Plasminogen and angiostatin interact with heat shock proteins

Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.     

Modularity and homology: modelling of the titin type I modules and their interfaces

Titin is a giant muscle protein with a highly modular architecture consisting of multiple repeats of two sequence motifs, named type I and type II. Type I motifs are homologous to members of the fibronectin type 3 (Fn3) superfamily, one of the motifs most widespread in modular proteins. Fn3 domains are thought to mediate protein-protein interactions and to act as spacers. In titin, Fn3 modules are present in two different super-repeated patterns, likely to be involved in sarcomere assembly through interactions with A-band proteins. Here, we discuss results from homology modelling the whole family of Fn3 domains in titin. Homology modelling is a powerful tool that will play an increasingly important role in the post-genomic era. It is particularly useful for extending experimental structure determinations of parts of multidomain proteins that contain multiple copies of the same motif. The 3D structures of a representative titin type I domain and of other extracellular Fn3 modules were used as a template to model the structures of the 132 copies in titin. The resulting models suggest residues that contribute to the fold stability and allow us to distinguish these from residues likely to have functional importance. In particular, analysis of the models and mapping of the consensus sequence onto the 3D structure suggest putative surfaces of interaction with other proteins. From the structures of isolated modules and the pattern of conservation in the multiple alignment of the whole titin Ig and Fn3 families, it is possible to address the question of how tandem modules are assembled. Our predictions can be validated experimentally.     

Binding of vitronectin and plasminogen to Helicobacter pylori

Abstract We have studied how some extracellular matrix proteins, fibronectin, fibrinogen, collagen type I and type IV, plasminogen and vitronectin bind to Helicobacter pylori . Radiolabelled vitronectin and plasminogen bound to the haemagglutinating H. pylori strain 17874 at a high level (53% and 32%, respectively), type IV collagen showed an intermediate level of binding (16%), while binding by ¹²⁵I-labelled fibrinogen, fibronectin and collagen type I remained at a low level (5–7%). Both ¹²⁵I-vitronectin and plasminogen showed a dose-dependent binding to cells of H. pylori 17874. Plasminogen binding by this strain was specific since the binding was inhibited by nonlabelled plasminogen, but not by highly glycosylated glycoproteins such as fetuin and orosomucoid or by a variety of monosaccharides. We have previously shown that ¹²⁵I-vitronectin shows a specific and saturable binding to H. pylori 17874, and that sialic acid-rich glycoproteins such as fetuin and orosomucoid drastically reduced binding. We now report that a simultaneous incubation of ¹²⁵I-vitronectin and ¹²⁵I-plasminogen with cells of H. pylori 17874 showed a total binding approximately similar to the level of binding when either ¹²⁵I-plasminogen, or ¹²⁵I-vitronectin only were incubated with the bacterial cells. Nonlabelled vitronectin inhibited the binding of ¹²⁵I-plasminogen by H. pylori , but nonlabelled plasminogen had no effect on the binding of ¹²⁵I-vitronectin. Our findings suggest that there are different but probably closely localized binding sites for vitronectin and plasminogen on H. pylori 17874.     

Calcium binding to leptospira outer membrane antigen LipL32 is not necessary for its interaction with plasma fibronectin, collagen type IV, and plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca(2+). Recent crystal structures have been obtained for the protein in the apo- and Ca(2+)-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca(2+) and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca(2+) binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca(2+) affinity as the wild-type protein. We then evaluated if Ca(2+) binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca(2+) ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.     

Origin of fibronectin type II (FN2) modules: structural analyses of distantly-related members of the kringle family idey the kringle domain of neurotrypsin as a potential link between FN2 domains and kringles

Analysis of complete genome sequences has made it clear that fibronectin type II (FN2) modules are present only in the vertebrate lineage, raising intriguing questions about the origin of this module type. Kringle domains display many similarities to FN2 domains; therefore it was suggested previously that they are highly divergent descendants of the same ancestral protein-fold. Since kringles are present in arthropodes, nematodes, and invertebrate chordates as well as in vertebrates, it is suggested that the FN2 domain arose in the vertebrate lineage through major structural modification of the more ancestral kringle fold. To explore this structural transition, in the present work we compare key structural features of two highly divergent kringle domains (the kringle of Caenorhabditis elegans Ror receptor tyrosine kinase and the kringle of rat neurotrypsin) with those of plasminogen kringles and FN2 domains. Our NMR conformation fingerprinting analysis indicates that characteristic (1)H-NMR markers of kringle or FN2 native folding, such as the dispersion of Trp aromatic connectivities and shifts of the Leu(46)/Thr(16) methyl signals, both decrease in the order kringles > neurotrypsin kringle > FN2 domains. These results suggest that the neurotrypsin kringle may represent an intermediate form between typical kringles and FN2 domains.     

Evolutionary origin of numerous kringles in human and simian apolipoprotein(a)

Human apolipoprotein(a) has a great size heterogeneity and consists of 38 kringle domains in the amino terminal and a serine protease domain in the carboxyl terminal. All but one kringle of apolipoprotein(a) are homologous to the fourth kringle of plasminogen. However, the 38th kringle resembles the fifth kringle of plasminogen and its seems to have been deleted in simian species. The phylogenetic trees suggest that an ancestral apolipoprotein(a) may have started with a duplicate of a plasminogen type protein. It also implies that deletion of the three kringles in the amino terminus followed, and that one of the remaining two kringles was duplicated in both human and simian species and the other was processed by a deletion in simian species after species separation. Thus, the number of kringles in other mammals not yet studied may vary considerably from species to species.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.     

A structural assessment of the apo[a] protein of human lipoprotein[a].

Apolipoprotein[a], the highly glycosylated, hydrophilic apoprotein of lipoprotein[a] (Lp[a]), is generally considered to be a multimeric homologue of plasminogen, and to exhibit atherogenic/thrombogenic properties. The cDNA-inferred amino acid sequence of apo[a] indicates that apo[a], like plasminogen and some zymogens, is composed of a kringle domain and a serine protease domain. To gain insight into possible positive functions of Lp[a], we have examined the apo[a] primary structure by comparing its sequence with those of other proteins involved in coagulation and fibrinolysis, and its secondary structure by using a combination of structure prediction algorithms. The kringle domain encompasses 11 distinct types of repeating units, 9 of which contain 114 residues. These units, called kringles, are similar but not identical to each other or to PGK4. Each apo[a] kringle type was compared with kringles which have been shown to bind lysine and fibrin, and with bovine prothrombin kringle 1. Apo[a] kringles are linked by serine/threonine- and proline-rich stretches similar to regions in immunoglobulins, adhesion molecules, glycoprotein Ib-alpha subunit, and kininogen. In comparing the protease domains of apo[a] and plasmin, apo[a] contains a region between positions 4470 and 4492 where 8 substitutions, 9 deletions, and 1 insertion are apparent. Our analysis suggests that apo[a] kringle-type 10 has a high probability of binding to lysine in the same way as PGK4. In the only human apo[a] polymorph sequenced to date, position 4308 is occupied by serine, whereas the homologous position in plasmin is occupied by arginine and is an important site for proteolytic cleavage and activation. An alternative site for the proteolytic activation of human apo[a] is proposed.     

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.     

The voltage-dependent anion channel is a receptor for plasminogen kringle 5 on human endothelial cells

Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1-3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1-3 or 4 has been identified as cell surface-associated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltage-dependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr252-Lys283, which is homologous to the primary sequence of VDAC residues Tyr224-Lys255. Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (Kd of 28 nm) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca2+ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5.     

Secondary structure predictions of human plasminogen and the bovine prothrombin kringle loops

Secondary structural predictions, based upon the statistical methodology of Chou and Fasman, for the kringle loops of human plasminogen and bovine prothrombin suggest a "winding staircase" pattern of beta-turns, spaced by short regions of ordered and coil structures. Analysis of the predicted structures of the regions containing the two His (113 and 387) and Asp (136 and 410) residues in plasminogen kringles 1 and 4, which have been found to be important in binding the ligand, epsilon-aminocaproic acid, shows that all are localized at the same positions on beta-turns. In addition, both of the two Asp residues occur at the end of homologous nonapeptide regions common to all of the five human plasminogen and two bovine prothrombin kringles, indicating evolutionary conservation to preserve biologically critical conformations. Examination of the protein conformation in the region of Asn288, the residue which is glycosylated in one of the two circulating variants of human plasminogen, shows that it most likely exists in a position which may present topographical hindrance to post-translational attachment of carbohydrate, thus, possibly, explaining the incomplete glycosylation of human plasminogen with complex-type carbohydrate.     

Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides.     

Analysis of the aromatic 1H-NMR spectrum of the kringle 5 domain from human plasminogen. Evidence for a conserved kringle fold

A kringle 5 domain fragment from human plasminogen has been investigated by 1H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acid/base-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in 1H2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.     

Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins.

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.     

Angiostatin and plasminogen share binding to endothelial cell surface actin.

Previous studies from this laboratory have demonstrated that plasminogen binds to endothelial cell surface-associated actin via its kringles in a dose-dependent and specific manner. The purpose of this study was to determine whether angiostatin, a proteolytic fragment of plasminogen, shares binding properties with plasminogen. Our results indicated that like plasminogen, angiostatin bound to actin in a time-, concentration-, and kringle-dependent manner. Furthermore, this binding was significantly inhibited by excess plasminogen, suggesting that both proteins shared binding motifs on the actin molecule. Fluorescence studies demonstrated that angiostatin bound to intact endothelial cells through its kringles, and this binding was also inhibited by plasminogen but not by unrelated proteins. Ligand blot analyses on endothelial cell lysates indicated that angiostatin interacted with a 42 kDa protein, which was identified as actin. Furthermore, an anti-actin antibody inhibited binding of angiostatin to endothelial cells by approximately 25%. These results suggest that angiostatin and plasminogen share binding to endothelial cell surface actin and, therefore, that angiostatin has the potential to inhibit plasmin-dependent processes such as cell migration-movement.     

Aminoalkyl affinity matrices.

Aminoalkyl matrices are used in affinity chromatography of amine oxidases and other proteins with affinity for amino groups. Under appropriate circumstances chromatography on aminoalkyl matrices may yield purification factors around 100 to 1000, and they have been used in affinity purification of many members of the amine oxidase family. Other proteins with affinity for aminoalkyl matrices include thiol ester proteins, lactoferrin, and proteins with lysine-binding kringles (plasminogen, plasminogen activator, apolipoprotein A). The affinity of thiol ester proteins for aminoalkyl matrices is abolished after inactivation of the thiol ester group by reaction with low molecular weight amines including ammonia. Due to this, an ammonium sulphate precipitation step should be included in purification schemes for amine oxidases. The affinity of lactoferrin for aminoalkyl matrices stems from an affinity for the repeating amino groups in glycosaminoglycans, and this explains why lactoferrin requires diamines for efficient elution. The affinity of plasminogen for aminoalkyl groups is exploited in a one-step purification from plasma, and is also utilised in purification schemes for angiostatin, an angiogenesis-inhibiting fragment of plasminogen. Apolipoprotein A is homologous to plasminogen, and also has affinity for aminohexyl columns. The common binding motif for these proteins are lysine-binding kringles. Due to the properties of the amino group itself, aminoalkyl matrices will inevitably also function as anion exchangers, and this must be taken into consideration in the choice of conditions for sample loading, column washing and elution of bound proteins. Depending on the length of the alkyl chain, the matrices also have a potential for hydrophobic interactions. This property has been exploited in the purification of several proteins but must be minimized during affinity chromatography of amine oxidases. In conclusion, aminoalkyl matrices are valuable tools for affinity chromatography of several different proteins, and simple variations of sample pretreatment, sample loading, and column washing and elution conditions allow efficient selective purification of proteins with different affinities for the matrices.     

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.     

Role of the Two Structural Domains from the Periplasmic Escherichia coli Histidine-binding Protein HisJ

Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.     

Integration of human plasminogen or streptokinase into stable complexes with oxidoreductases and pyruvate kinase

Summary The formation of stable equimolar complexes of streptokinase or plasminogen with muscle lactate dehydrogenase or pyruvate kinase, heart mitochondrial malate dehydrogenase and hepatic catalase at pH 7.4, 3.0 and 10.0 was first detected by differential spectroscopy methods. All complexes, except those of plasminogen with dehydrogenases, were resistant to 6 M urea. Judging from circular dichroism spectra, tertiary and secondary structures were considerably changed in the complexes. These changes were significantly dependent upon the nature of interacting proteins; in some cases their structures were more ordered. NAD (but not NADH) hampered the formation of streptokinase complexes with dehydrogenases. The plasminogen-activating function of streptokinase and the ability of plasminogen to be activated by streptokinase in the complexes with oxidoreductases were essentially unchanged. Pyruvate kinase induced a moderate (by 35%) increase in the streptokinase activating function. It is assumed that the formation of complexes of streptokinase or plasminogen with enzymes may serve as a link in metabolic regulation and/or intercellular interactions.