11 and FN2-14 produced results indistinguishable from unpurified fragments. Sedimentation coefficients in high salt were similar for the three fragments but in low salt were substantially increased for FN2-15+V and FN2-14 but only moderately increased for FN2-11. Therefore, the presence of the FN12-14 domain within proteolytic fragments of FN2-14 and FN2-15+V was required to produce a substantial salt-dependent increase in sedimentation coefficient.

                              
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Table I
Velocity sedimentation of bovine fibronectin proteolytic fragments
Measurements were obtained from 15-40% glycerol gradients containing 1 mM PIPES, pH 7.0, and either 20 mM NaCl (low salt) or 200 mM NaCl (high salt).

FN12-14 Is Required for Dimerization of FN2-14 and Interacts with FN2-3-- To determine the role of FN12-14 in the increased sedimentation coefficient of FN2-14, a selection of recombinant fragments within the FN2-14 region was produced in bacteria using the pET system. Sedimentation coefficients of the fragments (FN2-14, FN4-14, FN7-14, FN12-14, and FN2-11) in high and low salt glycerol gradients are shown in Table II. Recombinant FN2-14 showed a substantial change in s (Delta s = 1.3), similar to its proteolytic counterpart (Delta s = 2.1). This indicates that glycosylation is not required to produce the salt-induced sedimentation change; however, the smaller Delta s value for recombinant FN2-14 suggests that glycosylation may enhance the salt effect. Similar to proteolytic FN2-11, recombinant FN2-11 showed only a small change in s (Delta s = 0.5) compared with FN2-14. FN12-14 alone showed no salt-induced change in sedimentation properties. Likewise, recombinant fragments having incremental deletion from the amino terminus of FN2-14 exhibited only modest salt-induced changes in sedimentation coefficient. FN7-14 had a Delta s of 0.7 whereas FN4-14 displayed a Delta s of 0.5. These data suggest that FN2-3, like FN12-14, is required to produce the sedimentation change seen in FN2-14.

                              
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Table II
Velocity sedimentation of bacterially expressed recombinant human fibronectin fragments without the alternatively spliced A domain
Measurements were obtained from 15-40% glycerol gradients containing 1 mM PIPES, pH 7.0, and either 20 mM NaCl (low salt) or 200 mM NaCl (high salt).

The salt-induced sedimentation change shown by recombinant FN2-14 could be explained by two mechanisms. First, FN2-14 may fold upon itself via an intramolecular interaction or second, may undergo intermolecular dimerization. To determine which mechanism was operating, the molecular weight of FN2-14 in low and high salt was determined by equilibrium sedimentation in 1 mM PIPES, pH 7.0, containing either 500 mM NaCl (high salt) or 20 mM NaCl (low salt) (Fig. 2). In high salt, FN2-14 had a molecular mass of 129 kDa, in good agreement with the expected molecular mass of a FN2-14 monomer. In low salt, FN2-14 had a molecular mass of 262 kDa, consistent with a dimer. Therefore, FN2-14 undergoes intermolecular bonding rather than folding upon itself in low salt. In contrast to FN2-14, the molecular masses of FN7-14 and FN2-11 indicated monomers at both high and low salt (Fig. 2).


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Fig. 2.   Equilibrium sedimentation of recombinant FN2-14, FN2-11, and FN7-14 in high and low salt. The apparent molecular masses of recombinant FN2-14 (A and B), FN7-14 (C and D), and FN2-11 (E and F) were determined by computer modeling as a function of concentration (absorbance at 230 nm; A230) across the cell. Equilibrium sedimentation was performed in 1 mM PIPES, pH 7.0, containing either 500 mM NaCl (A, C, and E; high salt) or 20 mM NaCl (B, D, and F; low salt). In high salt, the apparent molecular mass of FN2-14 remained constant as a function of concentration at 129 kDa. In low salt, the apparent molecular mass of FN2-14 increased as a function of concentration, with an average of 262 kDa. Apparent molecular masses of FN7-14 and FN2-11 were approximately those of the monomer and were not significantly influenced by the salt concentration.

Because our velocity sedimentation had suggested the dimerization was due to an interaction of FN2-3 with FN12-14 we tried to demonstrate this by sedimenting a mixture of FN2-11 and FN7-14. However sedimentation equilibrium showed no indication of heterodimers at either high or low salt. We then decided to use an affinity column to look for weaker associations. Fig. 3 shows an experiment passing a mixture of FN7-10 and FN7-14 over an affinity matrix of FN2-11. In very low salt (20 mM Tris), the FN7-14 bound completely to the column whereas FN7-10 did not. The FN7-14 was eluted by 500 mM NaCl. A later experiment showed that 50 mM NaCl was sufficient to completely elute FN7-14. We also found that FN12-14 bound completely to the FN2-11 column in 20 mM Tris and was eluted by 50 mM NaCl (data not shown). We conclude that a single FN12-14 can bind to FN2-11, presumably to the FN2-3 segment, in low salt, but the interaction is substantially weaker than the dimerization of full-length FN2-14.


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Fig. 3.   FN2-11 affinity column. Starting material (SM) containing purified FN7-14 and FN7-10 in 20 mM Tris, pH 8.0, was passed over an FN2-11 affinity column equilibrated in 20 mM Tris, pH 8.0. Whereas FN7-10 came out in the flow-through (FT) and subsequent washing with 20 mM Tris, pH 8.0, FN7-14 bound to the FN2-11 column and was eluted with 20 mM Tris, pH 8.0, containing 500 mM NaCl. Later experiments showed that FN7-14 could be eluted at 50 mM NaCl. Proteins in the collected fractions were separated using SDS-PAGE and visualized by Coomassie Blue staining.

FN12-14 Is Required to Produce the Compact Conformation of Fibronectin-- Because the previous experiments were accomplished with portions of the fibronectin molecule, it remained to be determined what role FN12-14 performed in mediating the compact conformation within the context of the entire fibronectin molecule. Because Type I and II domains contain a number of disulfide bonds, we chose to express full-length and FN12-14-deleted fibronectin in baculovirus. Unlike native fibronectin in which only one subunit contains the V domain, this baculovirus expression produces fibronectin having V in both subunits.

Two constructs with FN12-14 deleted as well as full-length "wild-type" (rFN wild-type) were produced. In the first construct (Delta FN12-14), the amino acid sequence "TS" (rendered from an inserted SpeI site) replaced FN12-14. In the second construct (FNTNA1-A3), the segment FN12-14 was replaced with type III domains A1 to A3 from human tenascin-C. Following baculovirus expression, all samples were analyzed by silver stain SDS-PAGE under reducing or nonreducing conditions (Fig. 4). They all showed a single sharp band in the reduced lane; in nonreduced lanes, the most prominent band was at the position of a dimer. Each of the recombinant proteins showed a small fraction of monomer in the nonreduced lanes, but this was judged to be less than one-fourth of the total. These results indicate that fibronectins expressed in baculovirus were mostly intact dimers. FNTNA1-A3 migrated significantly slower on SDS-PAGE than either bovine plasma fibronectin or rFN wild-type. This is likely due to anomalous migration of FNTNA1-A3, because PCR and restriction analysis of the expression construct indicated that only one A1-A3 segment was inserted (data not shown).


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Fig. 4.   Silver-stained SDS-PAGE of baculovirus-expressed fibronectins under reducing and nonreducing conditions. Fibronectins were expressed and purified as described under "Experimental Procedures." Proteins were run on a 5% polyacrylamide gel with or without 5% 2-mercaptoethanol as a reducing agent and visualized by silver staining. Under both reducing and nonreducing conditions, the majority of baculovirus-expressed fibronectins migrated similarly to a bovine plasma fibronectin control, indicating that the baculovirus-expressed fibronectins were predominantly purified as dimers. Lanes: A, bovine plasma fibronectin; B, Delta FN12-14; C, FNTNA1-A3; D, rFN wild-type.

These recombinant fibronectins were sedimented through glycerol gradients, and fractions were analyzed by silver stain SDS-PAGE and by immunoblotting for fibronectin. Each sample was sedimented with standards in the same tube, and the s value of fibronectin was estimated by linear interpolation between catalase (11.3 S) and bovine serum albumin (4.6 S). Sedimentation coefficients are given in Table III. Like plasma fibronectin, rFN wild-type showed the characteristic shift from 9.5 S in high salt to 12.6 S in low salt. The two mutant constructs with deleted or substituted FN12-14 domains sedimented at 8.4-8.6 S in high salt, suggesting that they have a more open conformation than rFN wild-type. Moreover, these constructs showed only a small (Delta FN12-14) or no (FNTNA1-A3) increase in s at low salt. We conclude that the compact conformation is substantially or completely inhibited for FN missing the FN-III domains 12-14 or having them replaced with FN-III domains from tenascin.

                              
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Table III
Velocity sedimentation of recombinant fibronectins expressed in baculovirus
Measurements were obtained from 15-40% glycerol gradients containing 1 mM PIPES, pH 7.0, and either 20 mM NaCl (low salt) or 200 mM NaCl (high salt).

The Alternatively Spliced A Domain Interacts with FN12-14-- The experiments of Manabe et al. (20), suggesting an influence of the A domain on fibronectin conformation, prompted us to examine the effect of A on the sedimentation of fibronectin fragments. For these experiments, fibronectin fragments FNA, FN12-14+A, FN7-14+A, and FN2-14+A were expressed in bacteria. Velocity sedimentation data of the fragments is shown in Table IV. In high salt the fragments containing A sedimented the same as or slightly faster than the corresponding fragments without A (compare Tables II and IV). Remarkably, all fragments containing both FN12-14 and FNA domains showed a salt-induced change in s value that was much larger than for the same segments without A. However, FNA by itself did not show any s value shift. The velocity sedimentation data suggested that the A domain of one subunit is binding to FN12-14 of the other to form a dimer. To address this issue, equilibrium sedimentation of FN7-14+A was performed. At a protein concentration of 30 µM in the presence of 150 mM NaCl, FN7-14+A formed dimers with weak associations (KD of 100 µM); at this salt and protein concentration, FN2-14 failed to associate (Table V). We conclude that FN fragments containing FN12-14+A form dimers and that this association appears to be of higher affinity than that between FN2-3 and FN12-14.

                              
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Table IV
Velocity sedimentation of bacterially expressed recombinant human fibronectin fragments containing the alternatively spliced A domain
Measurements were obtained from 15-40% glycerol gradients containing 1 mM PIPES, pH 7.0, and either 20 mM NaCl (low salt) or 200 mM NaCl (high salt).

                              
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Table V
Equilibrium sedimentation of recombinant FN2-14 and FN7-14+A in 150 mM NaCl
Equilibrium sedimentation was performed in 1 mM PIPES, pH 7.0, 150 mM NaCl at a protein concentration of 30 µM. Apparent molecular masses and dissociation constants (KD) were determined by computer modeling.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results indicate that the compact conformation of plasma fibronectin is maintained by a small number of electrostatic associations between distant segments of the molecule. A proposed structural model indicating the important associations is shown in Fig. 5. From the crystal structure of FN7-10 (3), each subunit is proposed to be relatively rigid with only a few hinge points located at specific domain interfaces. An intersubunit electrostatic bond between FN2-3 and FN12-14 brings together the two subunits of plasma fibronectin and effectively produces a compact conformation. Because our data result from a deletional analysis, however, we cannot exclude the involvement of domains near FN2-3 and FN12-14 (such as FN4 and FN11) contributing to the binding sites. The model also includes an interaction between the amino-terminal heparin binding (Hep1) domain and FN12-14, as suggested by Homandberg et al. (16). This interaction would reign in the amino termini of the plasma fibronectin subunits. Although two interactions assigned to the FN12-14 segment would appear to be hindered sterically, the interactions of FN2-3 and Hep1 with FN12-14 may occur on opposing faces of FN12-14 or use different FN-III domains within FN12-14.


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Fig. 5.   Models of proposed interactions producing the FN2-14 homodimer (A), the compact conformation of the plasma fibronectin dimer (B), and an altered conformation of cellular fibronectin containing the A domain (C). FN12-14 (hatched domains) interacts with FN2-3 (dark domains) and the amino-terminal Hep1 domain to form the compact conformation of plasma fibronectin. The FN12-14/FN2-3 contact is an intersubunit interaction (arrows with solid lines). The FN12-14/Hep1 contact is also shown as an intersubunit interaction (arrowheads). A pronounced hinge point at the interface of FN9-10 (straight-lined domains) is also displayed as a bend in the compact conformation. This bend may inactivate the integrin binding site, or alternatively, the compact conformation may bury the integrin binding site in the interior. In cellular fibronectin, interaction of the A domain (shaded) with FN12-14 may disrupt the compact conformation by competing with FN2-3 and Hep1 (interaction denoted by arrows with dashed lines). Therefore, inclusion of A is hypothesized to free FN9-10 from the function-blocking effects of the compact conformation.

FN2-14 formed a dimer detectable by sedimentation equilibrium, whereas the separate fragments FN2-11 and FN7-14 did not. The interaction of FN2-11 and FN7-14 could be demonstrated on an affinity column, but it was too weak to form stable dimers in the centrifuge. The stronger dimerization of full-length FN2-14 may be due to cooperativity. The separate fragments can only form a single electrostatic bond, but the full-length FN2-14 may be able to form two electrostatic bonds, with an FN2-3/FN12-14 pair at each end (Fig. 5). The cooperativity of forming two bonds simultaneously can substantially enhance the affinity (28).

It also seems that the interaction between FN2-14 fragments is stronger within an intact fibronectin dimer than it is for subunits free in solution. Thus, the fibronectin dimer remains in the compact conformation at 150 mM NaCl, whereas we found that FN2-14 was dimeric at 20 mM NaCl but monomeric at 150 mM NaCl. We suggest that the enhanced interaction within the dimer results from a higher effective concentration of FN2-14 binding epitopes and to a restricted, favorable orientation of the epitopes. If the epitopes are about 30 nm apart in the fibronectin dimer, this would give an effective concentration of 15 µM for one epitope relative to the other. This concentration is higher than the 2 µM concentration of free subunits in our experiments but perhaps not enough to explain the full enhancement. Favorable orientation of the epitopes toward each other could enhance this interaction further.

Our experiments do not rule out additional contacts between FN12-14 and fibronectin domains outside of FN2-14. Indeed, an interaction between Hep1 and FN12-14 plasma fibronectin proteolytic fragments has previously been reported (16, 29), and we have included this contact in our model. In fact, this interaction would appear to bring fibronectin's amino termini toward the central portion of the compact structure, further compressing the fibronectin molecule.

The electrostatic nature of the interactions is consistent with the charges associated with the domains, as shown in Fig. 1. FN12-14 and FN5 are the only domains with an excess of positive charges, whereas FN2-3 and FNA have the highest negative charge. The electrostatic bonds that we observe here may involve FN2-3 or FNA, both highly negatively charged, bonding to the positively charged FN12-14. The heparin binding site in FN13 comprises a cluster of positively charged amino acids that form a cationic cradle on one face of the domain (30). This heparin binding site may be involved in the electrostatic interaction with FN2-3, but it is likely not the only contributor because heparin was found not to induce the extended conformation (31).

As discussed in the introduction, fibronectin's compact conformation may suppress its interaction with integrins. Two structural mechanisms can be envisioned to suppress the interaction of plasma fibronectin (via FN9-10) with integrin: 1) alteration of the spatial relationship of FN9 relative to FN10 and 2) rendering FN9-10 inaccessible to integrin by burying this domain within the core of fibronectin's compact conformation. The current model of a fibronectin molecule is a series of rigid domain-domain interfaces interrupted by a small number of flexible interfaces (3). Remarkably, the interface between FN9 and FN10, which synergize to form a high affinity integrin binding site (32, 33), is very small (3), suggesting that this interface is particularly flexible. If the compact conformation utilizes the FN9 and FN10 interface as a hinge point, the bending may eliminate the possibility of FN9 and FN10 to simultaneously bind to the integrin. In support of this hypothesis, spatial disruption of FN9 relative to FN10 by insertion of a short linker sequence reduces its cell binding, spreading, and focal adhesion kinase phosphorylation activity (34).

Inclusion of the alternatively spliced A domain is known to augment fibronectin's adhesion to integrin and its cell binding and spreading activity (20, 35). The increased activity was not due to the binding of A to cells. Rather, the data suggested that incorporation of A alters the conformation of fibronectin, producing a conformation favoring integrin binding. Manabe et al. (20) proposed that inclusion of A opens the compact conformation of fibronectin by inducing a rotation in the fibronectin subunit without disrupting any long range domain-domain interactions.

Our data suggest an alternative mechanism by which A could produce an open fibronectin conformation. A proposed model of fibronectin containing the alternatively spliced A domain is shown in Fig. 5C. In our model, FN12-14+A binds to FN12-14+A of the other subunit, displacing the interactions between FN12-14 and FN2-3/Hep1. This displacement produces an open fibronectin conformation freeing the integrin binding site from the constraints of the compact conformation. The data from Manabe et al. (20) and Hino et al. (35) are also consistent with such a model. In support of our model we show that FN7-14+A can form dimers whereas FN7-14 does not dimerize. Furthermore, FN12-14+A (but not FNA or FN12-14) exhibits a salt-induced change in sedimentation, consistent with dimerization. Because FNA does not interact with itself, the interaction between FN12-14+A fragments probably involves A binding to one or two domains within FN12-14. The interaction between FNA and FN12-14 appears to be of higher affinity than that between FN2-3 and FN12-14 because FN7-14+A dimers are maintained in physiological salt, whereas FN2-14 does not form dimers in physiological salt.

    ACKNOWLEDGEMENTS

We thank Dr. Francisco Baralle, Dr. Luciano Zardi, Dr. Jean Schwarzbauer, and Dr. Albert Kornblihtt for their generous gift of fibronectin cDNAs.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA47056.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Current address: Dept. of Pathology and Laboratory Medicine, Brown University Medical School, Providence, RI 02912.

parallel To whom correspondence should be addressed: 365 Sands Bldg., Box 3011, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6385; Fax: 919-684-3687; E-mail: h.erickson{at}cellbio.duke.edu.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; FN-III, fibronectin type III domain; FN7-10, fibronectin type III domains 7 through 10; rFN wild-type, full-length recombinant fibronectin; Delta FN12-14, fibronectin with FN12-14 deleted; FNTNA1-A3, fibronectin with FN12-14 replaced with tenascin C FN-III domains A1-A3; PAGE, polyacrylamide gel electrophoresis; PIPES, piperazine-N,N'-bis-(2-ethanesulfonic acid); Delta s, change in sedimentation coefficient; Hep1, heparin binding domain nearest to fibronectin's amino terminus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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