Crystal structure of the GalNAc/Gal-specific agglutinin from the phytopathogenic ascomycete Sclerotinia sclerotiorum reveals novel adaptation of a beta-trefoil domain

Abstract

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of approximately 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-beta1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 A resolution, respectively. SSA adopts a beta-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site alpha. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the beta-trefoil domain in the evolution of fungal lectins.

Figure 1

Overall view of the structure of SSA. (A) Overall structure of the SSA monomer showing the three subdomains, denoted α, β and γ viewed in two orientations rotated by 90°, and colored green, cyan and orange, respectively. β-strands of a subunit are labeled β1 to β12. Dashed lines indicate the disordered β8-β9 loop region in the apo SSA structure. (B) Structure-based sequence alignment of the three individual subdomains of SSA; residues involved in carbohydrate binding at site α are shown by (H).

Figure 2

Carbohydrate binding site at site α. (A) Overall view of the dimer with bound Galβ1,3GalNAc in each subunit. (B) Close-up views of the carbohydrate binding site in (left) the apo form with bound glycerol (orange) and (middle) the complex with bound Galβ1,3GalNAc (orange), viewed in a similar orientation. The 1.95 Å resolution 2mFo-DFc electron density map (cyan) is contoured at 1 σ. Molecular surface (right) showing the shallow binding site. The position of the docked α-GalNAc (green) is displayed and overlaid with bound Galβ1,3GalNAc. Side chains that directly interact with the disaccharide are labeled. (C) Overall view (left) of the Marasmius oreades agglutinin (MOA) dimer with bound blood group B Galα1,3(Fucα1,2)Gal trisaccharide (green) (accession code 3EF2). The N- and C-terminal domains from each subunit are colored in yellow and green, respectively. Close-up stereo view (right) of the overlay of the carbohydrate binding sites in SSA and MOA. (D) Overall view (left) of the EW29 lectin with bound GalNAc (blue) (accession code 2ZQO). Close-up stereo view (right) of the overlay of the carbohydrate binding sites, oriented as in C, in SSA and EW29. Side chains that directly interact with the bound carbohydrate in EW29 and MOA are labeled

Figure 3

Sequence alignment of SSA with homologs from Botryotinia fuckeliana (Bf-SSA, accession EDN28997.1), Pyrenophora tritici-repentis (Ptr-SSA, accession EDU42915.1) Cochliobolus heterostrophus (Ch-SSA, accession FK679805.1), as putative representative members of this new class of fungal lectins. Identical residues are indicated with a black background while similar residues are boxed. Amino acids involved in carbohydrate binding and dimer assembly of SSA are indicated by (H) and (σ), respectively. Secondary structure elements are indicated above the sequence.