Domain organization of the 39-kDa receptor-associated protein.

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds to the low density lipoprotein receptor-related protein (LRP) as well as certain members of the low density lipoprotein receptor superfamily and antagonizes ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denaturation of RAP is highly reversible and occurs in a broad temperature range with two well resolved heat absorption peaks. A good fit of the endotherm was obtained with four two-state transitions suggesting these many cooperative domains in the molecule. A number of recombinant fragments of RAP were expressed in bacteria, and their domain composition and stability were characterized by DSC, circular dichroism, and fluorescence spectroscopy. The results confirmed that RAP is composed of four independently folded domains, D1, D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-323, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent molecule. Isolated D3 was partially degraded during isolation from bacterial lysates. The isolated D4 was capable of binding with high affinity to LRP whereas neither D1 nor D2 bound. At the same time a fragment containing both D1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containing D1 and D2 indicate that these two domains interact with each other and that the proper folding of the second domain into a native-like active conformation requires presence of the first domain.

Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen.

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

Domain structure, stability and interactions in streptokinase.

The structural organization of streptokinase was established through detailed study of its denaturation by differential scanning calorimetry. Streptokinase exhibited a complex endotherm whose shape was sensitive to changing pH. In all cases the endotherms were easily described by four two-state transitions indicating unambiguously the presence of four independently folded domains in the molecule. Two of them were slightly destabilized by lowering pH from 7.0 to 3.8 while the other two were stabilized in this pH range. Two proteolytic fragments of streptokinase were examined, a 37-kDa fragment beginning at Ile1 with a cleavage following Phe62, and a 17-kDa fragment beginning at Lys 147. At pH 8.5, three two-state transitions were observed in the former and two in the latter indicating this many domains in each and suggesting that the fragments are formed by a step-wise removal of individual domains from the parent molecule. Comparison of the melting of these fragments with that of streptokinase allowed the first two transitions in the parent protein to be assigned to the melting of two NH2-terminal domains and the two higher-temperature transitions to the melting of the two COOH-terminal domains. The latter two domains strongly interact with each other since the absence of the most stable extreme COOH-terminal domain in both fragments resulted in a strong destabilization of its neighbor whose melting occurred with a midpoint near room temperature. The two NH2-terminal domains seem to be more independent. One of them melts similarly in the parent protein and both fragments while the other, formed by the 1-146 region, is less stable in the 37-kDa fragment. This destabilization is most probably due to the cleavage after Phe62 which, based on the sequence similarity of streptokinase with serine proteases, may be part of a surface-oriented loop.

Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen.

Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

Thermal stability of individual domains in platelet glycoprotein IIbIIIa.

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin alpha IIb beta 3) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and inactive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low-temperature heat-absorption peak in the range of 40-65 degrees C followed by a broad peak stretching over 65-110 degrees C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low-temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15 degrees C in the presence of EDTA, suggesting that these domains are formed by the 243-468 region of the IIb subunit which contains four Ca(2+)-binding motifs. It was further stabilized by 3 degrees C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca(2+)-binding region in the formation of the ligand-binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa NH2-terminal portion of the IIIa subunit disulfide-linked to its 50-kDa COOH-terminal portion including the cysteine-rich core, exhibited a fluorescence-detected Ca(2+)-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa.

Localization of factor IXa and factor VIIIa interactive sites.

The contribution of the catalytic and noncatalytic domains of factor IXa to the interaction with its cofactor, factor VIIIa, was evaluated. Two proteolytic fragments of factor IXa, lacking some or all of the serine protease domain, failed to mimic the ability of factor IXa to enhance the reconstitution of factor VIIIa from isolated A1/A3-C1-C2 dimer and A2 subunit. Both fragments, however, inhibited this factor IXa-dependent activity. Selective thermal denaturation of the factor IXa serine protease domain eliminated its effect on factor VIIIa reconstitution. Modification of factor IXa with dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-IXa) stabilized this domain, and heat-treated DEGR-IXa retained its ability to enhance factor VIIIa reconstitution. These results indicate the importance of the serine protease domain as well as structures residing in the factor IXa light chain (gamma-carboxyglutamic acid and/or epidermal growth factor domains) for cofactor stabilizing activity. In the presence of phospholipid, the A1/A3-C1-C2 dimer produced a saturable increase in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa (Fl-FFR-IXa). This effect was inhibited by a factor IXa fragment comprised of the gamma-carboxyglutamic acid and epidermal growth factor domains. The difference in Fl-FFR-IXa anisotropy in the presence of A1/A3-C1-C2 dimer (delta r = 0.043) compared with factor VIIIa (delta r = 0.069) represented the contribution of the A2 subunit, A peptide corresponding to factor VIII A2 domain residues 558-565 decreased the factor VIIIa dependent-anisotropy of Fl-FFR-IXa to a value similar to that observed with the A1/A3-C1-C2 dimer. These results support a model of multiple interactive sites in the association of the enzyme-cofactor complex and localize sites for the A1/A3-C1-C2 dimer and the A2 subunit to the factor IXa light chain and serine protease domain, respectively.

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.

Thermal stability and domain-domain interactions in natural and recombinant protein C.

Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.

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.

Domain structure, stability and domain-domain interactions in recombinant factor XIII.

The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that the intact protein melts in two distinct temperature regions reflecting unfolding of different parts of the molecule with different stability. The less stable structures unfold in a low temperature transition with a tm of 69 degrees C or lower depending on conditions. Unfolding of the more stable structures was observed at extremely high temperatures, tm > 110 degrees C at acidic pH < 3.5 and tm = 90 degrees C at pH 8.6 with 2 M GdmCL. Thermodynamic analysis of the low and high temperature DSC-obtained heat absorption peaks indicated unambiguously that the first represents melting of three thermolabile independently folded domains while two thermostable domains melt in the second one giving a total of five domains in each a subunit of rFXIII. Both 24 kDa and 12 kDa fragments exhibited a sigmoidal spectral transition at comparatively high temperature where the thermolabile structures are already denatured, indicating that two thermostable domains are formed by the C-terminal portion of rFXIII and correspond to the two beta-barrels revealed by crystallography. The remaining 56 kDa portion forms three thermolabile domains, one of which corresponds to the N-terminal beta-sandwich and the other two to the catalytic core. Fast accessible surface calculations of the X-ray model of rFXIII confirmed the presence of two structural subdomains in the core region with the boundary at residue 332. The thermolabile domains appear to interact with each other intra- and/or intermolecularly resulting in dimerization the a subunits. At acidic pH, where all domains became destabilized but still remained folded, interdomainial interactions seemed to be abolished, resulting in the reversible dissociation of the dimer as revealed by ultracentrifugation analysis.

Role of the alpha C domains of fibrin in clot formation.

The role of the carboxyl-terminal portion of the alpha chains of fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to fibrin monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of fibrin monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.

The NH2-terminal fibrin-binding site of fibronectin is formed by interacting fourth and fifth finger domains. Studies with recombinant finger fragments expressed in Escherichia coli.

The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five finger modules of fibronectin (F1-5) binds reversibly to fibrin and facilitates cross-linking by Factor XIII. To narrow down the fibrin-binding site within this region, we have used recombinant technology to express a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and their pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli. These recombinant fragments were separated from the carrier maltose-binding protein by digestion with human factor Xa or other proteases, and their structural integrity was confirmed by spectroscopic and calorimetric methods. The recombinant F1 and F4-5 exhibited fluorescence-detected melting transitions of the same magnitude and with the same midpoint (Tm) as their natural analogues prepared from Fib-1 by proteolysis. Differential scanning calorimetry experiments further demonstrated that these fragments are properly folded and have compact structures identical to the natural ones. Isolated rF4 melts at a much lower temperature than rF5 or the bimodular fragment rF4-5, indicating the loss of a stabilizing interaction between fingers 4 and 5. Comparison of fluorescence spectra of individual rF4 and rF5 with that of rF4-5 was also consistent with an interaction that affects the environment of Trp residue(s). rF2 also melts at a lower temperature than rF3 or rF2-3, suggesting a stabilizing interaction between the second and third fingers as well. When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind. All other fragments, including individual fingers 4 and 5, failed to bind. Thus, fibrin binding is not a common property of all fingers. The results indicate that a recognition site for fibrin is located within fingers 4 and 5. The interaction between these neighboring domains may play an important role in proper orientation of the residues forming this site.

Ca(2+)-dependent interactions between Gla and EGF domains in human coagulation factor IX.

The Ca(2+)-induced interaction between the Gla and EGF domains of human factor IX was investigated by means of three fragments: 6-kDa Gla, 19-kDa (EGF)2, and 25-kDa Gla-(EGF)2. Size-exclusion chromatography and spectroscopic measurements revealed that the Gla-EGF interaction is rather strong; it can be reconstituted by mixing the 6-kDa and 19-kDa fragments which form a stable 1:1 heterocomplex in the presence of Ca2+. By itself, the 6-kDa Gla self-associates in these conditions. The Gla-EGF interaction can be disrupted in 5 M urea where the compact structure of both domains is preserved. Binding of Ca2+ to 19-kDa (EGF)2 occurred with a Kd of 71 microM in the absence and 108 microM in the presence of 5 M urea and stabilized the first EGF domain, increasing its Tm by 12 degrees C. Addition of Ca2+ to the 6-kDa and 25-kDa fragments produced biphasic changes in their fluorescence; the intensity increased slightly at low Ca2+ concentration and then decreased in a monotonic manner. In 5 M urea, only the decrease occurred, with apparent Kds of 0.33 and 0.30 mM for 6-kDa Gla and 25-kDa Gla-(EGF)2, respectively. Thus, in 5 M urea in the presence of Ca2+, the isolated Gla domain has a compact structure and Ca2+ binding properties similar to those in the 25-kDa fragment. In the absence of urea, the Gla domain interacts either with itself, when isolated, or with the first EGF domain when present, as in the 6-kDa/19-kDa heterocomplex, in the 25-kDa fragment and presumably intact factor IX.

Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases.

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.

Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated alpha C fragments on polymerization.

The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.

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.

Domain structure and domain-domain interactions in human coagulation factor IX.

Coagulation factor IX has the modular composition Gla-(EGF)2-SP, where Gla, EGF, and SP represent the gamma-carboxy-Glu-rich, epidermal growth factor-like, and serine protease modules, respectively. The protein melts in two distinct temperature regions. The SP module melts at lower temperature between 42 and 55 degrees C, depending on the pH, with irreversible loss of esterolytic activity. The endotherm corresponding to this transition is readily described by a two-state transition indicating the melting of a single cooperative unit. A thrombin-generated 12-kDa fragment representing the COOH-terminal half of the SP module and a 45-kDa fragment containing the NH2-terminal half of the SP module and the rest of the molecule can be separated by exclusion chromatography in 3 M urea and recombined in its absence. Both fragments retain a compact structure as evidenced by melting transitions near 60 degrees C at neutral pH. This indicates that the SP module contains two independently folded domains that strongly interact with each other and seem to merge into one cooperative unit in the intact protein. At low pH at high temperature a second peak appears which is also observed in a 19-kDa fragment containing the EGF modules. Thermodynamic analysis of this second peak showed that the two EGF modules are independently folded and provided evidence for a weak interaction between them. Fluorescence and CD measurements indicated that the secondary structure of the isolated 6-kDa Gla fragment is substantially increased in the presence of Ca2+. The Ca2+-loaded Gla fragment undergoes a sigmoidal unfolding transition as revealed by fluorescence and CD measurements. This same transition in a 25-kDa Gla-(EGF)2 fragment was stabilized by more than 10 degrees C, indicating a strong interaction between the Ca(2+)-loaded Gla and EGF domains. Thus, factor IX consists of five independently folded interacting domains.

Domain structure and domain-domain interactions of recombinant tissue plasminogen activator.

The melting of recombinant tissue plasminogen activator (rtPA) has been investigated by differential scanning calorimetry and fluorescence spectroscopy. At neutral pH, rtPA melts with only partial reversibility in a single sharp peak that can be deconvoluted into four transitions. By contrast, at acidic pH the melting process is spread over a broad range of temperature and is highly reversible. Under these conditions five transitions are resolved by deconvolution analysis. Additional measurements in 6 M guanidinium chloride reveal a sixth transition representing an extremely stable domain. Comparison of the melting curves of several fragments with those of the parent protein allowed all of the transitions to be assigned. The results indicate that rtPA is comprised of six independently folded domains. Two of these domains correspond to the two kringle modules whose thermodynamic properties are similar to those of the kringles in plasminogen. Two additional domains are formed by the epidermal growth factor (EGF)-like and finger modules, the latter of which is extremely stable, requiring the presence of a chemical denaturant for its melting to be observed. The serine protease module contains two more domains which at neutral pH melt cooperatively in a single transition but at low pH melt independently, accounting for the greater number of transitions observed there. Measurements with a 50-kDa fragment lacking the C-terminal half of the serine protease module and with a variant lacking the finger and EGF domains indicate that the serine protease domains interact strongly with and are stabilized by the finger and/or EGF domains in the intact protein. This interaction between domains located at opposite ends of the rtPA molecule produces a more compact structure. A better understanding of such interactions may enhance efforts to engineer plasminogen activators with improved thrombolytic properties.