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The Structural Flexibility of PR-10 Food Allergens

Führer, Sebastian, Unterhauser, Jana, Zeindl, Ricarda, Eidelpes, Reiner, Fernández-Quintero, Monica L., Liedl, Klaus R., Tollinger, Martin·2022·International Journal of Molecular Sciences
DOI 10.3390/ijms23158252PMC PMC9330593
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Abstract

PR-10 proteins constitute a major cause of food allergic reactions. Birch-pollen-related food allergies are triggered by the immunologic cross-reactivity of IgE antibodies with structurally homologous PR-10 proteins that are present in birch pollen and various food sources. While the three-dimensional structures of PR-10 food allergens have been characterized in detail, only a few experimental studies have addressed the structural flexibility of these proteins. In this study, we analyze the millisecond-timescale structural flexibility of thirteen PR-10 proteins from prevalent plant food sources by NMR relaxation-dispersion spectroscopy, in a comparative manner. We show that all the allergens in this study have inherently flexible protein backbones in solution, yet the extent of the structural flexibility appears to be strikingly protein-specific (but not food-source-specific). Above-average flexibility is present in the two short helices, α1 and α2, which form a V-shaped support for the long C-terminal helix α3, and shape the internal ligand-binding cavity, which is characteristic for PR-10 proteins. An in-depth analysis of the NMR relaxation-dispersion data for the PR-10 allergen from peanut reveals the presence of at least two subglobal conformational transitions on the millisecond timescale, which may be related to the release of bound low-molecular-weight ligands from the internal cavity.

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Figure 1 Sequence-identity matrix of thirteen selected PR-10 allergens from birch pollen (Betula verrucosa, Bet v 1), apple (Malus domestica, Mal d 1), golden kiwi fruit (Actinidia chinensis, Act c 8), green kiwi fruit (Actinidia deliciosa, Act d 8), plum (Prunus domestica, Pru d 1), peach (Prunus persica, Pru p 1), peanut (Arachis hypogaea, Ara h 8), and hazelnut (Corylus avellana, Cor a 1). The structure of Bet v 1 (PDB: 4A88, isoform Bet v 1.0101) is displayed above. Secondary-structure elements and the nine connecting loops (L1–L9) are labeled, and N- and C-termini are indicated. The main entrance (ε1) to the inner cavity of the protein is indicated by an arrow.
Figure 1 Sequence-identity matrix of thirteen selected PR-10 allergens from birch pollen (Betula verrucosa, Bet v 1), apple (Malus domestica, Mal d 1), golden kiwi fruit (Actinidia chinensis, Act c 8), green kiwi fruit (Actinidia deliciosa, Act d 8), plum (Prunus domestica, Pru d 1), peach (Prunus persica, Pru p 1), peanut (Arachis hypogaea, Ara h 8), and hazelnut (Corylus avellana, Cor a 1). The structure of Bet v 1 (PDB: 4A88, isoform Bet v 1.0101) is displayed above. Secondary-structure elements and the nine connecting loops (L1–L9) are labeled, and N- and C-termini are indicated. The main entrance (ε1) to the inner cavity of the protein is indicated by an arrow.CC BY · PubMed Central
Figure 2 Clustering based on the charge profiles and hydrogen-bond donor/acceptor capacities of the thirteen PR-10 allergens. Cluster 1 comprises the proteins Cor a 1.0401, Cor a 1.0404, Mal d 1.0101, and Pru p 1.0101; Cluster 2 comprises Ara h 8.0101, Act d 8.0101, Bet v 1.0101, Cor a 1.0402, Cor a 1.0403, Pru d 1.0101, Pru p 1.0201, and Pru p 1.0301; Cluster 3 only contains Act c 8.0101. The surfaces are color-coded following the YRB highlighting scheme of Hagemans et al. [25]. The color-coding of positively and negatively charged residues, hydrophobic residues, and residues with hydrogen-bond capacities is specified. All three clusters are shown in the same orientation, with the C-terminal α-helix facing to the front, with its C-terminus pointing down.
Figure 2 Clustering based on the charge profiles and hydrogen-bond donor/acceptor capacities of the thirteen PR-10 allergens. Cluster 1 comprises the proteins Cor a 1.0401, Cor a 1.0404, Mal d 1.0101, and Pru p 1.0101; Cluster 2 comprises Ara h 8.0101, Act d 8.0101, Bet v 1.0101, Cor a 1.0402, Cor a 1.0403, Pru d 1.0101, Pru p 1.0201, and Pru p 1.0301; Cluster 3 only contains Act c 8.0101. The surfaces are color-coded following the YRB highlighting scheme of Hagemans et al. [25]. The color-coding of positively and negatively charged residues, hydrophobic residues, and residues with hydrogen-bond capacities is specified. All three clusters are shown in the same orientation, with the C-terminal α-helix facing to the front, with its C-terminus pointing down.CC BY · PubMed Central
Figure 3 Structural flexibility of thirteen selected PR–10 allergens. (A) Bar plot showing the percentages of residues with backbone amide 15N relaxation-dispersion amplitudes (∆R2,eff) exceeding 1 s–1 at 700 MHz. Bars are color-coded according to ranges of ∆R2,eff values. (B) Residue-specific mean values of ∆R2,eff over all thirteen PR–10 proteins are plotted on the structure of Bet v 1.0101 (PDB: 4A88), color-coded from rigid (white) to flexible (red). (C) Heat map comparing backbone amide 15N RD amplitudes vs. residue numbers, colored according to the shown gradient. Proteins are ordered as in (A). Grey squares indicate sequence positions for which experimental data are not available (i) due to gaps in a specific PR–10 protein sequence, (ii) because of the resonance overlap of backbone amides, (iii) for prolines, or (iv) in cases where resonance assignments are not available.
Figure 3 Structural flexibility of thirteen selected PR–10 allergens. (A) Bar plot showing the percentages of residues with backbone amide 15N relaxation-dispersion amplitudes (∆R2,eff) exceeding 1 s–1 at 700 MHz. Bars are color-coded according to ranges of ∆R2,eff values. (B) Residue-specific mean values of ∆R2,eff over all thirteen PR–10 proteins are plotted on the structure of Bet v 1.0101 (PDB: 4A88), color-coded from rigid (white) to flexible (red). (C) Heat map comparing backbone amide 15N RD amplitudes vs. residue numbers, colored according to the shown gradient. Proteins are ordered as in (A). Grey squares indicate sequence positions for which experimental data are not available (i) due to gaps in a specific PR–10 protein sequence, (ii) because of the resonance overlap of backbone amides, (iii) for prolines, or (iv) in cases where resonance assignments are not available.CC BY · PubMed Central
Figure 4 Backbone amide NMR resonance assignments and relaxation-dispersion data for Ara h 8.0101. (A) The 700 MHz 1H–15N–HSQC spectrum of Ara h 8.0101 (0.5 mM) in 20 mM sodium phosphate (pH: 6.9), supplemented with 10% D2O, at 25 °C. Assignments are shown using single–letter codes. Horizontal lines indicate asparagine and glutamine NH2 side–chain resonances. The resonance labeled by one asterisk indicates a residue below the intensity cutoff, the signal labeled by two asterisks indicates the aliased signal of the single arginine sidechain ε–NH. (B) Structure of Ara h 8.0101 (PDB: 4MA6) with backbone amides that were used in global fits of the 15N relaxation-dispersion data in the β-sheet and three α-helices, highlighted in orange and blue, respectively. (C,D) Backbone amide 15N relaxation-dispersion data for eight representative amino acid residues in the β-sheet (C) and in helices α1–α3 (D). Exchange contributions (Rex), which are calculated as R2,eff – R2,eff (νCPMG = ∞), are shown, along with the best fit-lines, which were obtained separately from the global fits of all the residues in (C,D), respectively.
Figure 4 Backbone amide NMR resonance assignments and relaxation-dispersion data for Ara h 8.0101. (A) The 700 MHz 1H–15N–HSQC spectrum of Ara h 8.0101 (0.5 mM) in 20 mM sodium phosphate (pH: 6.9), supplemented with 10% D2O, at 25 °C. Assignments are shown using single–letter codes. Horizontal lines indicate asparagine and glutamine NH2 side–chain resonances. The resonance labeled by one asterisk indicates a residue below the intensity cutoff, the signal labeled by two asterisks indicates the aliased signal of the single arginine sidechain ε–NH. (B) Structure of Ara h 8.0101 (PDB: 4MA6) with backbone amides that were used in global fits of the 15N relaxation-dispersion data in the β-sheet and three α-helices, highlighted in orange and blue, respectively. (C,D) Backbone amide 15N relaxation-dispersion data for eight representative amino acid residues in the β-sheet (C) and in helices α1–α3 (D). Exchange contributions (Rex), which are calculated as R2,eff – R2,eff (νCPMG = ∞), are shown, along with the best fit-lines, which were obtained separately from the global fits of all the residues in (C,D), respectively.CC BY · PubMed Central
Figure 5 NMR spectroscopic analysis of binding of (–)–epicatechin to Ara h 8.0101. (A) Binding curves for two representative amino acid residues (Ile56 in strand β3, and Ile145 in helix α3). Site-specific KD values were derived from ligand-induced 1H and 15N chemical-shift perturbations (Δδobs) by nonlinear least-squares fitting [33]. (B) Structure and numbering scheme of (–)–epicatechin. (C) X-ray structure of Ara h 8.0101 in complex with (–)–epicatechin (PDB: 4MA6). Amino acid residues in red show backbone amides with Δδobs > 0.1 ppm upon (-)-epicatechin binding that were used to determine mean value and standard deviation of KD. (D) Ligand-observed 1H–CPMG relaxation-dispersion profiles, recorded at 600 MHz, for (-)-epicatechin binding to Ara h 8.0101. Data are shown for protons 1H–6/8 (indicated in green in panel (B)), and for increasing saturation (0–12%) of the ligand by protein. Solid lines represent the global fit to the data, yielding the off-rate (koff) of the (-)-epicatechin from the complex.
Figure 5 NMR spectroscopic analysis of binding of (–)–epicatechin to Ara h 8.0101. (A) Binding curves for two representative amino acid residues (Ile56 in strand β3, and Ile145 in helix α3). Site-specific KD values were derived from ligand-induced 1H and 15N chemical-shift perturbations (Δδobs) by nonlinear least-squares fitting [33]. (B) Structure and numbering scheme of (–)–epicatechin. (C) X-ray structure of Ara h 8.0101 in complex with (–)–epicatechin (PDB: 4MA6). Amino acid residues in red show backbone amides with Δδobs > 0.1 ppm upon (-)-epicatechin binding that were used to determine mean value and standard deviation of KD. (D) Ligand-observed 1H–CPMG relaxation-dispersion profiles, recorded at 600 MHz, for (-)-epicatechin binding to Ara h 8.0101. Data are shown for protons 1H–6/8 (indicated in green in panel (B)), and for increasing saturation (0–12%) of the ligand by protein. Solid lines represent the global fit to the data, yielding the off-rate (koff) of the (-)-epicatechin from the complex.CC BY · PubMed Central

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Metadata

Authors
Führer, S., Unterhauser, J., Zeindl, R., Eidelpes, R., Fernández-Quintero, M. L., Liedl, K. R., & Tollinger, M.
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2022-07-26
Volume
23
Issue
15
Pages
8252
ISSN
1422-0067
DOI
10.3390/ijms23158252
PMC
PMC9330593
Cited by
24
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Austrian Science Fund FWF (P26849 to M.T., P30737 to K.R.L., 858017)
  • Austrian Research Promotion Agency FFG (P26849 to M.T., P30737 to K.R.L., 858017)

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Führer, S., Unterhauser, J., Zeindl, R., Eidelpes, R., Fernández-Quintero, M. L., Liedl, K. R., & Tollinger, M. (2022). The Structural Flexibility of PR-10 Food Allergens. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms23158252