Β-trefoil Domain Research Articles

A nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation.

Fascin-1-mediated actin-bundling activity is central to the generation of plasma membrane protrusions required for cell migration. Dysregulated formation of cellular protrusions is observed in metastatic cancers, where they are required for increased invasiveness, and is often correlated with increased Fascin-1 abundance. Therefore, there is interest in generating therapeutic Fascin-1 inhibitors. We present the identification of Nb 3E11, a nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation. The crystal structure of the Fascin-1/Nb 3E11 complex reveals the structural mechanism of inhibition. Nb 3E11 occludes an actin-binding site on the third β-trefoil domain of Fascin-1 that is currently not targeted by chemical inhibitors. Binding of Nb 3E11 to Fascin-1 induces a conformational change in the adjacent domains to stabilize Fascin-1 in an inhibitory state similar to that adopted in the presence of small-molecule inhibitors. Nb 3E11 could be used as a tool inhibitor molecule to aid in the development of Fascin-1 targeted therapeutics.

The Crystal Structure of Bacillus thuringiensis Tpp80Aa1 and Its Interaction with Galactose-Containing Glycolipids.

Tpp80Aa1 from Bacillus thuringiensis is a Toxin_10 family protein (Tpp) with reported action against Culex mosquitoes. Here, we demonstrate an expanded target range, showing Tpp80Aa1 is also active against the larvae of Anopheles gambiae and Aedes aegypti mosquitoes. We report the first crystal structure of Tpp80Aa1 at a resolution of 1.8 Å, which shows Tpp80Aa1 consists of two domains: an N-terminal β-trefoil domain resembling a ricin B lectin and a C-terminal putative pore-forming domain sharing structural similarity with the aerolysin family. Similar to other Tpp family members, we observe Tpp80Aa1 binds to the mosquito midgut, specifically the posterior midgut and the gastric caecum. We also identify that Tpp80Aa1 can interact with galactose-containing glycolipids and galactose, and this interaction is critical for exerting full insecticidal action against mosquito target cell lines.

The choanoflagellate pore-forming lectin SaroL-1 punches holes in cancer cells by targeting the tumor-related glycosphingolipid Gb3

Choanoflagellates are primitive protozoa used as models for animal evolution. They express a large variety of multi-domain proteins contributing to adhesion and cell communication, thereby providing a rich repertoire of molecules for biotechnology. Adhesion often involves proteins adopting a β-trefoil fold with carbohydrate-binding properties therefore classified as lectins. Sequence database screening with a dedicated method resulted in TrefLec, a database of 44714 β-trefoil candidate lectins across 4497 species. TrefLec was searched for original domain combinations, which led to single out SaroL-1 in the choanoflagellate Salpingoeca rosetta, that contains both β-trefoil and aerolysin-like pore-forming domains. Recombinant SaroL-1 is shown to bind galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as the glycosphingolipid Gb3, when embedded in giant unilamellar vesicles or cell membranes. Crystal structures of complexes with Gb3 trisaccharide and GalNAc provided the basis for building a model of the oligomeric pore. Finally, recognition of the αGal epitope on glycolipids required for hemolysis of rabbit erythrocytes suggests that toxicity on cancer cells is achieved through carbohydrate-dependent pore-formation.

Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins.

Leptospirosis is a globally important neglected zoonotic disease. Previous data suggest that a family of virulence-modifying (VM) proteins (PF07598) is a distinctive feature of group I pathogenic Leptospira that evolved as important virulence determinants. Here, we show that one such VM protein, LA3490 (also known as Q8F0K3), is expressed by Leptospira interrogans serovar Lai, as a secreted genotoxin that is potently cytotoxic to human cells. Structural homology searches using Phyre2 suggested that VM proteins are novel R-type lectins containing tandem N-terminal ricin B-chain-like β-trefoil domains. Recombinant LA3490 (rLA3490) and an N-terminal fragment, t3490, containing only the predicted ricin B domain, bound to the terminal galactose and N-acetyl-galactosamine residues, asialofetuin, and directly competed for asialofetuin-binding sites with recombinant ricin B chain. t3490 alone was sufficient for binding, both to immobilized asialofetuin and to the HeLa cell surface but was neither internalized nor cytotoxic. Treatment of HeLa cells with rLA3490 led to cytoskeleton disassembly, caspase-3 activation, and nuclear fragmentation, and was rapidly cytolethal. rLA3490 had DNase activity on mammalian and bacterial plasmid DNA. The combination of cell surface binding, internalization, nuclear translocation, and DNase functions indicate that LA3490 and other VM proteins evolved as novel forms of the bacterial AB domain-containing toxin paradigm.

Extending Janus lectins architecture: Characterization and application to protocells

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being exploited for producing tools with precise topology and specificity. We developed the concept of engineered chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the β-propeller domain from Ralstonia solanacearum and the β-trefoil domain from fungus Marasmius oreades, is specific for fucose and α-galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The engineered protein recognizes and binds H1299 human lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.

NAD+ Bound N‐terminal Domain of CARDS Toxin Increases IL‐1β Secretion

Mycoplasma pneumoniae (Mp) is an atypical bacterium that is linked to various respiratory diseases such as walking pneumonia and asthma. Upon infection, Mp produces a 591‐aa virulence factor known as Community Acquired Respiratory Distress Syndrome Toxin (CARDS TX). The crystal structure of this 591‐aa cytotoxin reveals a triangular molecule comprised of an N‐terminal ADP‐ribosylating domain and a C‐terminal tandem β‐trefoil domain that is responsible for vacuolation of the host's cells. Based on structural and sequence homology to other ADP ribosylating toxins, the NAD+ binding site has been predicted, yet none of the published structures of the CARDS toxin contain NAD+. The loop connecting the N− and the C‐termini is anchored using a disulfide bond that surrounds the NAD+ binding site which might sterically hinder NAD+ binding. An N‐terminal truncation mutant (NTTM) lacking the disulfide bond has been cloned and the protein was expressed, purified and crystallization trials are underway. The NTTM was also used to treat U937 cells in presence and absence of NAD+ to test the effect of the NTTM on cytokine secretion. The data show that NTTM causes an increase in the secretion of IL‐1β and TNF‐α in presence of NAD+ suggesting an increase in the inflammatory response to the NTTM in presence of NAD+.Support or Funding InformationSt. Mary's University Biaggini Funds and The San Antonio Livestock Expedition FundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vibrio cholerae hemolysin: The β-trefoil domain is required for folding to the native conformation

Vibrio cholerae cytolysin/hemolysin (VCC) is a 65kDa β-pore-forming toxin causing lysis and death of eukaryotic cells. Apart from the core cytolysin domain, VCC has two lectin domains with β-trefoil and β-prism folds. The β-prism domain binds to cell surface carbohydrate receptors; the role of the β-trefoil domain is unknown. Here, we show that the pro-VCC mutant without the β-trefoil domain formed aggregates highly susceptible to proteolysis, suggesting lack of a properly folded compact structure. The VCC variants with Trp532Ala or Trp534Ala mutation in the β-trefoil domain formed hemolytically inactive, protease-resistant, ring-shaped SDS-labile oligomers with diameters of ~19nm. The Trp mutation induced a dramatic change in the global conformation of VCC, as indicated by: (a) the change in surface polarity from hydrophobic to hydrophilic; (b) movement of core Trp residues to the protein-water interface; and (c) decrease in reactivity to the anti-VCC antibody by >100-fold. In fact, the mutant VCC had little similarity to the wild toxin. However, the association constant for the carbohydrate-dependent interaction mediated by the β-prism domain decreased marginally from ~3×108 to ~5×107M−1. We interpret the observations by proposing: (a) the β-trefoil domain is critical to the folding of the cytolysin domain to its active conformation; (b) the β-prism domain is an autonomous folding unit.

Glycan Specificity of the Vibrio vulnificus Hemolysin Lectin Outlines Evolutionary History of Membrane Targeting by a Toxin Family

Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs.

Structure and Glycan-Binding Properties of the Vibrio Vulnificus Hemolysin B-Trefoil Lectin

Pore forming toxins (PFTs) are secreted by bacterial pathogens as water-soluble monomers that oligomerize on the cell surface to form transmembrane channels. To increase their affinity for the membrane, many PFTs utilize proteins, carbohydrates and/or lipids as receptors. Previous studies suggest that a PFT secreted by Vibrio cholerae, V. cholerae cytolysin (VCC), recognizes cell surface glycans in its mode of action. However, while VCC contains two domains whose structures resemble known carbohydrate binding motifs, a β-trefoil and β-prism fold, only the β-prism domain actively binds carbohydrate ligands. This presents the question of why an organism would make a protein with an inactive lectin domain. Sequence analysis identified that orthologs of VCC contain either both of the lectin-like domains, or only the β-trefoil domain. Here, we utilize functional and structural approaches to investigate the carbohydrate-binding activities of a VCC ortholog, Vibrio vulnificus hemolysin (VVH), which contains only a β-trefoil lectin-like domain. Our studies identify that the VVH β-trefoil lectin domain binds a broad range of branched sugars containing multiple N-acetyllactosamine (LacNAc) repeats, with micromolar affinity. This is in contrast to the β-prism domain of VCC, which binds a narrower range of complex N-glycans with nanomolar affinity. We solved the X-ray crystal structure of the VVH β-trefoil domain identifying two interesting traits. First, the β-trefoil lectin domain alone forms a heptameric ring characteristic of many PFTs. Second, VVH contains a unique loop and coordinating residues absent in VCC. We hypothesize an evolutionary relationship between VVH and VCC such that upon gaining the more specific β-prism domain, activity of the β-trefoil domain was lost.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two β-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-α-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

BEL -trefoil: A novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

Determination of the Carbohydrate-Binding Specificity of Lectin-Like Domains in Vibrio Cholerae Cytolysin

Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin that contains two putative sugar-binding domains: the β-trefoil domain and the β-prism domain. Previous hemagglutinating studies suggest that VCC binds glycoproteins with terminal β1-galactosyl moieties [1]; however, crystallization of monomeric VCC with β-octyl glucoside suggests the β-prism domain may have an affinity for glucosides. We aim to characterize the carbohydrate-binding specificity of the β-trefoil and β-prism domains. The crystal structure of VCC indicates that Asp617 within the β-prism domain forms hydrogen bonds with the bound glucoside moeity. Hemolysis assays demonstrated that the removal of the β-prism domain causes a 1000-fold decrease in lytic activity toward rabbit erythrocytes, while conversion of Asp617 to alanine and lysine decreases the hemolytic activity100-fold, indicating that the sugar-binding activity of VCC may play a role in cell lysis. The β-prism domain was expressed as a separate construct and isothermal titration calorimetry (ITC) was used to determine the binding affinity for the interaction of the β-prism domain with monosaccharides. The binding of the isolated β-prism domain reflected the binding affinities observed using ITC for full length VCC. The β-prism domain showed an affinity for mannose, but not galactose, indicating that recognition of terminal β1-galactosyl moieties does not occur at the β-prism domain. The carbohydrate-binding activity of the β-prism domain may explain its role in cell lysis.[1] Saha, N; Banerjee, K.K. Carbohydrate-mediated Regulation of Interaction of Vibrio cholerae Hemolysin with Erythrocyte and Phospholipid Vesicle, J. Biol. Chem. (1997) 272, 162-167.

Horizontal gene transfer contributed to the evolution of extracellular surface structures

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication

Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the β-trefoil domain of C-terminus heavy chain (Hcβtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcβtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcβtre-Ad2F alone also induced elevated antibody production in contrast to Hcβtre alone. Plasma from Hcβtre + CT- and Hcβtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcβtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcβtre + CT-immunized mice, which only showed ∼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.

Mechanism of Actin Filament Bundling by Fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

Stability and Sugar Recognition Ability of Ricin-like Carbohydrate Binding Domains

Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two β-trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1α and site 2γ recognition.

Structure, Conformational Dynamics, and Evolutionary Conservation of Human Fascin-1

Cells express a range of actin-binding proteins to crosslink filamentous actin (F-actin) into compact ordered bundles ranging from stereocilia and microvilli to filopodia. Human fascin-1 is found nearly exclusively in filopodia, which protrude from the leading edge of migrating cells in normal and cancerous cells. Previously, we and others have found that fascin-1 confers unusual mechanical integrity to these actin bundles, which play a role in sensing the extracellular environment. Here, we examine the packing, conformational flexibility, and evolutionary sequence conservation of full length Homo sapiens fascin-1. Unlike the ubiquitous crosslinking proteins fimbrin and alpha-actinin that consist of dual calponin homology domain pairs, fascin-1 consists of four β-trefoil domains organized into a double-lobe-like structure. Sequence analysis suggests that these β-trefoil domains are stabilized by bulky hydrophobic residues in the core of each domain, and that interfacial residues between the two lobes of fascin play an important role in stabilizing the molecule. Conformational dynamics analysis reveals an allosteric coupling between highly conserved surface patches near the putative actin-binding sites of the molecule, with potential implications on its ability to crosslink F-actin tightly and confer unusual mechanical properties to F-actin bundles. Future mutational and structural studies motivated by this work will further elucidate the molecular basis for the unique function of fascin in human cells.

FRET-Based Mapping of Three Structural Domains within the Full-Length Ryanodine Receptor Type 1

The type 1 ryanodine receptor (RyR1) mediates excitation contraction coupling in skeletal muscle by releasing intracellular calcium in response to cell depolarization. Understanding the structure and conformational dynamics of RyR1 is challenging due to its size (homotetrameric subunit Mr∼560 kDa) and its association with numerous regulatory proteins. However, recent X-ray crystallographic studies indicate that a fragment comprised of the first 200 amino acids of RyR1 folds into a compact β-trefoil domain. Based on structural homology with the inositol trisphosphate receptor, the next 400 amino acids of RyR1 are predicted to fold into a second β-trefoil and an alpha helical domain. In this report, the relative orientation of these three domains (termed here as β1, β2 and α1) within the full-length RyR1 protein has been investigated using a novel FRET-based technique. This method monitors the relative proximity of a GFP fluorescent donor fused into RyR1 and a fluorescent acceptor, Cy3NTA, targeted to poly-histidine (His) tags inserted into the primary sequence of RyR1. In this study, GFP was fused to single positions within β1, β2 and α1 and FRET was then measured to Cy3NTA targeted to 6 His tags singly introduced into these three domains. The results of these measurements provide a detailed picture of the relative orientation of these three sub-domains within full-length RyR1 expressed in a cellular context. Further structural studies using this technique can now be undertaken to determine how these domains move during channel gating. (Supported by NIH grant R21ARO56406).

Adenovirus F protein as a delivery vehicle for botulinum B

BackgroundImmunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within β-trefoil domain (Hcβtre), we hypothesize that immunization with the Hcβtre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination.ResultsThe Hcβtre serotype B immunogen was genetically fused to Ad2F (Hcβtre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcβtre-Ad2F relative to Hcβtre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcβtre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcβtre and Hcβtre-Ad2F protected mice against 2.0 LD50.ConclusionThese results demonstrate that Hcβtre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.