NMR resonance assignments of a hypoallergenic isoform of the major birch pollen allergen Bet v 1

In Northern America and Europe a great number of people are suffering from birch pollen allergy and pollen related food allergies. The trigger for these immunological reactions is the 17.5 kDa major birch pollen allergen Bet v 1, which belongs to the family of PR-10 (pathogenesis-related) proteins. In nature, Bet v 1 occurs as a mixture of various isoforms that possess different immunological properties despite their high sequence identities. Bet v 1.0102 (Bet v 1d), which is investigated here, is a hypoallergenic isoform of Bet v 1 and a potential candidate for allergen-specific immunotherapy. We assigned the backbone and side chain 1H, 13C and 15N resonances of this protein and predicted its secondary structure. The NMR-chemical shift data indicate that Bet v 1.0102 is composed of three α-helices and a seven stranded β-sheet, in agreement with the known structure of the hyperallergenic isoform Bet v 1.0101 (Bet v 1a). Our resonance assignments create the foundation for detailed characterization of the dynamic properties of Bet v 1 isoforms by NMR relaxation measurements.

immunotherapy (Valenta et al. 2016). Very recent studies hypothecate that these different immunological properties may result from a varying dynamical behavior and fold stabilities (Freier et al. 2015;Machado et al. 2016).
The three-dimensional structures of numerous Bet v 1 isoforms and mutants are available in the literature (Fernandes et al. 2013). Bet v onefolds into a curved seven-stranded antiparallel β-sheet, along with two short α-helices and an extended C-terminal helix, which create an expanded internal cavity, as it is typical for the PR-10 protein family. Of note, Bet v 1 structures that are available in the protein data base (PDB) exhibit only small differences, with backbone pair-wise rmsd values between 1.5 and 2 Å. For Bet v 1.0102 a three-dimensional structure has not been reported so far. Homologous PR-10 proteins found in fruits and vegetables with similar three-dimensional structures can elicit allergic cross-reactions with Bet v 1-specific IgE antibodies, including apples, cherries, peaches and others (Mills and Shewry 2004).
Here we present the solution NMR backbone and side chain assignment of hypoallergenic Bet v 1.0102 and a chemical shift-derived three-dimensional structure model of this protein created by CS-Rosetta. Chemical shift assignments of Bet v 1 are the prerequisite for future NMR dynamics studies of this protein.

Methods and experiments Protein expression and purification
For recombinant protein production the vector pET28b containing codon-optimized DNA of Bet v 1.0102 was used. The expression and purification protocol was adapted from Wallner et al. (2009). Protein expression in E. coli strain BL21 (DE3) Star was carried out in 1 l M9 minimal medium including 25 µg/ml kanamycin enriched with 15 NH 4 Cl (Cambridge Isotope Laboratories) and 13 C 6 -d-glucose (Sigma-Aldrich) for isotope labeling at 37 °C. After induction with 0.5 mM IPTG at OD 600 = 0.6-0.8, the culture was grown overnight at 16 °C and harvested by centrifugation at 2050×g. For cell resuspension a buffer containing 25 mM imidazole, 0.1% (v/v) Triton ×100 and 0.5 M urea was used. The cells were lysed by three cycles of freezing (liquid nitrogen) and thawing (water bath at 37 °C). First, DNAse I (1 µg/ml) and after 20 min of shaking, solid NaCl was added to a final concentration of 200 mM. The mixture was centrifuged at 9500×g for 30 min and the supernatant was collected. Under constant stirring on ice solid NaCl and NaH 2 PO 4 were slowly added to a concentration of 1 and 0.5 M, respectively. After 2 h of stirring the precipitated proteins were collected at 9500×g for 30 min and the supernatant containing Bet v 1.0102 was filtered through a 45 µm filter. Bet v 1.0102 was purified by hydrophobic interaction  N57δ2 N82δ2 * δ δ chromatography using 3 × 5 ml phenylsepharose columns (HiTrap™ Phenyl FF, GE Healthcare) at a flow rate of 2 ml/ min. After the protein solution was loaded, a linear gradient of 30 ml with 0.2 M NaH 2 PO 4 and 1 M NaCl, pH 4.2 was performed to remove urea. For protein elution, a linear gradient of 180 ml using 25 mM TrisHCl pH 9.3, 8% (v/v) 2-propanol was applied. Bet v 1.0102 containing fractions were collected and concentrated by Amicon Ultra Centrifugal Filters with a cut-off of 3000 Da (Millipore). As final purification step size exclusion chromatography was performed using a 120 ml gel filtration column (HiLoad 16/600 Superdex 75 prep grade, GE Healthcare) with 5 mM sodium phosphate buffer, pH 8.0 at a flow rate of 1 ml/min. An ÄKTAprime™ plus chromatography system (GE Healthcare) was used for all chromatographic steps with an integrated UV-detector for protein detection at 280 nm. The protein concentration was determined using a NanoPhotometer ® (Implen). Expression and purification of Bet v 1.0102 were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% gels. Purified Bet v 1.0102 was dialyzed against 50 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0 for NMR resonance assignment experiments.

Assignment and secondary structure
The 1 H-15 N-HSQC spectrum of Bet v 1.0102 is shown in Fig. 1. Backbone amide 1 H-15 N resonance assignment was obtained for 143 out of 151 non-proline residues, which corresponds to 94.7%. In addition, 96.9% of Cα, 96.9% of Cβ and 94.0% of CO resonances were assigned, and assignment of side chain protons is 87.4% complete. Furthermore, side-chain 13 C resonances beyond β-positions and side-chain amides in Asn and Glu ( 15 N and 1 H) were assigned partially. The chemical shift data of Bet v 1.0102 have been deposited at the Biological Magnetic Resonance Data Bank (BMRB) with the accession number 27040.
Using the Bet v 1.0102 chemical shifts (HN, N, Cα, Cβ and CO), the secondary structure elements of the protein were predicted by TALOS+ (Shen et al. 2009) (Fig. 2a). Our data show that this protein contains seven β-strands (β1−β7) and two short helices (α1−α2), as well as a long C-terminal helix (α3). In addition, a three-dimensional model of the The secondary structure probability is reflected by the height of the bars (blue β-strands, red α-helices). Unassigned backbone amide NH resonance are indicated by asterisks. For comparison, the secondary structure elements of Bet v 1.0101 (α1-α3 and β1-β7) are shown above. B Lowest energy CS-ROSETTA structure model of Bet v 1.0102. Amino acids that are different in Bet v 1.0102 compared to Bet v 1.0101 are highlighted in red (T7I, F30V, S57N, I91V, S112C, I113V, D125N) protein was generated using CS-ROSETTA (Lange et al. 2012) (Fig. 2b). In this structure, Bet v 1.0102 consists of a long C-terminal helix that is embraced by a curved, seven-stranded antiparallel β-sheet. Helices α1 and α2 form a V-shaped support for helix α3, creating a large internal cavity, consistent with the canonical PR-10 fold (Fernandes et al. 2013).
Taken together, the Bet v 1.0102 chemical shift data indicate no significant structural difference between the hypoallergenic isoform Bet v 1.0102 and the hyperallergenic isoform Bet v 1.0101 (Gajhede et al. 1996). However, the observed variability in resonance intensities is an indication for the dynamic nature of this protein. A detailed NMR spectroscopic investigation of the dynamic properties of Bet v 1 proteins in the context of their observed immunologic features is currently being performed at our laboratory. In addition, the NMR chemical shift assignments of Bet v 1.0102 build the basis for epitope mapping and ligand binding studies.