Functional characterization of ECP-heparin interaction: a novel molecular model

• Published in: PloS one Vol. 8; no. 12; p. e82585
• Main Authors: Hung, Ta-Jen, Tomiya, Noboru, Chang, Tse-Hao, Cheng, Wen-Chi, Kuo, Ping-Hsueh, Ng, Sim-Kun, Lien, Pei-Chun, Lee, Yuan-Chuan, Chang, Margaret Dah-Tsyr
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11-12-2013; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Amino acids; Anticoagulants; Asthma; B cells; Binding; Binding proteins; Binding Sites; Biochemistry; Bioinformatics; Calorimetry; Catalysis; Cellular biology; Eosinophil cationic protein; Eosinophil Cationic Protein - chemistry; Eosinophil Cationic Protein - genetics; Eosinophil Cationic Protein - metabolism; Eosinophils; Eosinophils - enzymology; Fibroblasts; Gene Expression; Granulocytes; Growth factors; Heparan sulfate; Heparin; Heparin - chemistry; Heparin - metabolism; Humans; Inflammatory diseases; Leukocytes (eosinophilic); Mathematical models; Models, Molecular; Molecular Sequence Data; Oligosaccharides - chemistry; Oral contraceptives; Predictions; Protein Binding; Protein Conformation; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Residues; Ribonuclease; Ribonuclease A; Sequence Alignment; Structure-function relationships; Thermodynamics; Titration; Titration calorimetry; United States > US; Maryland; China; Baltimore Maryland; Taiwan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0082585

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.