Results:
Commonα-hairpin fold provides a
structural basis for IgE cross-reactivity
Examining the A1LS sequence
reveals a single VBP motif with an additional pair of cysteines at both
N and C-terminals, which likely fold to form a single unit. In contrast,
the JR2LS contains three VBP motifs (Figure 1A and 1B). The J2LS
degrades into a series of 5-7 kDa fragments corresponding to the
individual α-hairpins [16]. Based on this background, the three J2LS
VBP motifs (J2.1, J2.2, J2.3) were produced recombinantly and their
structures analyzed individually. Circular dichroism (CD) spectra of all
four VBPs (A1LS, J2.1, J2.2, J2.3) show distinct minima at 222 and 210
nm consistent with predominantly α-helical secondary structure (S1).
Further studies on the full-length J2LS using small-angle X-ray
scattering (SAXS) yielded a radius of gyration of 25±2Å; significantly
greater than the 20Å expected for a globular protein of equivalent
molecular weight. Additionally, the shape of the corresponding Kratky
plot suggested a high degree of flexibility. This data supports the
proposed model in which J2.1, J2.2, and J2.3 could exist as
independently folded hairpins separated by unstructured linker regions
(S2), and validates our approach in examining each of these LSs as
separate entities rather than part of a larger globular system.
The structural similarity afforded by the common α-hairpin scaffold
could potentially give rise to cross-reactive epitopes despite the lack
of sequence identity (Figure 1C). To explore this possibility, the
structure of the WN and PN LSs was assessed using solution-NMR. Backbone
and side-chain assignments were obtained using standard triple-resonance
approaches. Analysis of the resulting chemical shifts using the TALOS
prediction algorithm reveals two α-helical regions centered on the CxxxC
motifs (S2). The downfield shift (>33 ppm) of the cysteine
Cb peaks, along with the presence of NOESY cross-peaks
between the Cb protons on opposite CxxxC repeats support
the presence of an α-hairpin disulfide bonding pattern (S3). The A1LS
contains an additional pair of cysteine residues located on either side
of the main CxxxC motif (Figure 1B), which were found to form a third
disulfide bond that was verified by mass-spectrometry (S1), explaining
the high stability and survival of the intact form in the seed. This
information, along with structural restraints derived from the available
NMR data was used to determine the 3D structure of all four LS
fragments. As shown in Figure 1A, all four constructs adopt an
unambiguous α-hairpin structure with low backbone RMSD values (S3). Few
long-range NOE interactions were detected beyond the disulfide bonded
cysteines in each of the 4 LS fragments; a rather unusual observation
for an ordered protein (S3). To further verify the calculated
structures, rotational correlation times (Tc) were
measured for all constructs (Figure 1A). The resulting values are
consistent with a >7 kDa globular protein and not with an
unfolded peptide. Taken together, this data suggests that A1LS, J2.1,
J2.2 and J2.3 each adopt a common α-hairpin structure maintained by the
conserved disulfide bonds. This architecture is common to the VBPs from
other plant species [14, 27, 28], and provides a consistent scaffold
that allows for permutations in amino acid sequence as shown by the low
sequence identity beyond the conserved CxxxC motifs.
Independently of the IgE binding analysis, the ability of these
α-hairpin structures to support cross-reactive epitopes was evaluated
using the SPADE computational tool developed by Dall’Antonia et
al. [29]. The A1LS amino acid sequence is shown (Figure 2A
and 2B) with previously identified epitopes and the location of the
overlapping peptides on the structure, respectively. The SPADE algorithm
takes into account the physical properties of each residue similar to
the PD metric employed for sequence analysis, but includes an additional
layer using the 3D structure to examine solvent accessible area and
spatial positioning from which regions of similarity corresponding to
putative cross-reactive 3D epitopes can be identified.
Comparing the structure of A1LS
with the three J2LS VBP’s reveal areas of high surface similarity (ΔSIM)
(Figure 2C), representing regions of potential cross-reactivity.
However, this similarity was markedly reduced in J2.3 despite the
conserved α-hairpin fold. The reverse comparison yields similar results
(Figure 3A), with JR2.3 once again displaying noticeably lower overall
(ΔSIM) values against A1LS than its stablemates. The amino acid sequence
of the J2LS VBPs and the location of the overlapping peptides are shown
in Figure 3B and 3C.