NMR structural determinants of eosinophil cationic protein binding to membrane and heparin mimetics.
Eosinophil cationic protein (ECP) is a highly stable, cytotoxic ribonuclease with the ability to enter and disrupt membranes that participates in innate immune defense against parasites but also kills human cells. We have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics at a residue level. We believe we have identified three Arg-rich surface loops and Trp(35) as crucial for membrane binding. Importantly, we have provided evidence that the interaction surface of ECP with heparin mimetics is extended with respect to that previously described (fragment 34-38). We believe we have identified new sites involved in the interaction for the first time, and shown that the N-terminal alpha-helix, the third loop, and the first and last beta-strands are key for heparin binding. We have also shown that a biologically active ECP N-terminal fragment comprising the first 45 residues (ECP1-45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP tertiary structure nor its high conformational stability are required for cytotoxicity.
García-Mayoral
María Flor
MF
Instituto de Química-Física "Rocasolano", CSIC, Madrid, Spain.
Moussaoui
Mohammed
M
de la Torre
Beatriz G
BG
Andreu
David
D
Boix
Ester
E
Nogués
M Victòria
MV
Rico
Manuel
M
Laurents
Douglas V
DV
Bruix
Marta
M
D032701:Biomimetics; D047150:Eosinophil Cationic Protein / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D006493:Heparin / Q000737:chemistry*; D008566:Membranes / Q000737:chemistry*; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010455:Peptides / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water / Q000737:chemistry
D008823:Micelles; D010455:Peptides; D011522:Protons; D014867:Water; D010767:Phosphorylcholine; C028810:dodecylphosphocholine; D014270:Trifluoroethanol; D006493:Heparin; D047150:Eosinophil Cationic Protein