Reversible unfolding of an isolated heparin and DNA binding fragment, the first type III module from fibronectin.

Several fragments containing all or part of the first type III homology unit of fibronectin were isolated and their folding properties examined by fluorescence spectroscopy and differential scanning calorimetry. Each fragment exhibits a reversible unfolding transition when heated or titrated with guanidinium chloride. This indicates that an isolated type III module can fold independently in the absence of neighboring modules. A comparison of the specific enthalpies of unfolding of these fragments with those of well-studied globular proteins suggests that this type III unit is composed of a stable core flanked by less compact or unstructured regions. Comparison of the heparin-binding properties of these fragments revealed that removal of 12 amino acids from the amino terminus of the largest one (Ile-585 to Val-675) increased its affinity for immobilized heparin such that it now binds at physiological ionic strength.

Interactions between type III domains in the 110 kDa cell-binding fragment of fibronectin.

Interactions between type III domains within the cell-binding region of fibronectin have been deduced through a study of the thermal stability of the 110 kDa cell-binding fragment and a variety of its subfragments by scanning calorimetry and fluorescence spectroscopy. Comparison of the melting profiles of different fragments demonstrated that all type III modules comprise independently folded domains and revealed that in the parent 110 kDa fragment, domains 2, 4, 5, 8, 9 and 11 are relatively labile (tm near 60 degrees C) while 3, 6, 7 and 10 are thermostable (tm above 110 degrees C). Three types of interactions were found: (1) stabilizing interactions, manifested by a decrease in the tm of one of the interacting domains when they are separated; (2) destabilizing interactions for which tm is elevated upon separation; and (3) cooperative interactions in which two adjacent domains tend to melt together in a single two-state transition. Examples of the latter include the pairs 4-5 and 8-9. Long range stabilizing interactions occurred primarily between thermostable domains, 3 with 6, 6 with 7 and 10, and 7 with 9. For example, module 9, which contains the synergistic cell-binding site, is destabilized by 16 degrees C in the absence of module 7. These long range interactions, some of which could be disrupted by alternative splicing, undoubtedly influence the supertertiary structure of the central region of fibronectin, rendering it more compact than previously appreciated and possibly playing a role in the regulation of matrix assembly or interactions with other molecules such as integrin receptors on cell surfaces.

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.

Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module.

The ninth type III module of murine fibronectin was expressed in E. coli and folded into a compact homogeneous monomer whose unfolding and refolding were then investigated by fluorescence, circular dichroism, calorimetry and electron microscopy. The isolated module is unusually labile under physiological conditions. When heated at 1 deg. C/minute it exhibits an irreversible endothermic transition between 35 and 42 degrees C depending on the protein concentration. The transition is accompanied by changes in secondary and tertiary structure with partial exposure of the single tryptophan and increased binding of the hydrophobic probe, 1,8-anilinonaphthalene-sulfonate. The partially unfolded intermediate undergoes rapid self-association leading to the formation of large stable multimers that, like the original monomer, contain substantial amounts of beta sheet structure. The multimers melt and dissociate reversibly in a second endothermic transition between 60 and 90 degrees C also depending on the protein concentration. This second transition destroys the remaining secondary structure and further exposes the tryptophan. Visualization of negatively stained specimens in the electron microscope reveals that partially unfolded rmIII-9 slowly forms amyloid-like fibrils of approximately 10 nm width and indeterminate length. A subdomain swapping mechanism is proposed in which beta strands from one partially unfolded molecule interact with complementary regions of another to form oligomers and polymers. The possibility that similar interactions could play a role in the formation of fibrils by fibronectin in vivo is discussed.

Domain organization of the terminal parts in the fibrinogen molecule.

Using limited proteolysis and scanning microcalorimetry it was shown that each terminal part of the fibrinogen molecule is constituted by four co-operative domains. Among these domains two strongly interacting domains are formed by the C-terminal part of the beta-chain, while the two other domains are formed by the C-terminal part of the gamma-chain.

The cleavage of beta-chain in bovine fibrinogen DH fragment (95 kDa) leads to a significant increase in its anticlotting activity.

It is shown that in the presence of Ca2+ plasmin converts bovine fibrinogen fragment DH (95 kDa) into DLA fragment by the cleavage of its beta-chain Arg372-Thr373 bond. DLA fragment consists of two components (82 and 12 kDa) held together by non-covalent bonds and has 3.5-fold higher anticlotting activity than DH fragment. The DH to DLA fragment conversion leads to the destabilization of thermolabile domains of the latter without the loss of their compact structure. The results obtained show that the activation of DH fragment by the cleavage of its Arg372-Thr373 bond bears some resemblance to the general activation of proenzyme into enzyme.

Localization of a fibrin polymerization site complementary to Gly-His-Arg sequence.

Dansyl-labeled tetrapeptide Gly-His-Arg-Pro which mimics the central fibrin polymerization site was used to investigate its binding to a number of fibrinogen fragments containing different numbers of domains. The tetrapeptide was found to bind to fragments DH(95 kDa), DL(82 kDa) and DY(63 kDa) but not to the TSD(28 kDa) fragment. The DY fragment differs from the TSD by the presence of beta and beta C domains. Therefore these domains, which are formed by the C-terminal part of the beta chain, possess a polymerization site complementary to the Gly-His-Arg containing counterpart.

[A model of proteolysis of the fragment DH (95 kD) of the fibrinogen molecule]. / Skhema proteoliza fragmenta DH (95 kDa) molekuly fibrinogena.

A detailed analysis of further proteolytic degradation of fibrinogen fragment DH (Mr 95 kDa) was performed. Two new proteolytic fragments DLA and DL derived from DH-fragment have been purified and analyzed. The results obtained allow to propose a general scheme of DH-fragment proteolysis by stepwise splitting of their individual domains. The borders and molecular masses of these domains were evaluated on the base of proteolysis data.

[Study on secondary structure and properties of alpha- and gamma-forms of human thrombin].

Secondary structure and enzymatic properties of human a-thrombin and its gamma-form (obtaining during autolysis of the native enzyme) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). According to DSC-data both alpha-thrombin and gamma-thrombin contained only one thermal transition peak at 58.5 and 53.3 degrees C, respectively. A comparison of these values suggested that gamma-form is less stable than initial a-thrombin. In contrast to that the thermogram of DIP-a-thrombin had two peaks (57.5 and 64.5 degrees C). CD spectra showed that conversion a- to gamma-thrombin influenced the secondary structure of the enzyme slightly. The study of the inhibitory effect of such polyanions as ATP and dextran sulfate (DS) upon thrombin-catalyzed cleavages of fibrinogen has shown that the growth of the negative charge of the polyanion molecule resulted in the increase of its inhibitory activity. The catalytically non-active DIP-alpha-thrombin, which retained the native anion-binding exosite 1, was shown to decrease the inhibitory power of the dextran sulfate. It was explained by competition of DS with the exosite 1 of both alpha- and DIP-alpha -thrombin. In contrast to that DIP-gamma-thrombin having exosite 1 destroyed neither competed nor influenced the anticoagulant capacity of dextran sulfate toward the native alpha-thrombin. In accordance with our data thrombin consists of two rather strong interacting domains. It was shown further that its anion-binding exosite 1 may play a significant role in the interaction of the enzyme with dextran sulfate.

Cryptic self-association sites in type III modules of fibronectin.

The first type III module of fibronectin (Fn) contains a cryptic site that binds Fn and its N-terminal 29 kDa fragment and is thought to be important for fibril formation (Morla, A., Zhang, Z., and Ruoslahti, E. (1994) Nature 367, 193-196; Hocking, D. C., Sottile, J. , and McKeown-Longo, P. J. (1994) J. Biol. Chem. 269, 19183-19191). A synthetic 31-mer peptide (NAPQ ... TIPG) derived from the middle of domain III1 was also shown to bind Fn, but the site of its interaction was not determined (Morla, A., and Ruoslahti, E. (1992) J. Cell Biol. 118, 421-429). By affinity chromatography on peptide-agarose, we tested a set of fragments representing the entire light chain of plasma Fn. Only 40-kDa Hep-2 (III12-15) failed to bind. The concentration of urea required for peak elution of Fn and the other fragments decreased in the order Fn > 42-kDa GBF (I6II1-2I7-9) > 19-kDa Fib-2 (I10-12) > 110-kDa CBF(III2-10) > 29-kDa Fib-1 (I1-I5). Neither Fn nor any of the fragments bound immobilized intact III1, confirming the cryptic nature of this activity. In an effort to detect interactions between other Fn domains, all fragments were coupled to Sepharose, and each fragment was tested on each affinity matrix before and after denaturation. The only interaction detected was that of fluid phase III1 with immobilized denatured 110-kDa CBF and 40-kDa Hep-2, both of which contain type III domains. Analysis of subfragments revealed this activity to be dominated by domains III7 and III15. Fn itself did not bind to the denatured fragments. Thus, domain III1 contains two cryptic "self-association sites," one that is buried in the core of the fold but recognizes many Fn fragments when presented as a peptide and another that is concealed in Fn but exposed in the native isolated domain and recognizes cryptic sites in two other type III domains. These interactions between type III domains could play an important role in assembly of Fn multimers in the extracellular matrix.

The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis.

The fibulins are an emerging family of extracellular matrix and blood proteins presently having two members designated fibulin-1 and -2. Fibulin-1 is the predominant fibulin in blood, present at a concentration of 30-40 micrograms/ml (approximately 1000-fold higher than fibulin-2). During the course of isolating fibulin-1 from plasma by immunoaffinity chromatography, a 340-kDa polypeptide was consistently found to co-purify. This protein was identified as fibrinogen (Fg) based on its electrophoretic behavior and reactivity with Fg monoclonal antibodies. Radioiodinated fibulin-1 was shown to bind to Fg transferred onto nitrocellulose filters after SDS-polyacrylamide gel electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound to Fg (and fibrin) adsorbed onto microtiter well plastic, and conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to fibulin-1 was also observed in surface plasmon resonance assays, and a dissociation constant (Kd) of 2.9 +/- 1.6 microM was derived. In addition, fluorescence anisotropy experiments demonstrated that the interaction was also able to occur in fluid phase, which suggests that complexes of fibulin-1 and Fg could exist in the blood. To localize the portion of Fg that is responsible for interacting with fibulin-1, proteolytic fragments of Fg were evaluated for their ability to promote fibulin-1 binding. Fragments containing the carboxyl-terminal region of the Bbeta chain (residues 216-468) were able to bind to fibulin-1. In addition, it was found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed fibrin-containing thrombi associated with human atherectomy specimens. The interaction between fibulin-1 and Fg highlights potential new roles for fibulin-1 in hemostasis as well as thrombosis.

Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen.

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.