Der p 5 crystal structure provides insight into the group 5 dust mite allergens

Abstract

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of approximately 3000 A(3) that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

FIGURE 1.

Asymmetric unit of the Der p 5 crystal structures. a, hexamer of Der p 5. There are six molecules of Der p 5 in the asymmetric unit of the crystal forming a trimer of dimers. The N terminus is designated, as well as the chain name of the molecules. b, alignment of the C-D and E-F dimers. c, alignment of the A-B and C-D dimers. The PDB code for the Der p 5 structure is 3MQ1.

FIGURE 2.

Cavity in the Der p 5 dimer. a, cavity in the A-B dimer is rendered with a solid surface, and the helices in the asymmetric unit are rendered as semi-transparent ribbons. b, zoomed region of a. Ordered density in the cavity was assumed to be either water or methylpentanediol; however, the electron density is poorly determined so it is rendered with a solid surface just to give an impression of the size of the cavity.

FIGURE 3.

Comparisons of Der p 5 with published Blo t 5 structures. The monomeric form of Der p 5 from chain A is displayed in a. b shows the first structure from 2JMH, and c is the first structure from 2JRK. Helix 1 is red; helix 2 is blue, and helix 3 is green. Circles at the bottom indicate the “handedness” of the structures, looking “down” the helical axis.

FIGURE 4.

Important residues for various inter-dimer interfaces. a shows the interface between A-B and C-D; b shows the interface between A-B and E-F, and c shows a crystallographic interface between C-D and E-F. Note that similar residues appear at each interface but in different conformations. Residues rendered with a thin line lack electron density and are modeled based on best allowed rotamer. F50_F has two conformations in the crystal structure.

FIGURE 5.

SAXS analysis. a shows the eight tested structures for fit to the SAXS data as described in the text. b, left panel, shows the theoretical fit of the SAXS data (red line) to the acquired scattering data (black squares with error bars) for the 2.5 mg/ml sample of Der p 5 and inset with the linear region of the Guinier fit. b, right panel, shows the P(r) plot for the same sample.

FIGURE 6.

Sequence comparisons. ClustalW alignments of Der p 21, Blo t 21, Blo t 5, and Der p 5 are shown. Color red are residues important for the Der p 5 intra-dimer interface; blue are residues important for inter-dimer interfaces, and green are residues involved in both types of interfaces. Asterisks indicate identity; colons indicate strong similarity, and periods indicate weaker similarity among the four proteins.