Alternaria alternata allergen Alt a 1: A unique β-barrel protein dimer found exclusively in fungi

Abstract

Background Alternaria is one of the most common molds associated with allergic diseases and 80% of Alternaria-sensitive patients produce IgE antibodies to a major protein allergen, Alt a 1. The structure and function of Alt a 1 is unknown. Objective To obtain a high resolution structure of Alt a 1 by X-ray crystallography and to investigate structural relationships between Alt a 1 and other allergens and proteins reported in the Protein Data Bank. Methods X-ray crystallography was used to determine the structure of Alt a 1 using a custom-designed set of crystallization conditions. An initial Alt a 1 model was determined by the application of a Ta6Br122+ cluster and Single-wavelength Anomalous Diffraction. Bioinformatic analyses were used to compare the Alt a 1 sequence and structure with other proteins. Results Alt a 1 is a unique β-barrel comprising 11 β-strands and forms a ‘butterfly-like’ dimer linked by a single disulfide bond, with a large (1345Å2) dimer interface. Intramolecular disulfide bonds are conserved among Alt a 1 homologs. Currently, the Alt a 1 structure has no equivalent in the Protein Data Bank. Bioinformatics analyses suggest that the structure is found exclusively in fungi. Four previously reported putative IgE binding peptides have been located on the Alt a 1 structure. Conclusions Alt a 1 has a unique, dimeric β-barrel structure that appears to define a new protein family with unknown function found exclusively in fungi. The location of IgE antibody binding epitopes is in agreement with the structural analysis of Alt a 1.The Alt a 1 structure will allow mechanistic structure/function studies and immunologic studies directed towards new forms of immunotherapy for Alternaria-sensitive allergic patients.

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“…Furthermore, the predicted structure suggests that the two remaining Cys residues, Cys24 and Cys29, which are absent from Alt a 1, may also form an intramolecular disulphide bond, given that they are located in close proximity to each other. This bond, however, would be in a different location to the second intramolecular disulphide bond of Alt a 1 (Cys104-Cys116) (Chruszcz et al 2012).…”

“…Three SSPs (Ecp20-1, Ecp20-2, and Ecp20-3) were consistently predicted to have structural homology to Alt a 1 (RCSB protein databank numbers 3V0R and 4AUD), an allergen protein with a b-barrel fold (Chruszcz et al 2012) from the broad host-range Dothideomycete fungal plant pathogen and saprophyte Alternaria alternata (Supplementary Table S2). Four SSPs (Ecp4, Ecp7, Ecp29 [CTR], and Ecp30) were consistently predicted to have structural homology to proteins with a b/g-crystallin fold, including the plant antimicrobial protein MiAMP1 from Macadamia integrifolia (1C01) (McManus et al 1999) and the yeast killer toxin WmKT from Williopsis mrakii (1WKT) (Antuch et al 1996).…”

“…For Ecp20-1 and Alt a 1, two conserved Cys residues at positions 50 and 65 (mature proteins), which are also present in Ecp20-2 and Ecp20-3, were identified. In Alt a 1, these Cys residues are known to form an intramolecular disulphide bond (Chruszcz et al 2012). Inspection of the predicted Ecp20-1 tertiary structure, which was modeled using Alt a 1 as a template in HHpred (MODELER) (Söding et al 2005;Webb and Sali 2002) and RaptorX (Källberg et al 2012), suggests that the conserved Cys50/Cys65 pair forms an intramolecular disulphide bond ( Supplementary Fig.…”

“…Remarkably, 10 of these SSPs were consistently predicted to have structural homology to proteins present in the RCSB protein databank. Of these, Ecp20-1, Ecp20-2, and Ecp20-3 were predicted to be structurally homologous to Alt a 1 from A. alternata, which adopts a b-barrel fold unique to fungi (Chruszcz et al 2012;De Vouge et al 1996). Recent studies have shown that Alt a 1 is an effector protein with multiple roles in promoting host colonization.…”

Alt a 1 from Alternaria interacts with PR5 thaumatin‐like proteins

“…The crystal structures recently determined for Alt a 1 [4] and Kiwi PR5 [22] proteins were used. Poisson-Boltzmann electrostatic potentials using AMBER atomic charges and radii assigned with PDB2PQR [23] were computed with the APBS program [24] to solve [26]) and PatchDock (bioinfo3d.cs.tau.ac.il/ PatchDock/ [27]) servers.…”