The human
eosinophil cationic protein (ECP), also known as
RNase 3, is an eosinophil secretion
protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial
lipopolysaccharides and
heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with
sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for
ligand binding studies. By exploring the
protein-
sulfate complex, we have defined the
sulfate binding site architecture. Three main sites (S1-S3) are located in the
protein active site; S1 and S2 overlap with the
phosphate binding sites involved in
RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated
ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of
ligand anionic groups. The location of additional
sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic
polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for
glycosaminoglycans and can assist in structure-based
drug design of inhibitors of the
protein cytotoxicity to host tissues during
inflammation.