Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.     

Integrity of refolded and reoxidized gelatin-binding fragments of fibronectin.

The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.     

Heparin-binding fibronectin fragments containing cell-binding domains and devoid of hep2 and gelatin-binding domains promote human embryo fibroblast proliferation.

The proliferation promoting activity of various proteolytic fragments of human plasma fibronectin was assayed. Study of this activity in fragments, purified by affinity chromatography, has shown that only heparin-binding fragments were capable of promoting fibroblast proliferation while gelatin- and fibrin-binding fragments were not. Heparin-binding fragments with high affinity for heparin were characterized by high activity levels while those with low heparin affinity were inactive. Heparin-binding fragments with the highest proliferation promoting activity contained the cell-binding domain and were virtually devoid of the hep2, hep1 and gelatin-binding domains.     

Solution structure of a pair of modules from the gelatin-binding domain of fibronectin

BACKGROUND: Fibronectin has a role in vital physiological processes such as cell migration during embryogenesis and wound healing. It mediates the attachment of cells to extracellular matrices that contain fibrous collagens. The affinity of fibronectin for native collagen and denatured collagen (gelatin) is located within a 42 kDa domain that contains four type 1 (F1) and two type 2 (F2) modules. A putative ligand-binding site has been located on an isolated F2 module, but the accessibility of this site in the intact domain is unknown. Thus, structural studies of module pairs and larger fragments are required for a better understanding of the interaction between fibronectin and collagen. RESULTS: The solution structure of the 101-residue 6F1 1F2 module pair, which has a weak affinity for gelatin, has been determined by multidimensional NMR spectroscopy. The tertiary structures determined for each module conform to the F1 and F2 consensus folds established previously. The experimental data suggest that the two modules interact via a small hydrophobic interface but may not be tightly associated. Near-random-coil 1H NMR chemical shifts and fast dynamics for backbone atoms in the linker indicate that this region is unlikely to be involved in the overall stabilisation of the module pair. CONCLUSIONS: The modules in the 6F1 1F2 module pair interact with each other via a flexible linker and a hydrophobic patch, which lies on the opposite side of the 1F2 module to the putative collagen-binding site. The intermodule interaction is relatively weak and transient.     

All six modules of the gelatin-binding domain of fibronectin are required for full affinity

The gelatin-binding sites of fibronectin are confined to a 42-kDa region having four type I and two type II modules in the following order: I(6)-II(1)-II(2)-I(7)-I(8)-I(9). To determine the relative importance of each module for recognition of gelatin, recombinant green fluorescent fusion proteins were prepared in which individual modules or groups of modules were deleted, and the resulting proteins were tested for binding to gelatin by analytical affinity chromatography. Deletion of both type II modules did not eliminate binding, confirming that at least some of the type I modules in this region are able to bind gelatin. It was found that deletion of type I module 6 tends to increase the affinity, whereas deletion of any other module decreases it. Deletion of module I(9) had a large effect but only if module II(2) was also present, suggesting an interaction between these two noncontiguous modules. Analysis of more than 20 recombinant fusion products led to the conclusion that all modules contribute to the interaction either directly by contacting the ligand or indirectly through module-module interactions.     

Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin

Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role.     

Identification of the heparin-binding determinants within fibronectin repeat III1: role in cell spreading and growth

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III1 repeat (FNIII1). In this study, we localized the heparin-binding activity of FNIII1 to a cluster of basic amino acids, Arg613, Trp614, Arg615, and Lys617. Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin- and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII1.     

The col-1 module of human matrix metalloproteinase-2 (MMP-2): structural/functional relatedness between gelatin-binding fibronectin type II modules and lysine-binding kringle domains

Human matrix metalloproteinase-2 (MMP-2) contains three in-tandem fibronectin type II (FII) repeats that bind gelatin. Here, we report the NMR solution structure of the first FII module of MMP-2 (col-1). The latter is described as a characteristic, globular FII fold containing two beta-sheets, a stretch of 3(1)-helix, a turn of alpha-helix, and an exposed hydrophobic surface lined with aromatic residues. We show that col-1 binds (Pro-Pro-Gly)6, a mimic of gelatin, with a Ka of approx. 0.42 mm(-1), and that its binding site involves a number of aromatic residues as well as Arg34, as previously found for the second and third homologous repeats. Moreover, the affinity of the in-tandem col-1+2 construct (col-12) toward the longer ligand (Pro-Pro-Gly)12 is twice that for (Pro-Pro-Gly)6, as expected from mass action. A detailed structural comparison between FII and kringle domains indicates that four main conformational features are shared: two antiparallel beta-sheets, a central 3(1)-helix, and the quasiperpendicular orientation of the two proximal Cys-Cys bonds. Structure superposition by optimizing overlap of cystine bridge areas results in close juxtaposition of their main beta-sheets and 31-helices, and reveals that the gelatin binding site of FII modules falls at similar locations and exhibits almost identical topological features to those of the lysine binding site of kringle domains. Thus, despite the minor (<15%) consensus sequence relating FII modules to kringles, there is a strong folding and binding site structural homology between the two domains, enforced by key common conformational determinants.     

Folding determinants of LDL receptor type A modules.

To investigate how three disulfide bonds and coordination of a calcium ion cooperate to specify the structure of an LDL-A module, we studied the interdependence of disulfide bond formation and calcium coordination in the folding of ligand-binding module 5 of the LDL receptor (LR5). In variants of LR5 containing only a single pair of cysteines normally disulfide-bonded in the native polypeptide, the addition of calcium does not alter the effective concentration of one cysteine for the other. LR5 only exhibits a calcium-dependent preference for formation of native disulfide bonds and detectable calcium-induced changes in structure when the two C-terminal disulfide bonds are present. Furthermore, when the conformation of this two-disulfide variant of LR5 is probed by NMR in the presence of calcium, only the C-terminal lobe of the module, which contains the calcium coordination site, acquires a near-native conformation; the N-terminal lobe appears to be disordered. These findings contrast with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is sufficient to drive the entire domain to acquire a stable, nativelike fold.     

Peptide ligands for the fibronectin type II modules of matrix metalloproteinase 2 (MMP-2)

The interaction of matrix metalloproteinase 2 (MMP-2) with gelatin is mediated by three repeats homologous to fibronectin type II (FN2) modules, which are inserted in the catalytic domain in proximity of the active site. We screened a random 15-mer phage display library to identify peptides that interact with the FN2 modules of MMP-2. Interestingly, the selected peptides are not gelatin-like and do not share a common, obvious sequence motif. However, they contain a high proportion of aromatic residues. The interactions of two peptides, WHWRH0RIPLQLAAGR and THSHQWRHHQFPAPT, with constructs comprising the in-tandem first and second and second and third FN2 modules of MMP-2 (Col-12 and Col-23, respectively) were characterized by NMR. Both peptides interact with Col-12 and Col-23 with apparent association constants in the mm(-1) range. Peptide binding results in perturbation of signals from residues located in the gelatin-binding pocket and flexible parts of the molecule. Although the former finding suggests that the gelatin-binding site is involved in the contact, the interpretation of the latter is less straightforward and may well reflect both the direct and indirect effects of the interaction.     

Tuning the mechanical stability of fibronectin type III modules through sequence variations

Cells can switch the functional states of extracellular matrix proteins by stretching them while exerting mechanical force. Using steered molecular dynamics, we investigated how the mechanical stability of FnIII modules from the cell adhesion protein fibronectin is affected by natural variations in their amino acid sequences. Despite remarkably similar tertiary structures, FnIII modules share low sequence homology. Conversely, the sequence homology for the same FnIII module across multiple species is notably higher, suggesting that sequence variability is functionally significant. Our studies find that the mechanical stability of FnIII modules can be tuned through substitutions of just a few key amino acids by altering access of water molecules to hydrogen bonds that break early in the unfolding pathway. Furthermore, the FnIII hierarchy of mechanical unfolding can be changed by environmental conditions, such as pH for FnIII10, or by forming complexes with other molecules, such as heparin binding to FnIII13.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

Uptake of homologous single-stranded fragments by superhelical DNA: II. Characterization of the reaction

We have studied the association of superhelical DNA (RFI)³ of phage G4 with defined single-stranded fragments isolated after cleavage of viral (+) strands by endonuclease R · HaeIII. The sedimentation rates of complexes formed by uptake of different single-stranded restriction fragments by G4 RFI were consistent with the view that base-pairing between the two components causes unwinding of superhelical turns, with one negative superhelical turn removed for every ten nucleotide residues of third strand taken up. The combining ratio of superhelical DNA and a single specific fragment was close to unity.At high concentrations of salt, nitrocellulose filters efficiently retained complexes of superhelical DNA and homologous fragments, which provided the basis for a rapid assay, and permitted the estimation of the thermodynamic and kinetic parameters of strand uptake in vitro. The reaction is reversible, with an apparent K_eq of approximately 10⁶m^?1. Apparent rate constants, k₁, for uptake of different fragments (85 to 1100 nucleotides long) varied about fourfold, with no obvious relationship to the length of the fragment. In 10 mm-Tris · HCl (pH 7.5), 200 mm-NaCl, fragments were taken up most rapidly at about 75 °C. Under these conditions, the apparent k₁ for a fragment 250 nucleotides long was approximately 600 m^?1s^?1, which is two or three orders of magnitude slower than the calculated rate of association of complementary strands of that length. At physiological temperatures, appreciable rates of strand uptake were seen only at low concentrations of salt (4 mm-Na⁺ in 10 mm-Tris · HCl), and were one or two orders of magnitude less than the rate at 75 °C in 200 mm-NaCl. At a given concentration of counterion a threshold temperature exists above which the rate of reaction rises sharply from an undetectable level.Thermodynamic calculations indicate that the reaction is entropically driven, and that the rate is limited by a step exhibiting a positive entropy and enthalpy of activation. The data are consistent with a model for strand uptake in which the rate-limiting step is the unstacking of a small number of base-pairs in the superhelical DNA. Stabilization and extension of the nucleus of base-pairs formed with the incoming strand is favored by the decrease in free energy associated with removal of superhelical turns.     

Immobilization of soluble fibrin on factor XIIIa-coated polystyrene beads mediated by N-terminal fibronectin fragments. II. Demonstration of covalent adducts of fibrin peptide chains and fibronectin fragments.

Previous experiments had shown that the free N-terminal fibronectin 30-kDa-domain mediates binding of soluble 125I-fibrin to transamidase-coated polystyrene beads (H?rmann et al., Biol. Chem. Hoppe-Seyler 368, 669-674, 1987). Now, the formation of covalent adducts of the N-terminal fragment with fibrin peptide chains is demonstrated. Binding of soluble 125I-fibrin was performed in presence of N-terminal fibronectin 30-kDa or 70-kDa fragments. The material adsorbed was removed from the beads under reducing conditions and analysed by dodecylsulfate gel electrophoresis followed by autoradiography. The 30-kDa fragment gave rise to bands of 80 kDa and 180-200 kDa which were lacking in the products of the 70-kDa compound. Instead, they showed bands at 120 kDa and ca. 280 kDa. Evidently, those bands represented covalent adducts of fibrin peptide chains or their dimers with the 30-kDa or the 70-kDa fragment, respectively. In addition, dimeric gamma-chains and alpha-chain polymers of fibrin were present indicating partial polymerization of bead-attached fibrin.     

Appearance and persistence of fibronectin in cartilage. Specific interaction of fibronectin with collagen type II

Binding of fibronectins (FN) to collagen types I-IV were studied using polyclonal antibodies against human and chicken FNs, proteoglycan monomers, collagen type II and monoclonal antibodies reacting with both soluble and insoluble forms of human FN. Plasma fibronectin and type II collagen were shown to interact specifically in a homologous system. Type II collagen, however, proved to be less effective in inhibition assays compared to other types of collagen. In high density cultures of chicken limb bud cells, fibronectin was first localized within the fibroblast-like cells of 4 hr cultures and an extensive extracellular filamentous network developed by the end of day 1. Fibronectin was present in the newly formed cartilage nodules although it seemed to disappear by day 6, when the proteoglycan accumulation became more intensive. Enzyme treatments (testicular hyaluronidase, chondroitinase ABC) helped to localize FN at this stage of development of chicken cartilage, in microdroplet high density cultures of human fetal chondrocytes and in articular cartilage. Fibronectin was localized only in the pericellular ring of intact human articular cartilage using monoclonal antibodies with the biotin-avidin system.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

Forced unfolding of fibronectin type 3 modules: an analysis by biased molecular dynamics simulations.

Titin, an important constituent of vertebrate muscles, is a protein of the order of a micrometer in length in the folded state. Atomic force microscopy and laser tweezer experiments have been used to stretch titin molecules to more than ten times their folded lengths. To explain the observed relation between force and extension, it has been suggested that the immunoglobulin and fibronectin domains unfold one at a time in an all-or-none fashion. We use molecular dynamics simulations to study the forced unfolding of two different fibronectin type 3 domains (the ninth, 9Fn3, and the tenth, 10Fn3, from human fibronectin) and of their heterodimer of known structure. An external biasing potential on the N to C distance is employed and the protein is treated in the polar hydrogen representation with an implicit solvation model. The latter provides an adiabatic solvent response, which is important for the nanosecond unfolding simulation method used here. A series of simulations is performed for each system to obtain meaningful results. The two different fibronectin domains are shown to unfold in the same way along two possible pathways. These involve the partial separation of the "beta-sandwich", an essential structural element, and the unfolding of the individual sheets in a stepwise fashion. The biasing potential results are confirmed by constant force unfolding simulations. For the two connected domains, there is complete unfolding of one domain (9Fn3) before major unfolding of the second domain (10Fn3). Comparison of different models for the potential energy function demonstrates that the dominant cohesive element in both proteins is due to the attractive van der Waals interactions; electrostatic interactions play a structural role but appear to make only a small contribution to the stabilization of the domains, in agreement with other studies of beta-sheet stability. The unfolding forces found in the simulations are of the order of those observed experimentally, even though the speed of the former is more than six orders of magnitude greater than that used in the latter.     

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.     

Interface characterization of the type II module pair from fibronectin

The lone (1)F2(2)F2 modular pair of fibronectin is found in the collagen-binding region. This exclusive localization suggests the (1)F2(2)F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the (1)F2(2)F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the (1)F2(2)F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. (15)N NMR relaxation data identify significant motion occurring in the linker region of the (1)F2(2)F2 pair, and analyses of the anisotropic diffusion properties of the (1)F2(2)F2 pair are consistent with the modules in the F2 pair tumbling independent of one another.