Figure Legends:
Figure 1:  PN and WN VBPaments adopt a common α-hairpin structure despite low sequence identity. A) NMR structures of J2LS and A1LS VBPs. Cartoon figures depict representative conformations of each structure. Inter-helix disulfides shown as sticks and highlighted in yellow. Complete NMR data tables are available in <blank>. B) Schematic view illustrating the disulfide bonding pattern for all three fragments. Trypsin cut sites identified by Downs et. al.  are indicated by the yellow arrows.  C) Identity matrix for the amino acid sequence of the VBPs.
Figure 2:  Potential PN cross-reactive regions on A1LS. A) Sequence of A1LS used in the peptide microarray analysis. Residues depicted in the solution-NMR structures are shown in capital letters. Residues from the expression system used to generate the NMR samples but are not present in the peptide microarrays are denoted with brackets. Residues highlighted in blue represent alpha-helices identified in the available NMR structures. Epitopes identified by Burks et. al are indicated in grey rectangles, whereas cross-reactive peptides identified are indicated by coloured rectangles. AEP cut site is indicated by the grey arrow. B) Structure of A1LS coloured by surface similarity (dSIM) values against J2.1, J2.2, and J2.3 as calculated using the SPADE surface comparison algorithm. Total dSIM (Σ[ΔSIM] ) for all residues is indicated. C) IgE-reactive peptides mapped onto the structure of A1LS. Peptides colour-coded as in (A).
Figure 3:  Potential PN cross-reactive regions on J2LS.A) Structure of J2.1, J2.2, and J2.3 coloured by surface similarity (dSIM) values against A1LS as calculated using the SPADE surface comparison algorithm. Total dSIM (Σ[ΔSIM] ) for all residues is indicated. B) Sequence of J2LS, showing the three repeat areas (Nesbit 2020), with helical areas highlighted in blue, residues used to obtain the 31 mer consensus sequence underlined, and the central 13mer consensus in bold letters. Residues included in the solution-NMR structures are capitalized; those included in the NMRsamples but not present in the peptide microarrays are denoted in parentheses. Epitopes identified by Maleki et. al 2011 are indicated in grey rectangles, with cross-reactive peptides identified in this study (Fig. 4B) shown in coloured rectangles labeled JR5-JR9. The AEP cut site is indicated by the grey arrow. C) IgE-reactive peptides mapped onto the structure of J2.1, J2.2, and J2.3. Peptides colour-coded as in (A). For the sake of simplicity, only JR6-JR9 are shown.
Figure 4: Comparison of IgE binding to the A1LS and J2LS among walnut (WN), peanut (PN), and dual allergic (PW) patients by microarray analysis . A) Modified z values were calculated from the microarray median signal-to-noise ratios (SNRs) and were used to determine the percentage of IgE binding within an amino acid range (shown in 2nd column) for each allergy group. B) The percentage of patients which bind IgE to peptides and major epitopes of A1LS and J2.1 and J2.2 is shown. A major epitope was identified as a peptide where 50% or more of the P, W or PW patients exhibited a positive binding event (z value of 3 or greater) for that peptide. The epitopes marked with an asterisk were recognized by all three allergy groups. The shared regions of overlapping peptides are underlined. Average z values are given as low (3 – 6), medium (6.1 – 9) and high (>9 and in red text). Amino acid composition is shown as percentage of hydrophobic (H), acidic (A), basic (B) or neutral (N) amino acids in the peptide sequence.
Figure 5: Property distance (PD)-graph showing the interrelatedness of the IgE binding epitopes of the LS to those of the 2S albumins of peanuts and tree nuts. The sequences of the LS peptides identified as IgE binding in this study (Figure 4B) are similar in their similar PCPs to the consensus and previously identified epitopes from PN and TN 2S albumins. The PD graph is the graphical depiction of the most similar sequences, which are connected by blue lines, and the lines descrease in intensity and width with increasing PD (lower similarity). Note that the previously determined PCP-consensus sequences (Nesbit et al. 2020) lie in the middle of the graph, and that the LS IgE reactive peptides show similarity to the 2S albumins.