Structural basis for saccharide binding by human RNase 2/EDN: Insights into its potential lectin-like properties

Human RNase 2, also named the eosinophil-derived neurotoxin (EDN), is a secretory protein of the vertebrate-specific RNase A superfamily involved in antiviral and proinflammatory cell response. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. By X-ray crystallography we first identified a conserved site at the protein surface binding to carboxylic anion molecules: malonate, tartrate and citrate. Searching for potential biomolecules rich in anion groups and considering previous report of EDN interaction to glycosaminoglycans, we explored the protein binding to saccharides. EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein bound ligands for glucose, fucose, mannose, galactose, trehalose, sucrose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main interaction site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid and mannose shared another close by protein surface region. Overall, we located 17 saccharide ligands that clustered into seven defined sites, outlining an extended interaction interface, which was further analysed by molecular modelling. Interestingly, within the RNase A superfamily, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first structural characterization of putative sugar binding sites for a mammalian RNase family member. The results highlight unique EDN residues that can mediate sugar interactions, of potential interest to further explore the protein physiological role.