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.     

Polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin

Gelatin-binding chymotryptic fragments from placental fibronectin contain polylactosamine carbohydrates (Zhu, B.C.R., Fisher, S.R., Pande, H., Calaycay, J. Shively, J.E., and Laine, R.A. (1984) J. Biol. Chem. 259, 3962-3970). We have separated polylactosamine-containing gelatin-binding fragments of placental fibronectin from their counterparts containing smaller "complex" N-linked saccharides using Sephadex G-200 gel permeation chromatography. The peptide portions of both fragments have similar amino acid composition and N-terminal sequence (see reference above). The strength of binding of these two glycosylation types of chymotryptic fragments to gelatin differs as shown by the following experiments. 1) Upon urea gradient elution of affinity-bound fibronectin fragments from gelatin-Sepharose chromatography, the apex of the elution peak for polylactosamine-containing fragments occurs at 2.0 M urea while the peak for complex N-linked carbohydrate-containing fragments maximized at 2.5 M urea indicating a tighter binding. Removal of polylactosamine sequences from the former glycopeptide by endo-beta-galactosidase digestion caused the elution peak for this fraction to change from 2.0 to 2.5 M, the same as for the complex N-linked carbohydrate-containing glycopeptide. 2) Competitive displacement experiments give an apparent dissociation constant of polylactosamine-containing fragments at 3 X 10(-9) M whereas this constant for complex carbohydrate-containing fragments is 1 X 10(-9) M. These results indicate that the binding of placental fibronectin to gelatin is weakened by the presence of high molecular weight polylactosamine carbohydrate. To our knowledge this is the first report that the type and extent of glycosylation of a glycoprotein can affect its binding affinity to a proteinacious ligand. Thus, fetal placental fibronectin may have different biological properties than fibronectins containing only the smaller N-linked complex carbohydrate.     

The PDC-109 protein from bovine seminal plasma is similar to the gelatin-binding domain of bovine fibronectin and a kringle domain of human tissue-type plasminogen activator

PDC-109, a protein of unknown function, is a major component of bovine seminal plasma. Using a computer program designed to detect evolutionary relationships between proteins, I find that the PDC-109 protein is similar to the gelatin-binding domain of bovine fibronectin and part of a kringle domain of human tissue-type plasminogen activator (t-PA). The computer-based comparison of the amino acid sequence of PDC-109 with that of the gelatin-binding domain of fibronectin and part of the second kringle domain of t-PA yields scores that are 15.5 standard deviations and 7.8 standard deviations higher, respectively, than were obtained with a comparison of randomized sequences of these proteins. The probability (p) of getting these scores by chance is less than 10(-50) and 3 X 10(-15), respectively. The similarity between the amino acid sequences of PDC-109 and the gelatin-binding domain in fibronectin and the kringle of t-PA suggests some approaches for identifying the functions of PDC-109. Both t-PA and the gelatin-binding domain of fibronectin have adhesive functions, and the gelatin-binding domain promotes viral transformation of fibroblasts in culture. These functions may be associated with the PDC-109 protein.     

Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin

We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions.     

Intrinsic tryptophan fluorescence of human serum proteins and related conformational changes

The unfolding of human serum proteins (HSP) was studied by measuring the intrinsic fluorescence intensity at a wavelength of excitation corresponding to tryptophan's or typosine's fluorescence and surface hydrophobicity. The maxima emission wavelengths (_max) of human serum albumin (HSA) and human serum globulin (HSG) before beer consumption (BC) were 336.0 and 337.0 nm and after BC shifted to 335.0 and 334.0 nm, respectively. The surface hydrophobicity slightly increased after BC. In a solution of 8 M urea the _max of BSA shifted to 346.4 and that of BSG to 342.5 nm. In contrast, in the same solution but after BC the _max positions of HSA and HSG shifted to 355.9 and 357.7 nm, respectively. A decrease in fluorescence intensity, a shift in the maximum of emission, and an increase in surface hydrophobicity which reflected unfolding of proteins were observed. Here we provide evidence that the loosening of the HSP structure takes place primarily in various concentrations of urea before and after beer consumption. Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption.     

Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides.     

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.     

The 14-3-3 protein affects the conformation of the regulatory domain of human tyrosine hydroxylase

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.     

Reversible unfolding of the gelatin-binding domain of fibronectin: structural stability in relation to function

Fibronectin, a large multidomain glycoprotein, binds denatured collagen (gelatin) and mediates cell attachment and spreading on collagen-coated surfaces. Despite the high affinity, binding to gelatin is disrupted by relatively mild conditions. We have examined the effects of denaturants on the structure and function of a 42-kDa gelatin-binding fragment (GBF) isolated from chymotryptic and thermolytic digests of the parent protein. Application of linear gradients to GBF-loaded gelatin-agarose columns resulted in peak elution of the fragment at pH 5.2 or 10.2, at 0.4 M dimethylformamide, 0.9 M GdmCl, or 2.0 M urea, conditions far short of those required to induce structural changes detectable by fluorescence or circular dichroism. Solvent perturbation, fluorescence quenching, and chemical modification experiments indicate that about half of the 8 tryptophans, one-third of the 21 tyrosines, and all of the 9 lysine residues are solvent-exposed in the native protein and that 1 or more of the latter are directly involved in binding to gelatin, most likely through a hydrogen-bonding mechanism. Titration with GdmCl produced a single unfolding transition centered near 2.5 M GdmCl as monitored by changes in fluorescence and circular dichroism. This transition was fully reversible with complete recovery of structural parameters and gelatin binding. Treatment with disulfide reducing agents caused rapid irreversible changes in structure similar to those produced by GdmCl with concomitant loss of gelatin binding. Thus, tertiary and secondary structures are important for binding, but binding can be disrupted without perturbing those structures.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

The single tryptophan residue of human placental lactogen. Effects of modification and cleavage on biologic activity and protein conformation

In order to define further the chemical features of the human placental lactogen (hPL) molecule responsible for its lactogenic activity, two derivatives of the hormone were prepared by treatment with BNPS-skatole (2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine). At a molar ratio of reagent to hPL of 7:1, a derivative was produced in which the single tryptophan was completely oxidized. At higher ratios, a second derivative was formed in which the peptide chain was cleaved at the tryptophan residue and the two resulting fragments remained bound by the disulfide bond between Cys53 and Cys165. Oxidation of the single tryptophan resulted in reduced immunologic activity, reduced helical content as measured by circular dichroism below 240 nm, and changes in the near-UV circular dichroic spectrum, each indicating a change in the conformation of the hPL molecule. Nevertheless, this derivative retained 20% of its ability to bind to lactogenic receptors and 40 to 50% of its ability to stimulate N-acetyllactosamine synthetase in vitro. Cleavage at the tryptophan was not complete, but the loss of immunologic and biologic activity was equivalent to the degree of cleavage, indicating that the cleaved derivative was completely inactive. In addition, separation of the cleaved fragments from intact hormone followed by recombination did not generate any immunologic or biologic activity. We conclude that the single tryptophan of hPL is not essential for the biologic activity of hPL. It is likely that the reduced activity associated with modification or cleavage at the tryptophan residue is due to changes in the conformation of the molecule.     

Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. Presence of polylactosamine glycopeptides and properties of a 44-kilodalton chymotryptic collagen-binding domain: difference from human plasma fibronectin

Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins.     

Experiments suggest that simulations may overestimate electrostatic contributions to the mechanical stability of a fibronectin type III domain

Steered molecular dynamics simulations have previously been used to investigate the mechanical properties of the extracellular matrix protein fibronectin. The simulations suggest that the mechanical stability of the tenth type III domain from fibronectin (FNfn10) is largely determined by a number of critical hydrogen bonds in the peripheral strands. Interestingly, the simulations predict that lowering the pH from 7 to approximately 4.7 will increase the mechanical stability of FNfn10 significantly (by approximately 33 %) due to the protonation of a few key acidic residues in the A and B strands. To test this simulation prediction, we used single-molecule atomic force microscopy (AFM) to investigate the mechanical stability of FNfn10 at neutral pH and at lower pH where these key residues have been shown to be protonated. Our AFM experimental results show no difference in the mechanical stability of FNfn10 at these different pH values. These results suggest that some simulations may overestimate the role played by electrostatic interactions in determining the mechanical stability of proteins.     

Heterogeneity of protein conformation in solution from the lifetime of tryptophan phosphorescence

The decay of Trp phosphorescence of proteins in fluid solutions was shown to provide a sensitive tool for probing the conformational homogeneity of these macromolecules in the millisecond to second time scale. Upon examination of 15 single Trp emitting proteins multiexponential decays were observed in 12 cases, a demonstration that the presence of slowly interconverting conformers in solution is more the norm rather than an exception. The amplitude of preexponential terms, from which the conformer equilibrium is derived, was found to be a sensitive function of solvent composition (buffer, pH, ionic strength and glycerol cosolvent), temperature, and complex formation with substrates and cofactors. In many cases, raising the temperature, a point is reached at which the decay becomes practically monoexponential, meaning that conformer interconversion rates have become commensurate with the triplet lifetime. Estimation of activation free energy barriers to interconversion shows that the large values of DeltaG* are rather similar among polypeptides and that the protein substates involved are sufficiently long-lived to display individual binding/catalytic properties.     

Photoconformational relaxation of protein structure from the data of tryptophan fluorescence

It was shown that the decrease of polarization degree with increasing the wavelength of protein tryptophane fluorescence lambda f was caused by the lengthening of the lifetime tau. A considerable rotational nanosecond mobility of the internal tryptophanyls was observed. This mobility is not spontaneous but caused by photoexcitation. The increase of tau with lambda f is observed not only for multitryptophan proteins but also for monotryptophan ones and for DL-tryptophane in glycerol. The increase of tau is caused by relaxation processes occurring in close proximity to tryptophane after its photoexcitation and reflects the photoinduced nanosecond mobility of the tryptophane microenvironment. Photoconformational relaxation of protein in the nanosecond time range and possible role of this process in photoinactivation of enzymes are discussed.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Actions of the functional upstream domain of protein F1 of Streptococcus pyogenes on the conformation of fibronectin

Fibronectin (Fn), discovered by Harvard's Plasma Protein Program as plasma "cold-insoluble globulin" in the 1940s, has attracted much interest over the past three decades. One of the most interesting features of Fn is its ability to change shape in response to various environmental conditions and interactions with other substances found in the extra-cellular space. Here we examine the potential of the functional upstream domain (FUD) of Streptococcus pyogenes protein F1 to bring about changes in structure of Fn. In particular, we investigate the accessibility of Fn's 10th type III module that contains the integrin binding RGD motif. By use of monoclonal antibodies in a competitive ELISA assay, we found that FUD interacts with the amino-terminal type I modules of Fn to unveil the cell-binding region of Fn. This conformational change was achieved at sub-equimolar ratios of FUD/Fn monomer. We discuss the functional relevance of the interaction for both Fn and S. pyogenes and correlate the results with a conformational model of Fn that arose out of a collaboration between our laboratory and that of John Ferry.     

Fluorescent fusion proteins derived from the tenth human fibronectin domain

Hybrid molecules of a new type bearing a red fluorescent protein mCherry and one of the alternative scaffold proteins, the 10th human fibronectin type III domain (¹⁰Fn3), which can be used for the construction of antibody mimics with various binding specificity, were obtained. Different variants of the gene encoding the hybrid fluorescent protein were constructed and their expression in Escherichia coli cells was studied. The mCherry N-terminal position and the modification of its N-terminal amino acid sequence proposed were shown to promote the efficient bacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construct a hybrid fluorescent protein ChIBF containing an α_Vβ₃-integrin binding variant of ¹⁰Fn3 was obtained, and its use for the visualization of α_Vβ₃-integrin at the surface of MDCK epithelial cells was demonstrated by confocal microscopy.