Antithrombin activity of the hirudin N-terminal core domain residues 1-43

A tetrahedral intermediate is the prominent feature of the generally accepted mechanism for aspartate transcarbamoylase. We have synthesized N-pyrophosphoryl-L-aspartate as a charged analogue of the postulated intermediate. Surprisingly, its affinity for the enzyme from Escherichia coli was substantially lower than that of the previously known inhibitor phosphonoacetyl-L-aspartate which contained a trigonal carbonyl group. Similar results were obtained with the corresponding mercaptosuccinate derivatives. We also tested a number of new pyrophosphate analogues as inhibitors. Our results cast doubt on some aspects of the current model for the mechanism of this enzyme.     

Antithrombin activity of the hirudin N-terminal core domain residues 1–43

Hirudin N-terminal core domain residues 1–43 (r-Hir^1–43) were prepared from limited proteolysis of recombinant hirudin by V8 Staphylococcal protease followed by purification with reversed-phase HPLC. r-Hir^1-43 lacks the putative reactive site of hirudin (Lys⁴⁷), but binds to thrombin (with K_i of 300 nM) and blocks the catalytic activity of the protease. The structural element which accounts for the thrombin inhibitory activity of r-Hir^1–43 is analyzed in this report.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.     

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.     

Fibronectin binds to amyloid P component. Localization of the binding site to the 31,000 dalton C-terminal domain

Fibronectin has been shown to play an important role in reticuloendothelial system functioning as well as in neutrophil and fibroblast migration to tissue injury sites. Fibronectin binds several macromolecules including components of the acute phase response. We have studied the interaction of fibronectin with the amyloid P component (AP). This glycoprotein, closely related to C-reactive protein, is deposited together with amyloid fibrils and is also a normal constituent of human fibronectin, its whole tryptic digest, and isolated fragments; fibronectin was retained by immobilized AP in a molar ratio fibronectin:AP of 1:5.8. In this paper we localized the binding site for AP in a tryptic 31 kDa fragment, near the C-terminal end of the fibronectin molecule. A shorter fragment of 22 kDa starting at position 82 of the 31 kDa domain and containing all the disulfide bridges present in the 31 kDa domain did not bind to AP; therefore the active site appears to be located within the 81 N-terminal residues of the 31 kDa fragment. To further support this conclusion, reduction and alkylation of either fibronectin or the 31 kDa fragment had no effect on their binding properties.     

Conserved C-terminal nascent peptide binding domain of HYPK facilitates its chaperone-like activity

Human HYPK (Huntingtin Yeast-two-hybrid Protein K) is an intrinsically unstructured chaperone-like protein with no sequence homology to known chaperones. HYPK is also known to be a part of ribosome-associated protein complex and present in polysomes. The objective of the present study was to investigate the evolutionary influence on HYPK primary structure and its impact on the protein’s function. Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying its importance in the structural and functional evolution. NPAA domain of human HYPK has unique amino acid composition preferring glutamic acid and happens to be more stable from a conformational point of view having higher content of α-helices than the rest. Cell biology studies indicate that overexpressed C-terminal human HYPK can interact with nascent proteins, co-localizes with huntingtin, increases cell viability and decreases caspase activities in Huntington’s disease (HD) cell culture model. This domain is found to be required for the chaperone-like activity of HYPK in vivo. Our study suggested that by virtue of its flexibility and nascent peptide binding activity, HYPK may play an important role in assisting protein (re)folding.     

Preparation of monoclonal antibodies to hirudin and hirudin peptides. A method for studying the hirudin--thrombin interaction.

A panel of four monoclonal antibodies was obtained against hirudin, a potent and specific inhibitor of thrombin, by immunizing three groups of mice with protein conjugates made of recombinant desulfatohirudin (group I) or two synthetic peptides representing the C-terminal sequences 40-65 (group II) and 52-65 (group III) of hirudin. Only the monoclonal antibody 4049-83-12, obtained from the group I of mice, showed high affinity for hirudin (Kd of 0.6 nM) and in vitro neutralizing properties. The anti-peptide monoclonal antibodies bound hirudin with lower affinity (Kd of 1.5-7 nM) and showed lower neutralizing capacities. An epitope analysis performed by competitive ELISA using various hirudin analogues and by limited proteolysis of the hirudin-antibody complex revealed that the binding domains of all the anti-peptide antibodies were located close to the C-terminus of hirudin, since the bond between Glu-61 and Glu-62 was not cleaved by the V8 staphylococcal protease in the presence of these antibodies. The epitope of the antibody 4049-83-12 was strictly conformation-dependent, it recognized neither S-carboxymethylated hirudin nor any peptides of hirudin. The cleavage of the bond between Glu-43 and Gly-44 by V8 protease, as well as the cleavage of the bond between Lys-47 and Pro-48 by lysyl endopeptidase, was prevented by the binding of the antibody 4049-83-12 to hirudin. The possibility that this epitope overlapped with a region of hirudin involved in the binding to thrombin is discussed.     

Alternative binding modes of proline-rich peptides binding to the GYF domain

Recognition of proline-rich sequences plays an important role for the assembly of multiprotein complexes during the course of eukaryotic signal transduction and is mediated by a set of protein folds that share characteristic features. The GYF (glycine-tyrosine-phenylalanine) domain is known as a member of the superfamily of recognition domains for proline-rich sequences. Recent studies on the complexation of the CD2BP2-GYF domain with CD2 peptides showed that the peptide adopts an extended conformation and forms a polyproline type-II helix involving residues Pro4-Pro7 [Freund et al. (2002) EMBO J. 21, 5985-5995]. R/K/GxxPPGxR/K is the key signature for the peptides that bind to the GYF domain [Kofler et al. (2004) J. Biol. Chem. 279, 28292-28297]. In our combined theoretical and experimental study, we show that the peptides adopt a polyproline II helical conformation in the unbound form as well as in the complex. From molecular dynamics simulations, we identify a novel binding mode for the G8W mutant and the wild-type peptide (shifted by one proline in register). In contrast, the conformation of the peptide mutant H9M remains close to the experimentally derived wild-type GYF-peptide complex. Possible functional implications of this altered conformation of the bound ligand are discussed in the light of our experimental and theoretical results.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

The C-terminal domain of the Arabidopsis AtMBD7 protein confers strong chromatin binding activity

The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.     

Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.     

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.     

Antibiotic activity of reversed peptides of alpha-helical antimicrobial peptide,P18

P18 (KWKLFKKIPKFLHLAKKF-NH(2)), an a-helical antimicrobial peptide designed from cecropin Amagainin 2 hybrid, was known to have potent antimicrobial activity against bacteria as well as fungi without hemolytic activity. To find the peptides comparable or superior to the antimicrobial activity of P18, the two reversed peptides (Rev-1 and Rev-2) of P18 were designed and synthesized. These peptides were found to have similar antimicrobial activity against bacterial and fungal cells without hemolytic activity as compared with P18. Furthermore, a reversed peptide, Rev-2 was shown to have a two-fold higher activity in killing some bacterial cells than P18. Therefore, these results suggested that Rev-2 peptide seems to be an excellent candidate for developing novel peptide antibiotics.     

A Nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding unmasks a C-terminal binding pocket.

In vivo function of the molecular chaperone Hsp90 is ATP-dependent and requires the full-length protein. Our earlier studies predicted a second C-terminal ATP-binding site in Hsp90. By applying direct biochemical approaches, we mapped two ATP-binding sites and unveiled the C-terminal ATP-binding site as the first example of a cryptic chaperone nucleotide-binding site, which is opened by occupancy of the N-terminal site. We identified an N-terminal gamma-phosphate-binding motif in the middle domain of Hsp90 similar to other GHKL family members. This motif is adjacent to the phosphate-binding region of the C-terminal ATP-binding site. Whereas novobiocin disrupts both C- and N-terminal nucleotide binding, we found a selective C-terminal nucleotide competitor, cisplatin, that strengthens the Hsp90-Hsp70 complex leaving the Hsp90-p23 complex intact. Cisplatin may provide a pharmacological tool to dissect C- and N-terminal nucleotide binding of Hsp90. A model is proposed on the interactions of the two nucleotide-binding domains and the charged region of Hsp90.     

The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.     

The N-terminal domain of CCL21 reconstitutes high affinity binding, G protein activation, and chemotactic activity, to the C-terminal domain of CCL19

CC chemokine receptor 7 (CCR7), which regulates the trafficking of leucocytes to the secondary lymphoid organs, has two endogenous chemokine ligands: CCL19 and CCL21. Although both ligands possess similar affinities for the receptor and similar abilities to promote G protein activation and chemotaxis, they share only 25% sequence identity. Here, we show that substituting N-terminal six amino acids of CCL21 (SDGGAQ) for the corresponding N-terminal domain of CCL19 (GTNDAE) results in a chimeric chemokine that exhibits high affinity binding and G protein activation of CCR7. These data demonstrate that despite dissimilar sequences, the amino terminal hexapeptide of these two chemokines is capable of performing similar roles resulting in receptor activation.     

Using directed evolution to improve the solubility of the C-terminal domain of Escherichia coli aminopeptidase P. Implications for metal binding and protein stability

There have been many approaches to solving problems associated with protein solubility. This article describes the application of directed evolution to improving the solubility of the C-terminal metal-binding domain of aminopeptidase P from Escherichia coli. During the course of experiments, the domain boundary and sequence were allowed to vary. It was found that extending the domain boundary resulted in aggregation with little improvement in solubility, whereas two changes to the sequence of the domain resulted in dramatic improvements in solubility. These latter changes occurred in the active site and abolished the ability of the protein to bind metals and hence catalyze its physiological reaction. The evidence presented here has led to the proposal that metals bind to the intact protein after it has folded and that the N-terminal domain is necessary to stabilize the structure of the protein so that it is capable of binding metals. The acid residues responsible for binding metals tend to repel one another - in the absence of the N-terminal domain, the C-terminal domain does not fold properly and forms inclusion bodies. Evolution of the C-terminal domain has removed the destabilizing effects of the metal ligands, but in so doing it has reduced the capacity of the domain to bind metals. In this case, directed evolution has identified active site residues that destabilize the domain structure.     

Interaction of heparin with synthetic peptides corresponding to the C-terminal domain of intestinal mucins

Unlike most other mucins described to date, two intestinal mucins, rat MLP (rat Muc2) and human MUC2 have a C-terminal tail that is enriched in cationic amino acids. The distribution of charge in each case resembles that of several well known heparin binding proteins. Peptides designated E20-14 and F13-15, corresponding to the C-terminal 14 amino acids of the two mucins, were synthesized and shown to bind³H-labelled heparin by a process that was saturable and mediated by strong electrostatic interactions, givingK _d values of 10^–7 to 10^–8 m. Using turbidometric analyses and native gel electrophoresis, we observed that peptide-heparin mixtures formed polydisperse aggregates that dissociated with a progressive increase in the concentration of heparin. Under certain conditions heparin protected the peptide from proteolysis by trypsin. Both heparin and dextran sulfate, the latter a highly sulfated synthetic polysaccharide, were potent inhibitors of³H-heparin binding to peptide E20-14, while less sulfated glycosaminoglycans were poorly- or non-inhibitory. Mucin in tissue dispersions and homogenates, or purified from rat intestine, did not bind to heparin, and failed to interact with an antibody specific for the peptide E20-14. Both mucin samples however, reacted with antibodies that recognize regions upstream of the C-terminal 14 amino acids. Immunofluorescent localization of E20-14 was confined to the basal perinuclear regions of goblet cells, whereas localization of an antibody to a flanking sequence on the N-terminal side of the C-tail, localized to mature mucin storage granules. These findings suggest that the heparin-binding C-tail of the mucin may be removed at an early stage of biosynthesis. Heparin-mucin complexes, if they formin vivo, are thus likely to be confined to the ER and/or Golgi compartments.     

The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein

The nucleoprotein of measles virus consists of an N-terminal moiety, N(CORE), resistant to proteolysis and a C-terminal moiety, N(TAIL), hypersensitive to proteolysis and not visible as a distinct domain by electron microscopy. We report the bacterial expression, purification, and characterization of measles virus N(TAIL). Using nuclear magnetic resonance, circular dichroism, gel filtration, dynamic light scattering, and small angle x-ray scattering, we show that N(TAIL) is not structured in solution. Its sequence and spectroscopic and hydrodynamic properties indicate that N(TAIL) belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The same epitopes are exposed in N(TAIL) and within the nucleoprotein, which rules out dramatic conformational changes in the isolated N(TAIL) domain compared with the full-length nucleoprotein. Most unstructured proteins undergo some degree of folding upon binding to their partners, a process termed induced folding. We show that N(TAIL) is able to bind its physiological partner, the phosphoprotein, and that it undergoes such an unstructured-to-structured transition upon binding to the C-terminal moiety of the phosphoprotein. The presence of flexible regions at the surface of the viral nucleocapsid would enable plastic interactions with several partners, whereas the gain of structure arising from induced folding would lead to modulation of these interactions. These results contribute to the study of the emerging field of natively unfolded proteins.