Structural and ligand binding analysis of the pet allergens Can f 1 and Fel d 7

Abstract

Introduction: Pet lipocalins are respiratory allergens with a central hydrophobic ligand-binding cavity called a calyx. Molecules carried in the calyx by allergens are suggested to influence allergenicity, but little is known about the native ligands.

Methods: To provide more information on prospective ligands, we report crystal structures, NMR, molecular dynamics, and florescence studies of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7.

Results: Structural comparisons with reported lipocalins revealed that Can f 1 and Fel d 7 calyxes are open and positively charged while other dog lipocalin allergens are closed and negatively charged. We screened fatty acids as surrogate ligands, and found that Can f 1 and Fel d 7 bind multiple ligands with preferences for palmitic acid (16:0) among saturated fatty acids and oleic acid (18:1 cis-9) among unsaturated ones. NMR analysis of methyl probes reveals that conformational changes occur upon binding of pinolenic acid inside the calyx. Molecular dynamics simulation shows that the carboxylic group of fatty acids shuttles between two positively charged amino acids inside the Can f 1 and Fel d 7 calyx. Consistent with simulations, the stoichiometry of oleic acid-binding is 2:1 (fatty acid: protein) for Can f 1 and Fel d 7.

Discussion: The results provide valuable insights into the determinants of selectivity and candidate ligands for pet lipocalin allergens Can f 1 and Fel d 7.

Keywords: allergen; cat; dog; lipocalin; structure.

Figure 1

Structures of Can f 1 and Fel d 7. (A) Structure of Can f 1 (C118S) (left) and WT Fel d 7 (right). (B) Can f 1 (C118S) and WT Fel d 7 were superimposed with RMSD value of 1.0 Å. (C) Sequence alignment of Can f 1 and Fel d 7. Key charged residues in the calyx R101 and K131 for Can f 1, and R102 and K132 for Fel d 7 are colored purple. Other residues lining the calyx are highlighted in yellow. Secondary elements are depicted as green cylinder for α-helix and blue arrows for β-strands.

Figure 2

Electrostatic surface of lipocalins. Electrostatic surface of (A) Can f 1, (B) Fel d 7, (C) Can f 2, (D) Can f 4, (E) Can f 6 are calculated using ABPS module in PyMol. The central calyx is shown with the arrow in A. Can f 1 and Fel d 7 show an open calyx while Can f 2, Can f 4, and Can f 6 show closed calyxes.

Figure 3

ANS binding to Can f 1 and Fel d 7. (A) The far-UV CD spectra of the wild-type and mutant proteins are shown. (B) The CD melting curve of wild-type and mutant proteins are measured at 210 nm in PBS buffer pH 7.4. The fluorescence increase upon ANS titration is shown for (C) WT Can f 1, Can f 1 (K131A), (D) WT Fel d 7, and Fel d 7 (K132A).

Figure 4

Can f 1 and Fel d 7 ligands. (A) WT Can f 1 and WT Fel d 7 are screened against a fatty acid library using ANS-displacement assay. The % reduction of fluorescence in the presence of 1 µM fatty acids is shown in blue for WT Can f 1 and red for WT Fel d 7 ligands, respectively. All measurements are duplicated, and the error bar indicate standard deviation. The labels on the x-axis correspond to the ligands in Supplementary Table S2. Arrows indicate A2, A4, D10, and H5. (B) The correlation plot shows a linear relationship between Can f 1 and Fel d 7 ligands. (C) The residual plot shows the difference between Can f 1 and Fel d 7 displacement for each ligand. (D) The % fluorescence reduction in the presence of the saturated fatty acids of certain carbon length. Note that 16 carbon fatty acid shows the largest % fluorescence reduction, suggesting the greatest binding capability. (E) The unsaturated fatty acids binding to WT Can f 1 and WT Fel d 7 is shown with the carbon length (first number) and the position of double bond (second number). (F) Further analysis shows the % fluorescence reduction for pinolenic acid with/without ester modification. Bar and whiskers plots indicate error bars.

Figure 5

Heteronuclear single quantum coherence (HSQC) spectra. The HSQC spectra of Can f 1 (A) and Fel d 7 (B) show chemical shift perturbations for hydrophobic residues in the absence (red) and presence (black) of 200 µM pinolenic acid for WT Can f 1. The similar trend is shown in the absence (orange) and presence (purple) of 200 µM pinolenic acid for the WT Fel d 7.

Figure 6

Molecular modeling of Can f 1 and Fel d 7. Models of Can f 1 with oleate (A & B), and 2 oleate molecules (C). Models of Fel d 7 with oleate (D), pinolenic acid (E), and 2 oleate molecules (F). Ligands are shown as sphere models, and key side chains of residues R101 and K 131 for Can f 1, and R102 and K132 for Fel d 7 are rendered as sticks.