1H, 15N and 13C resonance assignments of Ixolaris, a tissue factor pathway inhibitor from the tick salivary gland.

Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents sequence homology to TFPI (tissue factor pathway inhibitor). It binds to the coagulation enzyme factor Xa (FXa) or to its zymogen form, FX, and further inhibits tissue factor/FVIIa complex (extrinsic Xnase compex). Differently from TFPI, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. The Ixolaris-FXa/FX complex formation has been characterized by using a combination of biophysical and biochemical technics although no structural data is currently available. In this study, we reported the NMR chemical shift assignment of Ixolaris, as a first step to further establishing the structure, dynamics and function relationship for this protein.

Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.

Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D 1H and 2D 15N-1H NMR analysis yielded high quality 2D 15N-1H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions.

1H, 13C and 15N resonance assignments and second structure information of Fag s 1: Fagales allergen from Fagus sylvatica.

Fagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the (1)H, (15)N and (13)C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.

Epitope mapping by solution NMR spectroscopy.

Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X-ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations.

Bet v 1--a Trojan horse for small ligands boosting allergic sensitization?

BACKGROUND: Birch pollen allergy represents the main cause of winter and spring pollinosis in the temperate climate zone of the northern hemisphere and sensitization towards Bet v 1, the major birch pollen allergen, affects over 100 million allergic patients. The major birch pollen allergen Bet v 1 has been described as promiscuous acceptor for a wide variety of hydrophobic ligands. OBJECTIVE: In search of intrinsic properties of Bet v 1, which account responsible for the high allergenic potential of the protein, we thought to investigate the effects of ligand-binding on immunogenic as well as allergenic properties. METHODS: As surrogate ligand of Bet v 1 sodium deoxycholate (DOC) was selected. Recombinant and natural Bet v 1 were characterised physico-chemically as well as immunologically in the presence or absence of DOC, and an animal model of allergic sensitization was established. Moreover, human IgE binding to Bet v 1 was analysed by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Ligand-binding had an overall stabilizing effect on Bet v 1. This translated in a Th2 skewing of the immune response in a mouse model. Analyses of human IgE binding on Bet v 1 in mediator release assays revealed that ligand-bound allergen-induced degranulation at lower concentrations; however, in basophil activation tests with human basophils ligand-binding did not show this effect. For the first time, human IgE epitopes on Bet v 1 were determined using antibodies isolated from patients' sera. The IgE epitope mapping of Bet v 1 demonstrated the presence of multiple binding regions. CONCLUSIONS AND CLINICAL RELEVANCE: Deoxycholate binding stabilizes conformational IgE epitopes on Bet v 1; however, the epitopes themselves remain unaltered. Therefore, we speculate that humans are exposed to both ligand-bound and free Bet v 1 during sensitization, disclosing the ligand-binding cavity of the allergen as key structural element.

Structural basis for the interaction of human ß-defensin 6 and its putative chemokine receptor CCR2 and breast cancer microvesicles.

Human ß-defensins (hBDs) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the hBD6 by NMR spectroscopy that contains a conserved ß-defensin domain followed by an extended C-terminus. We use NMR to monitor the interaction of hBD6 with microvesicles shed by breast cancer cell lines and with peptides derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) possessing or not possessing sulfation on Tyr26 and Tyr28. The NMR-derived model of the hBD6/CCR2 complex reveals a contiguous binding surface on hBD6, which comprises amino acid residues of the α-helix and ß2-ß3 loop. The microvesicle binding surface partially overlaps with the chemokine receptor interface. NMR spin relaxation suggests that free hBD6 and the hBD6/CCR2 complex exhibit microsecond-to-millisecond conformational dynamics encompassing the CCR2 binding site, which might facilitate selection of the molecular configuration optimal for binding. These data offer new insights into the structure-function relation of the hBD6-CCR2 interaction, which is a promising target for the design of novel anticancer agents.

¹H, ¹³C and ¹5N resonance assignments and second structure information of Gad m 1: a ß-parvalbumin allergen from Atlantic cod (Gadus morhua).

Gad m 1 is the major allergen from Atlantic cod. It belongs to ß-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. ß-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of ß-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a ß-parvalbumin: Gad m 1. This report includes: (1)H, (13)C and (15)N resonance assignments of Gad m 1 as well as the second structure information based on the (13)C chemical shifts.

Evolutionary relationship between defensins in the Poaceae family strengthened by the characterization of new sugarcane defensins.

Plant defensins are small (45-54 amino acids), highly basic, cysteine-rich peptides structurally related to defensins of other organisms, including insects and mammals. Small putative proteins (MW < 10 kDa) containing eight cysteines were screened based on the sugarcane expressed sequence tag (EST) database. We selected ORFs that exhibited 25-100% similarity in primary sequence with other defensins in the NCBI database and that contained eight cysteines. This similarity is sufficient for folding prediction, but not enough for biological activity inference. Six putative defensins (Sd1-6) were selected, and activity assays showed that recombinant Sd1, Sd3 and Sd5 are active against fungi, but not against bacteria. Structural characterization, based on circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy showed that the structures of these Sds were compatible with alpha/beta proteins, a feature expected for plant defensins. Phylogenetic analysis revealed that sugarcane defensins could clearly be grouped within defensins from Poaceae family and Andropogoneae tribe. Our work demonstrates that defensins show strong conservation in the Poaceae family and may indicate that the same conservation occurs in other families. We suggest that evolutionary relationships within plant families can be used as a procedure to predict and annotate new defensins in genomes and group them in evolutionary classes to help in the investigation of their biological function.

Structural biology of membrane-acting peptides: conformational plasticity of anticoccidial peptide PW2 probed by solution NMR.

The bottleneck for the complete understanding of the structure-function relationship of flexible membrane-acting peptides is its dynamics. At the same time, not only the structure but also the dynamics are the key points for their mechanism of action. Our model is PW2, a TRP-rich, cationic peptide selected from phage display libraries that shows anticoccidial activity against Eimeria acervulina. In this manuscript we used a combination of several NMR techniques to tackle these difficulties. The structural features of the membrane-acting peptide PW2 was studied in several membrane mimetic environments: we compared the structural features of PW2 in SDS and DPC micelles, that were reported earlier, with the structure properties in different lipid vesicles and the peptide free in water. We were able to unify the structural information obtained in each of these systems. The structural constraints of the peptide free in water were fundamental for the understanding of plasticity necessary for the membrane interaction. Our data suggested that the WWR sequence is the region responsible for anchoring the peptide to the interfaces, and that this same region displays some degree of conformational order in solution. For PW2, we found that affinity is related to the aromatic region, by anchoring the peptide to the membrane, and specificity is related to the N- and C-termini, which are able to accommodate in the membrane due to its plasticity.

Implications of protein conformational diversity for binding and development of new biological active compounds.

The new generation of biologically active compounds developed during the 20(th) century relied on knowledge of enzymology and protein structure, and were based initially, on the understanding that protein-protein and small molecule-protein interactions occurred through a lock-and-key mechanism. Later, evidence suggested that this mechanism was usually followed by a conformational change, known as induced fit. Recent studies on protein dynamics, mainly by nuclear magnetic resonance (NMR) relaxation measurements, have shown that proteins are not structured in a unique conformation. Rather, they frequently have regions of conformational diversity. In the present review we will discuss a novel view of binding, put forward in by several research groups in the last 5 to 10 years. In the free state, protein regions displaying conformational diversity exhibit equilibria among pre-existing conformations. In the presence of a ligand, one of these conformations is stabilized, so that the ligand does not need to induce a new conformation. Upon ligand binding there is a population shift toward the bound conformational state. Conformational diversity of binding sites of several proteins has been measured and has important practical as well as thermodynamical consequences: binding sites can be mapped without prior knowledge of the ligand and also evolution of binding sites depends mostly on the free state, occurring at least partially independently of the ligand.

In-Cell NMR spectroscopy: inhibition of autologous protein expression reduces Escherichia coli lysis.

Structural studies by in-cell nuclear magnetic resonance are a developing new field of research, and their objective is to obtain structural information of proteins and other biological macromolecules in the cytoplasm of Escherichia coli cells. The major limitation of in-cell experiments is cell lysis that occurs during the experiments. In this article, we describe how inhibition of autologous expression by rifampicin at a high concentration decreases cell lysis in E. coli. We suggest that rifampicin is acting in the programmed cell death gene system MazEF, which is triggered by stress conditions and ultimately leads to cell lysis.

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis.

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas' disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2beta protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.

Structural studies of MS2 bacteriophage virus particle disassembly by nuclear magnetic resonance relaxation measurements.

In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle-the MS2 bacteriophage. MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli. At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5. Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0. At pH 4.5, two-dimensional (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum shows approximately 40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5. The (15)N linewidth is approximately 30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns. The average spin lattice relaxation time (T(1)) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate. The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage.

High-speed countercurrent chromatography as a valuable tool to isolate C-glycosylflavones from Cecropia lyratiloba Miquel.

A new apigeninglycoside, apigenin 6-C-galactosyl-6"-O-beta-galactopyranoside (1), isoorientin, and a mixture of orientin and isovitexin were isolated from leaves of Cecropia lyratiloba by high-speed countercurrent chromatography using a solvent system containing ethyl acetate, butanol, methanol and water. The structural elucidation of 1 was based on NMR spectroscopy.

cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris.

In the present work, we describe the cDNA cloning, expression in Pichia pastoris, purification, and characterization of the recombinant Pisum sativum defensin 1 (rPsd1), a novel Cys-rich protein presenting four disulfide bridges and high antifungal activity. Several parameters that affect the level of protein expression were assayed. The best condition yielded 13.8 mg/L (1.50 microg/10(8) cells) of active rPsd1. The recombinant rPsd1 was purified to homogeneity by cation exchange, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region. Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein has a very similar folding and a correct disulfide-bonding pattern when compared to native Psd1. Nevertheless, the rPsd1 presented a more species-specific antifungal activity. The importance of the N- and C-termini for Psd1 activity is pointed out.

Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units.

Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus are surrounded by a gelatinous layer rich in sulfated fucan. Shortly after fertilization this polysaccharide disappears, but 24 h later the embryos synthesize high amounts of dermatan sulfate concomitantly with the mesenchyme blastula-early gastrula stage when the larval gut is forming. This glycosaminoglycan has the same backbone structure [4-alpha-L-IdoA-1-->3-beta-D-GalNAc-1](n) as the mammalian counterpart but possesses a different sulfation pattern. It has a high content of 4-O- and 6-O-disulfated galactosamine units. In addition, chains of this dermatan sulfate are considerable longer than those of vertebrate tissues. Adult sea urchin tissues contain high concentrations of sulfated polysaccharides, but dermatan sulfate is restricted to the adult body wall where it accounts for approximately 20% of the total sulfated polysaccharides. In addition, sulfation at the 4-O-position decreases markedly in the dermatan sulfate from adult sea urchin when compared with the glycan from larvae. Overall, these results demonstrate the occurrence of dermatan sulfates with unique sulfation patterns in this marine invertebrate. The physiological implication of these oversulfated dermatan sulfates is unclear. One hypothesis is that interactions between components of the extracellular matrix in marine invertebrates occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with increased charge density.

Selectively labeling the heterologous protein in Escherichia coli for NMR studies: a strategy to speed up NMR spectroscopy.

Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.

Occurrence of heparin in the invertebrate styela plicata (Tunicata) is restricted to cell layers facing the outside environment. An ancient role in defense?

Heparin is an intracellular product of vertebrate mast cell currently used as exogenous anticoagulant. Despite of the potent biological activities of exogenous heparin, its physiological function has not been clearly established yet. Here, a heparin with similar structure and anticoagulant properties to the mammalian counterpart was shown to occur as the intracellular product of test cells, a cell monolayer that surrounds egg of the invertebrate Styela plicata (Chordata-Tunicata). As in the case of mammalian mast cells, heparin from the ascidian test cells is removed from the intracellular granules after incubation with compound 48/80. Following fertilization, the test cells surrounding the developing larva still retain heparin as metachromatic granulation. In the adult invertebrate, heparin occurs as intracellular granules at the apical tip of epithelial cells surrounding the lumen of both intestine and pharynx, in close contact with the external environment. This is the first description of the presence of heparin in cytoplasmic granules of epithelial-like cells around the lumen of sites exposed to external agents. This arrangement may reflect the participation of heparin in defense mechanisms in this invertebrate.

Structure and anticoagulant activity of sulfated galactans. Isolation of a unique sulfated galactan from the red algae Botryocladia occidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates.

We have characterized the structure of a sulfated d-galactan from the red algae Botryocladia occidentalis. The following repeating structure (-4-alpha-d-Galp-1-->3-beta-d-Galp-1-->) was found for this polysaccharide, but with a variable sulfation pattern. Clearly one-third of the total alpha-units are 2,3-di-O-sulfated and another one-third are 2-O-sulfated. The algal sulfated d-galactan has a potent anticoagulant activity (similar potency as unfractionated heparin) due to enhanced inhibition of thrombin and factor Xa by antithrombin and/or heparin cofactor II. We also extended the experiments to several sulfated polysaccharides from marine invertebrates with simple structures, composed of a single repeating structure. A 2-O- or 3-O-sulfated l-galactan (as well as a 2-O-sulfated l-fucan) has a weak anticoagulant action when compared with the potent action of the algal sulfated d-galactan. Possibly, the addition of two sulfate esters to a single alpha-galactose residue has an "amplifying effect" on the anticoagulant action, which cannot be totally ascribed to the increased charge density of the polymer. These results indicate that the wide diversity of polysaccharides from marine alga and invertebrates is a useful tool to elucidate structure/anticoagulant activity relationships.

Virus maturation targets the protein capsid to concerted disassembly and unfolding.

Many animal viruses undergo post-assembly proteolytic cleavage that is required for infectivity. The role of maturation cleavage on Flock House virus was evaluated by comparing wild type (wt) and cleavage-defective mutant (D75N) Flock House virus virus-like particles. A concerted dissociation and unfolding of the mature wt particle was observed under treatment by urea, whereas the cleavage-defective mutant dissociated to folded subunits as determined by steady-state and dynamic fluorescence spectroscopy, circular dichroism, and nuclear magnetic resonance. The folded D75N alpha subunit could reassemble into capsids, whereas the yield of reassembly from unfolded cleaved wt subunits was very low. Overall, our results demonstrate that the maturation/cleavage process targets the particle for an "off pathway" disassembly, because dissociation is coupled to unfolding. The increased motions in the cleaved capsid, revealed by fluorescence and NMR, and the concerted nature of dissociation/unfolding may be crucial to make the mature particle infectious.