The Crystal Structure of Eosinophil Cationic Protein in Complex with 2‘,5‘-ADP at 2.0 Å Resolution Reveals the Details of the Ribonucleolytic Active Site^,

Abstract

Eosinophil cationic protein (ECP) is a component of the eosinophil granule matrix. It shows marked toxicity against helminth parasites, bacteria single-stranded RNA viruses, and host epithelial cells. Secretion of human ECP is related to eosinophil-associated allergic, asthmatic, and inflammatory diseases. ECP belongs to the pancreatic ribonuclease superfamily of proteins, and the crystal structure of ECP in the unliganded form (determined previously) exhibited a conserved RNase A fold [Boix, E., et al. (1999) Biochemistry 38, 16794-16801]. We have now determined a high-resolution (2.0 A) crystal structure of ECP in complex with adenosine 2',5'-diphosphate (2',5'-ADP) which has revealed the details of the ribonucleolytic active site. Residues Gln-14, His-15, and Lys-38 make hydrogen bond interactions with the phosphate at the P(1) site, while His-128 interacts with the purine ring at the B(2) site. A new phosphate binding site, P(-)(1), has been identified which involves Arg-34. This study is the first detailed structural analysis of the nucleotide recognition site in ECP and provides a starting point for the understanding of its substrate specificity and low catalytic efficiency compared with that of the eosinophil-derived neurotoxin (EDN), a close homologue.

Post-translational Tyrosine Nitration of Eosinophil Granule Toxins Mediated by Eosinophil Peroxidase

“…The root mean square deviation between the EPO model and the template MPO prior to energy minimization and after energy minimization is 0.38 and 0.89 Å, respectively. For ECP, structure coordinates from Mohan et al (35) (PDB code 1H1H) were used for nitro-Tyr modeling at the nitro-Tyr 33 residue. Cytotoxicity Assays-Confluent monolayers of the human alveolar type II cell line A549 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were used for testing the toxicity of nitrated and non-nitrated eosinophil granule toxins.…”

“…However, upon nitration of Tyr 349 , the flexibility of this loop is lost due to steric hindrance and the net electronegativity of the nitrated Tyr 349 residue likely prevents the stacking interactions with Tyr 555 , forcing the nitro group of Tyr 349 to be permanently exposed to the surface of the molecule. The spatial orientation of the nitro-Tyr residues in ECP and EDN were deduced via a similar strategy based on the available crystal structure for these proteins (35,36). These assessments showed that Tyr 33 residues of ECP and EDN were both surface exposed (Fig.…”

Topography Studies on the Membrane Interaction Mechanism of the Eosinophil Cationic Protein

“…c The mean values of chains A and B. d PDB entry code 1DYT (35). e PDB entry code 1H1H (36). much more reduced quenching capacity.…”

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

“…In contrast, the direct identification of nitrations without affinity-extraction using HPLC-MS, such as in the auto-nitration site of eosinophilperoxidase (EPO) at Tyr 349 required highly purified protein and was only obtained after scrutinous HPLC separation of Figure 3. Identification of the tyrosine nitration site at Tyr −33 in eosinophil-derived neurotoxin by nano-ESI-FTICR-MS of the thermolysine peptide fragmnent, EDN (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). The modification denoted with X corresponds to carbamidomethylation at Cys-37.…”

“…Based on the available crystal structures of ECP and EDN [43,44], the topologies and orientation of the identified nitrated tyrosine structures were assessed using the BallView molecular modeling program (Figure 4). Accessible surface areas were calculated for both proteins using the Surface Racer program [45].…”