Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.

BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

Crystal structure of the human odorant binding protein, OBPIIa.

Human odorant-binding protein, OBPIIa , is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 Å resolution. OBPIIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight-stranded ß-barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand-binding site evoke a positive electrostatic potential. Human OBPIIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin.

Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

Structures of the germline-specific Deadhead and thioredoxin T proteins from Drosophila melanogaster reveal unique features among thioredoxins.

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and thioredoxin T (TrxT), that belong to the lethal(3)malignant brain tumor signature genes and to the 'survival network' of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here, the first structures of Dhd and TrxT are presented, unveiling new features of these two thioredoxins. Dhd has positively charged patches on its surface, in contrast to the negatively charged surfaces commonly found in most Trxs. This distinctive charge distribution helps to define initial encounter complexes with DNA/RNA that will lead to final specific interactions with cofactors to promote chromatin remodeling. TrxT contains a C-terminal extension, which is mostly unstructured and highly flexible, that wraps the conserved core through a closed conformation. It is believed that these new structures can guide future work aimed at understanding embryo development and redox homeostasis in Drosophila. Moreover, due to their restricted presence in Schizophora (a section of the true flies), these structures can help in the design of small-molecular binders to modulate native redox homeostasis, thereby providing new applications for the control of plagues that cause human diseases and/or bring about economic losses by damaging crop production.

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes.

Smad transcription factors are the main downstream effectors of the Transforming growth factor ß superfamily (TGFß) signalling network. The DNA complexes determined here by X-ray crystallography for the Bone Morphogenetic Proteins (BMP) activated Smad5 and Smad8 proteins reveal that all MH1 domains bind [GGC(GC)|(CG)] motifs similarly, although TGFß-activated Smad2/3 and Smad4 MH1 domains bind as monomers whereas Smad1/5/8 form helix-swapped dimers. Dimers and monomers are also present in solution, as revealed by NMR. To decipher the characteristics that defined these dimers, we designed chimeric MH1 domains and characterized them using X-ray crystallography. We found that swapping the loop1 between TGFß- and BMP- activated MH1 domains switches the dimer/monomer propensities. When we scanned the distribution of Smad-bound motifs in ChIP-Seq peaks (Chromatin immunoprecipitation followed by high-throughput sequencing) in Smad-responsive genes, we observed specific site clustering and spacing depending on whether the peaks correspond to BMP- or TGFß-responsive genes. We also identified significant correlations between site distribution and monomer or dimer propensities. We propose that the MH1 monomer or dimer propensity of Smads contributes to the distinct motif selection genome-wide and together with the MH2 domain association, help define the composition of R-Smad/Smad4 trimeric complexes.

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.

Smad transcription factors activated by TGF-ß or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-ß and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-ß and BMP pathways.

Protease recognition sites in Bet v 1a are cryptic, explaining its slow processing relevant to its allergenicity.

Despite a high similarity with homologous protein families, only few proteins trigger an allergic immune response with characteristic TH2 polarization. This puzzling observation is illustrated by the major birch pollen allergen Bet v 1a and its hypoallergenic protein isoforms, e.g., Bet v 1d. Given the key role of proteolytic processing in antigen presentation and T cell polarization, we investigated the recognition of Bet v 1 isoforms by the relevant protease cathepsin S. We found that at moderately acidic pH values Bet v 1a bound to cathepsin S with significantly lower affinity and was more slowly cleaved than its hypoallergenic isoform Bet v 1d. Only at pH values ≤ 4.5 the known proteolytic cleavage sites in Bet v 1a became accessible, resulting in a strong increase in affinity towards cathepsin S. Antigen processing and class II MHC loading occurs at moderately acidic compartments where processing of Bet v 1a and Bet v 1d differs distinctly. This difference translates into low and high density class II MHC loading and subsequently in TH2 and TH1 polarization, respectively.