Insights into the Glycosaminoglycan-Mediated Cytotoxic Mechanism of Eosinophil Cationic Protein Revealed by NMR

Abstract

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP’s toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-d-GlcNS6S-α(1–4)-l-IdoA2S-α(1–4)-d-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The 2S0 conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34–Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparin’s inhibition of ECP’s ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECP’s toxicity implicated in eosinophil pathologies.

Pharmacology of Heparin and Related Drugs

“…However, the catalytic triad of the RNase active site is involved in the interaction with heparin. As has been proposed for MBP, ECP may be concentrated at the cell surface by binding to HS, and the interaction may orient the protein in such a way as to aid destabilization of the cell membrane (García-Mayoral et al, 2013). Inappropriate release of such cytotoxic proteins during eosinophilic diseases such as asthma and rhinitis has been implicated in the damage to the respiratory epithelial cells characterizing these conditions.…”

“…Binds to catalytic amino acid triad, explaining heparin inhibition of ribonucleolytic activity (García-Mayoral et al, 2013) EMBP Crystal structure with disaccharide (2BRS.pdb) Calcium-independent binding to this C-type lectin (Swaminathan et al, 2001) Enzymes Glucuronyl C5-epimerase Crystal structure with heparin hexasaccharide (4PXQ.pdb)…”

“…Another eosinophil-granule protein, eosinophil cationic protein (ECP), also binds heparin (Fredens et al, 1991;Torrent et al, 2011) and the structure of a complex of ECP with a synthetic heparin trisaccharide has been determined by NMR spectroscopy and molecular dynamics (2LVZ) (García-Mayoral et al, 2013). ECP is a member of the RNase A superfamily, but its cytotoxicity is not a function of its weak RNase activity.…”

Current structural biology of the heparin interactome

“…Mammalian GAGs are said to be recognized as 'self' by the immune system, so that some bacteria have evolved 28 ], and human beta-defensins (HBDs) [29,30 ] were investigated by NMR titration experiments using heparin oligosaccharides. From these works, the preferable heparin binding sites were revealed, and the consensus BBXB binding motif as commonly seen in many HBPs, was shown to be present also in the immune-related proteins able to interact with GAGs.…”

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy

“…Although for many of the protein⅐GAG systems described in the literature, the determination of the GAG binding site was easily possible by measuring changes of the backbone NMR signals (65,66), the chemical shift perturbation data presented here can only provide a rather unclear binding model. Most of the affected IL-10 residues identified via chemical shift perturbation analysis are not solvent-exposed and should therefore be taken with caution as direct indicators of the primary site of interaction.…”