Conformational heterogeneity in the major birch pollen allergen

The structural flexibility of proteins is a prerequisite for their biological function. For example, the interconversion between different three-dimensional structures (conformers) of a protein that have diverse properties can be critical for ligand recognition, binding and enzymatic catalysis. With few exceptions, however, standard X-ray diffraction and nuclear magnetic resonance (NMR) spectroscopic studies of protein structures focus on the most stable conformer and can therefore not explain all experimental observations. The aim of this stand-alone project of the FWF is to provide a comprehensive description of the conformational heterogeneity of different isoforms and a genetically engineered version of the major birch pollen allergen Bet v 1. This protein is regarded as the main cause for allergic sensitization against birch pollen, affecting millions of people in North America and Western Europe. For Bet v 1 the exact structural determinants of allergenicity are, however, unclear. For example, various isoforms of this protein display drastically different immunological properties, yet virtually identical three-dimensional structures. A structure based description of the molecular mechanims of Bet v 1 allergens is thus not possible, and a functional contribution of structural flexibility is considered possible. It is known, for example, that dynamic global and/or local unfolding of proteins represents an effective mechanism for proteolytic cleavage via the transient exposure of potential cleavage sites to the surface of the three-dimensional protein structure. Proteolytic processing indeed plays a key role for allergic sensitization against Bet v 1 allergens. In our project we will use dynamic NMR spectroscopy to study conformational heterogeneity in Bet v 1 allergens. This technique enables us to directly monitor the transitions between different conformers at atomic resolution. Dynamic NMR spectroscopy will be complemented by a variety of biophysical techniques, ranging from isothermal calorimetry and structure analysis to systematic mutational studies. The integration of diverse sets of experimental data will enable us to provide a quantitative mechanistic and structure-based description of conformational heterogeneity in Bet v 1 allergens and create the basis for understanding the observed immunologic properties of these proteins.

In this project of the Austrian Science Fund FWF we characterized the birch pollen allergen Bet v 1 in detail. This protein is responsible for developing allergic reactions against birch pollen upon inhalation, affecting an estimated 100 million people worldwide. Bet v 1 exists in different isoforms in nature, which have distinguished immunological properties. Some isoforms are considered low allergenic, while others are categorized as being highly allergenic, despite the fact that they have virtually identical three-dimensional structures. A structural rationale for the differential immunological properties of Bet v 1 isoforms is thus missing. Using nuclear magnetic resonance (NMR) structure determination and relaxation techniques we characterized the structural flexibility of Bet v 1 isoforms in detail. Because NMR spectroscopy enables measurements at atomic resolution, a genuine description of the flexibility of these proteins can be provided. We showed that different isoforms of Bet v 1 can display striking disparities regarding the extent to which they fluctuate around their mean structural coordinates. Such fluctuations are of critical significance, as they allow for the access of proteases and subsequent cleavage of the protein backbone into smaller protein fragments. This is a necessary and critical step in allergic sensitization. Our data clearly establish that structural flexibility and immunogenicity are directly correlated. In addition, we investigated another intriguing aspect of these proteins. Bet v 1 is known to bind a variety of small molecules (ligands) to an internal cavity, which affects the level of immunological response in birch pollen allergic individuals. In our project we could show that ligand binding to Bet v 1 is again coupled to the structural flexibility of this protein, and that the protein scaffold becomes more compact upon binding a ligand molecule. Our results thus point towards the importance of experimental studies of protein flexibility for understanding interactions between allergens and ligands. Finally, we investigated proteins in food sources that elicit so-called cross-allergies in birch pollen allergic individuals. The three-dimensional structure of the major apple allergen Mal d 1 from Granny Smith apples was determined and ligand binding to this protein was studied in detail using NMR spectroscopy. These results provide the structural basis for the observed immunological cross-reactivity between birch pollen and apples.